The differential effects of 1,25-dihydroxyvitamin D3 on Salmonella-induced interleukin-8 and human beta-defensin-2 in intestinal epithelial cells.

Salmonellosis or Salmonella, one of the most common food-borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion of Salmonella by interleukin (IL)-8 and human ß-defensin-2 (hBD-2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25-dihydroxyvitamin D3 (1,25D3) on Salmonella-induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression of Salmonella-induced IL-8 but enhancement of hBD-2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhanced Salmonella-induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3-kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 on Salmonella-induced IL-8 expression, while knock-down of NOD2 by siRNA diminished the enhanced hBD-2 expression. These data suggest differential regulation of 1,25D3 on Salmonella-induced IL-8 and hBD-2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D-enhanced anti-microbial peptide in Salmonella-infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D-induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasive Salmonella infection.

[Predictive value of combination of MRI tumor regression grade and apparent diffusion coefficient for pathological complete remission after neoadjuvant treatment of locally advanced rectal cancer].

Objective: Pelvic high-resolution magnetic resonance imaging (MRI) has now become a standard method for evaluating the efficacy of neoadjuvant treatment for locally advanced rectal cancer (LARC). However, this traditional morphological qualitative assessment method based on T2-weighted imaging (T2WI) is not effective in predicting pathological complete remission (pCR). The purpose of this study is to investigate whether combining the magnetic resonance tumor regression grade (mrTRG) with apparent diffusion coefficient (ADC) can improve diagnostic value for pCR after preoperative neoadjuvant chemoradiotherapy (nCRT) of LARC. Methods: This was a diagnostic study. Clinicopathological data of 134 LARC patients who received nCRT and radical surgery in the First Affiliated Hospital of Kunming Medical University from January 2017 to December 2019 were retrospectively analyzed. All the patients underwent MRI which included T2WI and DWI sequences before and 8 weeks after nCRT. Two radiologists independently drew ROIs on T2WI and DWI to estimate mrTRG stage and calculate the mean ADC value. Receiver operating characteristics (ROC) method was applied to evaluate the predict value of mrTRG combined with mean ADC value for pCR. Results: Of 134 LARC patients, 85 were male and 49 were female with median age of 58 (28-82) years. After nCRT, MRI suggested 21 patients (15.7%) had clinical complete remission (cCR), e.g. mrTRG stage 1-2. Postoperative pathology revealed 31 (23.1%) patients had pCR. The evaluations of mrTRG and ADC value by the two readers were highly consistent, and the intra-group correlation coefficients were 0.83 (95% CI: 0.703-0.881) and 0.96 (95% CI: 0.989-0.996), respectively. There was a negative correlation between mrTRG and pCR (r(s)=-0.505, P<0.01), and a positive correlation between mean ADC value and pCR (r(s)=0.693, P<0.01). The ROC curve showed that mrTRG alone had a medium predictive value for pCR, with an area under the curve (AUC) of 0.832 (95% CI: 0.743-0.921); the mean ADC value had a higher predictive value for pCR, with AUC of 0.906 (95% CI: 0.869-0.962). The predictive value of the combined model of mrTRG and ADC value for pCR was significantly better than that of mrTRG alone (P=0.015), and the AUC was 0.908 (95% CI: 0.849-0.968). Conclusion: Both mrTRG and mean ADC value can be non-invasive methods to predict the efficacy of nCRT for LARC. Combining the mean ADC value with mrTRG can result in better pCR prediction.

Probiotics exert reciprocal effects on autophagy and interleukin-1ß expression in Salmonella-infected intestinal epithelial cells via autophagy-related 16L1 protein.

This study aimed to examine how probiotics affect autophagy and interleukin-1ß (IL-1ß) expression in Salmonella-infected intestinal epithelial cells (IECs). The original Caco-2 cells and ATG16L1 siRNA-transfected Caco-2 cells were pretreated or left untreated with probiotics, including Lactobacillus rhamnosus GG (LGG; ATCC 53103) and Bifidobacterium longum (BL; ATCC15697), and these cells were infected with wild-type Salmonella enterica serovar Typhimurium (S. Typhimurium strain, SL1344). Western blot analysis was used to detect the conversion of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I to LC3-II. Immunofluorescence was used to analyse LC3+ autophagosomes. Membrane proteins were analysed by western blot for protein (ATG16L1, NOD2), and total RNA by RT-PCR for mRNA expression [ATG16L1, vitamin D receptor (VDR)]. We demonstrated that probiotics enhanced both VDR mRNA, and nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) and autophagy-related protein 16-like 1 (ATG16L1) protein expression. The enhanced expression resulted in autophagic LC3-II protein expression and formation of LC3 punctae in Salmonella-infected Caco-2 cells. It was observed that ATG16L1 siRNA could attenuate this mechanism, and ATG16L1-mediated IL-1ß expression was suppressed by probiotics. These results suggest that probiotics enhