The immediate-type skin reaction and the emetic response in unsensitized monkeys on challenge with staphylococcal enterotoxin B (SEB) were studied to define the role of cysteinyl leukotrienes (LTs) in the action of the toxin. LY 171883, a selective LTD4/LTE4 receptor inhibitor, antagonized SEB-induced skin reactions and emetic responses completely. Inhibition of prostanoid formation by indomethacin, however, and pretreatment with BW 755C, a dual lipoxygenase and cyclooxygenase inhibitor, did not influence these reactions. The generation of endogenous cysteinyl LTs upon intragastric SEB administration was established in vivo. There was a tenfold increase in LTE4, the major biliary cysteinyl LT, and a novel cysteinyl LT metabolite in urine occurred, indicating strongly enhanced LT generation on SEB challenge. These results provide the first evidence that cysteinyl LTs may be important mediators in the pathophysiology of SEB-induced effects, as a model for pseudo-allergic reactions.

Astatine-211 is an alpha-emitter with a short half-life (7.2 hr). This paper discusses the potential of 211At targeted by antibodies for tumor therapy and the possible advantage of 211At over beta- and gamma-emitting radionuclides such as 131I currently employed in the field of radioimmunotherapy. Since the longest range alpha-particle from 211At is only 67 microns and the rate of energy loss is high (track averaged linear energy transfer LT approximately 120 keV/micron), a disintegration of 211At produces a large and extremely localized deposition of energy. A Monte-Carlo model has been developed for studying the stochastic fluctuation of alpha-particle hits and energy deposition in cell nuclei in an attempt to determine the efficacy of 211At-labeled antibodies for tumor cell inactivation. Calculations have been performed for 2 extreme conditions: (a) the case of 211At retained in the capillary, and (b) for a homogeneous distribution of 211At-labeled antibody in the tumor. The results of these two calculations represent the boundary conditions between which any real solution must lie. Finally, developments to the model to include antibody transport across the capillary membrane and through the tumor tissue are discussed.

Long term effects of in vivo treatment with human rIL-1 beta on diabetogenesis and thyroid disease were determined in the Biobreeding rat. Administration of high dose (10 micrograms/kg) IL-1 beta accelerated the onset of insulin-dependent diabetes mellitus compared to saline-injected controls. High dose treatment resulted in goiter development, pronounced LT, reduced serum T4 levels, and overall growth reduction. In contrast, low dose IL-1 beta (0.5 microgram/kg) administration significantly reduced the frequency of insulin-dependent diabetes mellitus (48%) compared to placebo (86%) and high dose IL-1 beta (93%) treatment groups. Rats protected by low dose IL-1 beta had unaffected growth rates and minimal to no pancreatic and thyroid pathology. Our results demonstrate that exogenous administration of IL-1 beta modulates Biobreeding rat idiopathic autoimmune diabetes and thyroid disease in a dose-dependent manner.

1. Factors associated with a lower rate of recurrent hepatitis B post-liver transplantation (LT) are negative hepatitis B e antigen and/or serum hepatitis B virus DNA pre-LT, hepatitis D virus superinfection, and fulminant hepatitis B. 2. Long-term intravenous hepatitis B immune globulin (HBIG) monotherapy can reduce the overall rate of recurrent hepatitis B to 20% to 35%. 3. Long-term lamivudine monotherapy is associated with a risk for drug resistance and overall 3-year rate of recurrent hepatitis B of 40% to 50%. 4. Combination prophylaxis with HBIG and lamivudine can reduce the overall rate of recurrent hepatitis B to 0% to 10%. 5. The dose and duration of HBIG therapy needed when used in combination with lamivudine may be lower, but the optimal regimen remains to be determined. 6. Lamivudine resistance before LT is associated with an increased risk for recurrent hepatitis B post-LT. 7. A cost-effective prophylactic regimen to prevent recurrent hepatitis B should be tailored according to risk.

Crosstalk between transcription factors and cytokines precisely regulates tissue homeostasis. Transcriptional intermediary factor 1γ (TIF1γ) regulates vertebrate hematopoietic development, can control transcription elongation, and is a component of the TGF-β signaling pathway. Here we show that deletion of TIF1γ in adult hematopoiesis is compatible with life and long-term maintenance of essential blood cell lineages. However, loss of TIF1γ results in deficient long-term hematopoietic stem cell (LT-HSC) transplantation activity, deficient short-term HSC (ST-HSC) bone marrow retention, and priming ST-HSCs to myelomonocytic lineage. These defects are hematopoietic cell-autonomous, and priming of TIF1γ-deficient ST-HSCs can be partially rescued by wild-type hematopoietic cells. TIF1γ can form complexes with TAL1 or PU.1-two essential DNA-binding proteins in hematopoiesis-occupy specific subsets of their DNA binding sites in vivo, and repress their transcriptional activity. These results suggest a regulation of adult hematopoiesis through TIF1γ-mediated transcriptional repression of TAL1 and PU.1 target genes.

Leukotrienes (LT) are potent lipid mediators of inflammation. 5-Lipoxygenase (5-LO) is the key enzyme in the conversion of arachidonic acid to LT. There are four LT: LTB(4), LTC(4), LTD(4) and LTE(4). LT have been extensively studied in airway inflammation but little is known about their roles in viral infection in the CNS. LTB(4) is a chemoattractant for neutrophils. In this work, we studied the roles of LT in acute vesicular stomatitis virus (VSV) encephalitis. Two methods were used to disrupt 5-LO activity: mice were treated with Zileuton, an enzyme antagonist, or 5-LO genetic knockout mice were used. We found that inhibition or deletion of 5-LO resulted in: (a) impaired process of neutrophil infiltration into the CNS early during viral infection; (b) fewer neurons expressed nitric oxide synthase-1 (NOS-1); (c) higher viral titers 1 day after viral infection; and (d) increased disruption of blood brain barrier (BBB). Our studies suggest that LT are important innate immune players during VSV pathogenesis and are beneficial to the host in early control of viral replication in the CNS.

This study describes the disposition and excretion of indole-3-carbinol (I3C), a natural dietary tumor modulator and candidate chemopreventive agent, in male Fisher 344 rats after continuous dietary or a single oral administration. Steady-state urinary and fecal excretion were attained 40 and 112 hr, respectively, after commencing continuous exposure. These two routes accounted for approximately 75% of the administered dose, of which 77% appeared in feces. After 7 days of 2,000 ppm dietary I3C, a mean of 1,154 microM I3C eq was found in liver, of which 17% was present as extractable, unbound I3C derivatives. Total equivalents in liver decreased to 643 and 411 microM 24 and 48 hr later, respectively, for animals returned to control diet. Mean levels of I3C eq in lung decreased from 436 to 219 microM, and blood levels decreased from 320 to 208 microM over the same 48-hr period. After administration of 1 mmol/kg radioinert I3C (a comparable daily dose as in the feeding study) for 6 days, animals were given 1 mmol/kg [3H]I3C. Mean liver levels were 257, 283, and 541 microM I3C eq at 1.5, 3, and 6 hr after dosing, and these levels represented 0.97%, 1.34%, and 2.45% of the total I3C dose administered, respectively. Concentrations of I3C eq changed little in blood, kidney, tongue, or lung over this time period. HPLC analysis of ethyl acetate extracts of liver from rats given an oral dose revealed 24 distinct [3H]I3C-derived peaks. Two of the predominant peaks were identified as 3,3'-diindolylmethane (I33', a linear dimer of I3C) and [2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LT, a linear trimer). A novel I3C metabolite was identified as 1-(3-hydroxymethyl)-indolyl-3-indolylmethane (HI-IM). Hepatic levels of these metabolites and three major, but unidentified, products were between 1.0 and 13.1 microM; highest levels were observed at 6 hr or, for HI-IM, at 1.5 hr postdosing. Parent I3C was not detected in liver extracts, whereas the potent Ah receptor agonist 3,2-b-indolocarbazole (ICZ) was estimated at 1.6 nM. These data suggest that neither I33', LT, or ICZ alone reach sufficient hepatic concentration to account for cytochrome P450IA induction by dietary I3C, or provide effective inhibition of microsomal bioactivation of the hepatocarcinogen aflatoxin B1; however, the total hepatic mixture of I3C derivatives may be sufficient to provide both modulatory responses in the rat.

OBJECTIVE

The anti-infectious activity of Bifidobacteria in combination with transgalactosylated oligosaccharides (TOS) against Salmonella enterica serovar Typhimurium LT-2 in an opportunistic antibiotic-induced murine infection model in mice was examined.

RESULTS

B. breve (strain Yakult) with natural resistance to streptomycin sulphate (SM, MIC:>> 4 mg ml(-1)), when given daily at a dose of 108 cfu/mouse orally under SM treatment was constantly excreted at 10(10) cfu g(-1) faeces so long as SM was administered, even at 2 weeks after discontinuing administration of B. breve. Explosive intestinal growth and subsequent extra-intestinal translocation of orally infected LT-2 under SM treatment were inhibited by B. breve colonization, and this anti-infectious activity was strengthened by synbiotic administration of TOS with B. breve. Comparison of anti-Salmonella activity among several Bifidobacterium strains with natural resistance to SM revealed that strains such as B. bifidum ATCC 15696 and B. catenulatum ATCC 27539T conferred no activity, even when they reached high population levels similar those of effective strains such as strain Yakult and B. pseudocatenulatum DSM 20439. Both the increase in the concentration of organic acids and the lowered pH in the intestine due to bifidobacterial colonization correlated with the anti-infectious activity. Moreover, the crude cecal extract of B. breve-colonized mice exerted growth-inhibitory activity against LT-2 in vitro, whereas that of the ineffective B. bifidum-colonized cecum showed much lower activity.

CONCLUSIONS

Intestinal colonization by bifidobacteria given exogenously together with TOS during antibiotic treatment prevents the antibiotic-induced disruption of colonization resistance to oral infection with S. enterica serovar Typhimurium, and the metabolic activity needed to produce organic acids and lower the intestinal pH is important in the anti-infectious activity of synbiotics against enteric infection with Salmonella.

CONCLUSIONS

These results indicate that certain bifidobacteria together with prebiotics may be used for the prophylaxis against opportunistic intestinal infections with antibiotic-resistant pathogens.

Hepatitis B is endemic in many regions of Asia, including China, Korea and India. This results in a heavy burden of hepatocellular carcinoma (HCC) because hepatitis B virus is a major risk factor in the development of the disease. In addition, the incidence of hepatitis-C-related HCC is on the rise in the United States. HCC patients with poor liver function reserve are not suitable candidates for resection, and liver transplantation (LT) has emerged as the treatment of choice for small unresectable HCCs. To treat more HCC patients with LT, the standard patient selection criteria have been expanded at a number of centers. Careful and well-considered selection of patients is the key to success in LT for HCC. Although tumor size and tumor number are used to predict whether transplantation is likely to be successful, the weighting that should be attached these two parameters has not been determined. In addition to the size and number of lesions, the morphology of HCC is also predictive of its behavior. Well-circumscribed lesions, in general, are less aggressive than those with poorly defined borders. On the waiting list for LT, HCC patients compete with liver failure patients. It is essential that the criteria used for selecting HCC patients for LT should be easily applicable and fair to other transplant candidates. In the face of the scarcity of deceased-donor livers and the inevitable risks for living liver donors, a predictably low rate of recurrence of HCC after LT is mandatory.

Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z-6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z-55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV-transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS)

The molecules that comprise the tumor necrosis factor ligand and receptor (TNF-L and TNF-R) families play important roles in tissue homeostasis and in multiple sclerosis (MS). For example, levels of the TNF ligand (TNF alpha; cachectin) correlate with disease progression and lymphotoxin (LT, TNF beta) has been localized in MS lesions. Members of the TNF-R family are typical signal sensors which upon binding with ligand aggregate and recruit signal transducers. To date, no TNF-R molecules have been reported in MS although TNF-RI and RII have been localized to oligodendrocytes in culture. In the present study, the expression of TNF, LT alpha (the soluble form of LT), LT beta (the beta chain of LT alpha beta, the membrane-bound form of LT), TNF-RI, TNF-RII, LT beta-R, FasL, and Fas receptor in MS lesions has been examined by immunohistochemistry for protein and by RT-PCR for mRNA. In addition, the TUNEL technique for DNA fragmentation was applied to detect apoptosis. The results have shown that contrarily to predictions, oligodendrocytes around active MS lesions frequently expressed TNF-R molecules belonging to the apoptotic cascade. However, these cells did not undergo apoptosis, as judged by TUNEL. On the other hand, lymphocytes (and a few microglial cells) in the same tissue displayed apoptosis. Microglial cells were the major effector cells in the CNS and expressed TNF, LT alpha and FasL. LT beta expression was seen on astrocytes and oligodendrocytes, and LT beta-R on astrocytes. We conclude that TNF-L and TNF-R molecules are extensively expressed in MS, that their expression occurs at high levels but is not specific for MS, and that oligodendrocytes are depleted by a cytolytic mechanism, not by apoptosis.

A plasmid encoding a mutant gene of heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, was induced by treatment of plasmid EWD 299 with hydroxylamine. A mutant strain of E. coli HB 101 carrying the mutant plasmid pTUH 6A produced a low toxic LT analogue (mutant LT), which was cross-reactive with anti-LT antibody. The mutant LT activity was less than 0.15 and 0.006% of the normal LT in the rabbit ileal loop test and in the rabbit skin permeability test, respectively. The amino acid composition of the mutant LT-B subunit was the same as that of the normal B subunit. Though the A2 fragment of the mutant LT was identical to normal LT by DNA analysis, the A1 fragment of the mutant LT differed from the normal A1 fragment in one amino acid at position 112; namely it had lysine instead of glutamic acid from the N terminus. These data suggest that glutamic acid at position 112 from the N terminus of the A1 fragment is important for the A subunit to express its biological activity.

The anthrax lethal toxin (LT) enters host cells and enzymatically cleaves MAPKKs or MEKs. How these molecular events lead to death from anthrax remains poorly understood, but published reports suggest a direct effect of LT on vascular permeability. We have found that LT challenge in mice disrupts signaling through Tie-2, a tonically activated receptor tyrosine kinase in the endothelium. Genetic manipulations favoring Tie-2 activation enhanced interendothelial junctional contacts, prevented vascular leakage, and promoted survival following a lethal dose of LT. Cleavage of MEK1/2 was necessary for LT to induce endothelial barrier dysfunction, and activated Tie-2 signaled through the uncleaved fraction of MEKs to prevent LT's effects on the endothelium. Finally, primates infected with toxin-secreting Bacillus anthracis bacilli developed a rapid and marked imbalance in the endogenous ligands that signal Tie-2, similar to that seen in LT-challenged mice. Our results show that B. anthracis LT blunts signaling through Tie-2, thereby weakening the vascular barrier and contributing to lethality of the disease. Measurement of circulating Tie-2 ligands and manipulation of Tie-2 activity may represent future prognostic and therapeutic avenues for humans exposed to B. anthracis.

The resonance-stabilized quinonoid 5-mercapto-2-nitrobenzoate (TNB) is a substrate for O-acetylserine sulfhydrylase-A (OASS-A) and -B (OASS-B), giving rise to the product S-(3-carboxy-4-nitrophenyl)-L-cysteine (S-CNP-cysteine) as confirmed by ultraviolet-visible and 1H NMR spectroscopies. A comparison of the kinetics of OASS-A and OASS-B indicates that the mechanism proceeds predominantly via a bi-bi ping pong kinetic mechanism as suggested by an initial velocity pattern consisting of parallel lines at low concentrations of reactants, but competitive inhibition by both substrates as the reactant concentrations are increased. Thus, in the first half-reaction, O-acetyl-L-serine (OAS) or beta-chloro-L-alanine (BCA) is converted to alpha-aminoacrylate in Schiff base with the active site pyridoxal 5'-phosphate, while in the second half-reaction cysteine (with sulfide as the reactant) or S-CNP-cysteine (with TNB as the reactant) is formed. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive. These data are consistent with acetate reversing the first half-reaction and producing more free enzyme to which acetate may also bind. Thus, there may be some randomness to the mechanism at high concentrations of the nucleophilic substrate.

Because the role of Kupffer cells (KCs) in liver transplantation (LT) tolerance is not well understood, we investigated their role in liver allograft acceptance in rats. Male Sprague-Dawley rats were randomly assigned to either an LT group or a transplantation group pretreated with GdCl(3) (Gd group). The rats were postoperatively sacrificed at indicated times for histology and assessment of KC function, nuclear factor kappa B (NF-kappaB) activity, and cytokine production. KCs and T cells (TCs) were isolated from allografts to assess Fas/Fas ligand (FasL) expression. Cytotoxicity of KCs against TCs was monitored by coculturing of (3)H-thymidine TCs with KCs at various effector-to-target ratios. The results were as follows. First, grafts were spontaneously accepted in the LT group with evident apoptosis of TCs; however, inhibition of KCs by pretreatment with GdCl(3) decreased TC apoptosis and shortened the survival of allografts. Second, KCs in the LT group had increased levels of FasL messenger RNA and protein with respect to that in the Gd group. Third, by in vitro cocultivation assays, KCs induced TC apoptosis though elevated expression of FasL, and this process could be blocked by anti-FasL antibody. Fourth, there was a positive correlation between activation of NF-kappaB and FasL expression in KCs and interleukin-4 production in the LT group, and the activation of NF-kappaB was inhibited by pretreatment with GdCl(3). In conclusion, KC-induced depletion of TCs via the Fas/FasL pathway might play a critical role in LT tolerance. However, the tolerance is abrogated by suppression of FasL and IL-4 expression via inhibition of NF-kappaB activity by GdCl(3).

Anthrax lethal toxin (LT) is an important virulence factor for Bacillus anthracis. In mice, LT lyses macrophages from certain inbred strains in less than 2h by activating the Nlrp1b inflammasome and caspase-1, while macrophages from other strains remain resistant to the toxin's effects. We analyzed LT effects in toxin-sensitive and resistant rat macrophages to test if a similar pathway was involved in rat macrophage death. LT activates caspase-1 in rat macrophages from strains harboring LT-sensitive macrophages in a manner similar to that in toxin-sensitive murine macrophages. This activation of caspase-1 is dependent on proteasome activity, and sensitive macrophages are protected from LT's lytic effects by lactacystin. Proteasome inhibition also delayed the death of rats in response to LT, confirming our previous data implicating the rat Nlrp1 inflammasome in animal death. Quinidine, caspase-1 inhibitors, the cathepsin B inhibitor CA-074Me, and heat shock also protected rat macrophages from LT toxicity. These data support the existence of an active functioning LT-responsive Nlrp1 inflammasome in rat macrophages. The activation of the rat Nlrp1 inflammasome is required for LT-mediated rat macrophage lysis and contributes to animal death.

BACKGROUND

Tumor necrosis factor alpha (TNF-alpha) is often considered the main proinflammatory cytokine induced by lipopolysaccharide (LPS) and consequently the critical mediator of the lethality associated with septic shock.

METHODS

We used mice carrying a deletion of both the lymphotoxin alpha (LT-alpha) and TNF-alpha genes to assess the role of TNF in the cytokine cascade and lethality induced by LPS.

RESULTS

Initial production of IL-1 alpha, IL-1 beta, IL-6, and IL-10 is comparable in wild-type and mutant mice. However, at later times, expression of IL-1 alpha, IL-1 beta, and IL-10 is prolonged, whereas that of IL-6 decreases in mutant mice. Expression of IFN-gamma is almost completely abrogated in mutants, which is in agreement with a more significant alteration of the late phase of the cytokine cascade. We measured similar LD50 (600 micrograms) for the intravenous injection of LPS in mice of the three genotypes (+/+, +/-, -/-), demonstrating that the absence of TNF does not confer long-term protection from lethality. However, death occurred much more slowly in mutant mice, who were protected more efficiently from death by CNI 1493, an inhibitor of proinflammatory cytokine production, than were wild-type mice.

CONCLUSIONS

Thus, while TNF-alpha is not required for the induction of these cytokines by LPS, it modulates the kinetics of their expression. The lethality studies simultaneously confirm a role for TNF as a mediator of early lethality and establish that, in the absence of these cytokines, other mediators take over, resulting in the absence of long-term protection from LPS toxicity.

OBJECTIVE

To determine whether an elevated neutrophil-lymphocyte ratio (NLR) is negatively associated with tumor recurrence in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) after liver transplantation (LT), and to determine the optimal predictive NLR cut-off value.

METHODS

The data of HCC patients who had undergone LT came from the China Liver Transplant Registry database. We collected data from 326 liver cancer patients who had undergone LT at our medical center. We divided the patients into groups based on their NLRs (3, 4 or 5). We then compared the clinicopathological data and long-time survival between these groups. Meanwhile, we used receiver operating characteristic analysis to determine the optimal NLR cut-off.

RESULTS

Of 280 HCC patients included in this study, 263 were HBV positive. Patients with an NLR < 3 and patients with an NLR ≥ 3 but < 4 showed no significant differences in overall survival (OS) (P = 0.212) or disease-free survival (DFS) (P = 0.601). Patients with an NLR ≥ 4 but < 5 and patients with an NLR ≥ 5 also showed no significant differences in OS (P = 0.208) or DFS (P = 0.618). The 1-, 3- and 5-year OS rates of patients with an NLR < 4 vs an NLR ≥ 4 were 87.8%, 63.8% and 61.5% vs 73.9%, 36.7% and 30.3%, respectively (P < 0.001). The 1-, 3- and 5-year DFS rates of patients with an NLR < 4 vs NLR ≥ 4 were 83.9%, 62.9% and 60.7% vs 64.9%, 30.1% and 30.1%, respectively (P < 0.001). Univariate and multivariate analyses demonstrated that three factors, including NLR ≥ 4 (P = 0.002), were significant predictors of tumor recurrence in HCC patients after LT.

CONCLUSIONS

A preoperative elevated NLR significantly increased the risk for tumor recurrence in HCC patients after LT.

<A<em>b</em>stractText>EGFR-mutant lung cancers are clinically and genomically heterogeneous with concurrent R<em>B</em> transcriptional corepressor 1 (R<em>B</em>1)/tumor protein p53 (TP53) a<em>lt</em>erations identifying a su<em>b</em>set at increased risk for small cell transformation. The genomic a<em>lt</em>erations that induce lineage plasticity are unknown.</A<em>b</em>stractText><A<em>b</em>stractText>Patients with EGFR/R<em>B</em>1/TP53-mutant lung cancers, identified <em>b</em>y next-generation sequencing from 2014 to 2018, were compared to patients with untreated, metastatic EGFR-mutant lung cancers without <em>b</em>oth R<em>B</em>1 and TP53 a<em>lt</em>erations. Time to EGFR-tyrosine kinase inhi<em>b</em>itor discontinuation, overall survival, SCLC transformation rate, and genomic a<em>lt</em>erations were evaluated.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Patients with EGFR/R<em>B</em>1/TP53-mutant lung cancers represented 5% (43 of 863) of EGFR-mutant lung cancers <em>b</em>ut were uniquely at risk for transformation (7 of 39, 18%), with no transformations in EGFR-mutant lung cancers without <em>b</em>aseline TP53 and R<em>B</em>1 a<em>lt</em>erations. Irrespective of transformation, patients with EGFR/TP53/R<em>B</em>1-mutant lung cancers had a shorter time to discontinuation than EGFR/TP53- and EGFR-mutant -only cancers (9.5 versus 12.3 versus 36.6 months, respectively, p = 2 × 10^-9). The triple-mutant population had a higher incidence of whole-genome dou<em>b</em>ling compared to NSCLC and SCLC at large (80% versus 34%, p &<em>lt</em>; 5 × 10^-9 versus 51%, p &<em>lt</em>; 0.002, respectively) and further enrichment in triple-mutant cancers with eventual small cell histology (seven of seven pre-transformed plus four of four <em>b</em>aseline SCLC versus 23 of 32 never transformed, respectively, p = 0.05). Activation-induced cytidine deaminase/apolipoprotein <em>B</em> mRNA editing enzyme, catalytic polypeptide-like mutation signature was also enriched in triple-mutant lung cancers that transformed (false discovery rate = 0.03).</p><A<em>b</em>stractText>EGFR/TP53/R<em>B</em>1-mutant lung cancers are at unique risk of histologic transformation, with 25% presenting with de novo SCLC or eventual small cell transformation. Triple-mutant lung cancers are enriched in whole-genome dou<em>b</em>ling and Activation-induced cytidine deaminase/apolipoprotein <em>B</em> mRNA editing enzyme, catalytic polypeptide-like hypermutation which may represent early genomic determinants of lineage plasticity.</A<em>b</em>stractText>

Intranasal (i.n.) administration of an Escherichia coli-expressed chimeric VP6 protein from the EDIM strain of murine rotavirus to adult BALB/c (H-2(d)) mice along with LT(R192G), an attenuated mutant of the mucosal adjuvant E. coli heat-labile toxin, has been found to consistently stimulate ca. 99% reductions in rotavirus shedding after subsequent EDIM challenge. This study was designed to determine the robustness of this protection, i.e. can VP6 immunization consistently protect against shedding in this model, thus, providing an indication of its potential as a vaccine. Intranasal immunization with two 8.8 microg doses of EDIM VP6 and 10 microg of LT(R192G) was found to stimulate 99% reductions in EDIM shedding in four additional strains of inbred mice belonging to three haplotypes, i.e. DBA/2 (H-2(d)), C57BL/6 (H-2(b)), 129 (H-2(b)) and C3H (H-2(k)). Protection stimulated against EDIM antigen shedding following i.n. immunization with VP6 from the human CJN strain was less (P=0.02) than induced by EDIM VP6 (86% versus 99%), but no further loss of protection was observed when the dose of CJN VP6 was reduced 100-fold. Protection against EDIM shedding was also maintained after i.n. immunization of three strains of outbred mice (CF-1, CD-1 and Swiss Webster) with either EDIM or CJN VP6, i.e. EDIM VP6 immunization reduced EDIM shedding by 99% while CJN VP6 immunization produced reductions of 86-96%. Protection stimulated by oral immunization of BALB/c mice with two 8.8 microg doses of either VP6 chimera plus LT(R192G) was not significantly different from that induced by i.n. immunization. Finally, protection found after either oral or i.n. immunization with EDIM or CJN VP6 was no different when the mice were challenged with McN, another strain of murine rotavirus. These results support further evaluation of VP6 as a vaccine.

<A<em>b</em>stractText>Patients with acute myeloid leukemia (AML) in remission remain at risk for relapse even after allogeneic hematopoietic cell transplantation (alloHCT). AML measura<em>b</em>le residual disease (MRD) status <em>b</em>efore alloHCT has <em>b</em>een shown to <em>b</em>e prognostic. Whether modulation of the intensity of the alloHCT conditioning regimen in patients with AML who test positive for MRD can prevent relapse and improve survival is unknown.</A<em>b</em>stractText><A<em>b</em>stractText>U<em>lt</em>ra-deep, error-corrected sequencing for 13 commonly mutated genes in AML was performed on preconditioning <em>b</em>lood from patients treated in a phase III clinical trial that randomly assigned adu<em>lt</em> patients with myeloid malignancy in morphologic complete remission to myeloa<em>b</em>lative conditioning (MAC) or reduced-intensity conditioning (RIC).</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>No mutations were detected in 32% of MAC and 37% of RIC recipients; these groups had similar survival (3-year overall survival [OS], 56% <i>v</i> 63%; <i>P</i> = .96). In patients with a detecta<em>b</em>le mutation (next-generation sequencing [NGS] positive), relapse (3-year cumulative incidence, 19% <i>v</i> 67%; <i>P</i> &<em>lt</em>; .001) and survival (3-year OS, 61% <i>v</i> 43%; <i>P</i> = .02) was significantly different <em>b</em>etween the MAC and RIC arms, respectively. In mu<em>lt</em>ivaria<em>b</em>le analysis for NGS-positive patients, adjusting for disease risk and donor group, RIC was significantly associated with increased relapse (hazard ratio [HR], 6.38; 95% CI, 3.37 to 12.10; <i>P</i> &<em>lt</em>; .001), decreased relapse-free survival (HR, 2.94; 95% CI, 1.84 to 4.69; <i>P</i> &<em>lt</em>; .001), and decreased OS (HR, 1.97; 95% CI, 1.17 to 3.30; <i>P</i> = .01) compared with MAC. Models of AML MRD also showed <em>b</em>enefit for MAC over RIC for those who tested positive.</p><A<em>b</em>stractText>This study provides evidence that MAC rather than RIC in patients with AML with genomic evidence of MRD <em>b</em>efore alloHCT can resu<em>lt</em> in improved survival.</A<em>b</em>stractText>

<A<em>b</em>stractText>Sodium glucose cotransporter 2 inhi<em>b</em>itors may reduce kidney hyperfi<em>lt</em>ration, there<em>b</em>y preventing dia<em>b</em>etic kidney disease progression, which may in turn reduce cardiovascular risk, including heart failure. However, the mechanisms that regulate renal function responses to sodium glucose cotransporter 2 inhi<em>b</em>ition are not yet fully understood. We explored the renal protective effects of sodium glucose cotransporter 2 inhi<em>b</em>ition with empagliflozin, with a focus on glomerular hemodynamic effects and tu<em>b</em>uloglomerular feed<em>b</em>ack using in vivo mu<em>lt</em>iphoton microscopy imaging techniques.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>C57BL/6 mice and spontaneously dia<em>b</em>etic Ins2^+/Akita mice were studied. The mice were treated with empagliflozin (20 mg·kg^-1·d^-1) and insulin for 4 weeks, and the single-nephron glomerular fi<em>lt</em>ration rate was measured using mu<em>lt</em>iphoton microscope. A neuronal nitric oxide synthase inhi<em>b</em>itor (7-nitroindazole, 20 mg·kg^-1·d^-1) or a cyclooxygenase-2 inhi<em>b</em>itor (SC58236, 6 mg/L), or an A1 adenosine receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine, 1 mg·kg^-1·d^-1) was administered to elucidate the mechanisms of tu<em>b</em>uloglomerular feed<em>b</em>ack signaling and single-nephron glomerular fi<em>lt</em>ration rate regulation.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>The urinary excretion of adenosine, nitric oxide meta<em>b</em>olites, and the prostanoid prostaglandin E2 was also quantified. The single-nephron glomerular fi<em>lt</em>ration rate in the Ins2^+/Akita group was higher than in controls (C57BL/6; 4.9±1.3 nL/min versus Ins2^+/Akita; 15.8±6.8 nL/min) and lower in Ins2^+/Akita /empagliflozin to 8.0±3.3 nL/min (P&<em>lt</em>;0.01). In vivo imaging also revealed concomitant afferent arteriolar dilation (P&<em>lt</em>;0.01) and increased glomerular permea<em>b</em>ility of al<em>b</em>umin in the Ins2^+/Akita group. Empagliflozin ameliorated these changes (P&<em>lt</em>;0.01). Urinary adenosine excretion in the Ins2^+/Akita/empagliflozin group was higher than in Ins2^+/Akita (Ins2^+/Akita; 3.4±1.4 nmol/d, Ins2^+/Akita/empagliflozin; 11.2±3.0 nmol/d, P&<em>lt</em>;0.05), whereas nitric oxide meta<em>b</em>olites and prostaglandin E2 did not differ. A1 adenosine receptor antagonism, <em>b</em>ut not neuronal nitric oxide synthase or cyclooxygenase-2 inhi<em>b</em>ition, <em>b</em>locked the effect of empagliflozin on renal function. Empagliflozin increased urinary adenosine excretion and reduced hyperfi<em>lt</em>ration via afferent arteriolar constriction, effects that were a<em>b</em>olished <em>b</em>y A1 adenosine receptor <em>b</em>lockade.</p><A<em>b</em>stractText>Adenosine/A1 adenosine receptor pathways play a pivotal role in the regulation of the single-nephron glomerular fi<em>lt</em>ration rate via tu<em>b</em>uloglomerular feed<em>b</em>ack mechanisms in response to sodium glucose cotransporter 2 inhi<em>b</em>ition, which may contri<em>b</em>ute to renal and cardiovascular protective effects reported in clinical trials.</A<em>b</em>stractText>

Prophylactic or therapeutic immunomodulation is an antigen-independent strategy that induces nonspecific immune system activation, thereby enhancing host defense to disease. In this study, we investigated the effect of prophylactic immunomodulation on the outcome of influenza virus infection using three bacterially derived immune-enhancing agents known for promoting distinct immunological profiles. BALB/c mice were treated nasally with either cholera toxin (CT), a mutant form of the CT-related Escherichia coli heat-labile enterotoxin designated LT(R192G), or CpG oligodeoxynucleotide. Mice were subsequently challenged with a lethal dose of influenza A/PR/8/34 virus 24 h after the last immunomodulation treatment and either monitored for survival or sacrificed postchallenge for viral and immunological analysis. Treatment with the three immunomodulators prevented or delayed mortality and weight loss, but only CT and LT(R192G) significantly reduced initial lung viral loads as measured by plaque assay. Analysis performed 4 days postinfection indicated that prophylactic treatments with CT, LT(R192G), or CpG resulted in significantly increased numbers of CD4 T cells, B cells, and dendritic cells and altered costimulatory marker expression in the airways of infected mice, coinciding with reduced expression of pulmonary chemokines and the appearance of inducible bronchus-associated lymphoid tissue-like structures in the lungs. Collectively, these results suggest that, despite different immunomodulatory mechanisms, CT, LT(R192G), and CpG induce an initial inflammatory process and enhance the immune response to primary influenza virus challenge while preventing potentially damaging chemokine expression. These studies provide insight into the immunological parameters and immune modulation strategies that have the potential to enhance the nonspecific host response to influenza virus infection.

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (<em>LT</em>) <em>B</em>4 (<em>LT</em><em>B</em>4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that <em>LT</em><em>B</em>4 control of MyD88 expression involves the generation of microRNAs. Our data show that <em>LT</em><em>B</em>4, via its receptor <em>B</em> leukotriene receptor 1 (<em>B</em><em>LT</em>1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. <em>LT</em><em>B</em>4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the <em>LT</em><em>B</em>4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and <em>LT</em>-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR <em>B</em><em>LT</em>1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.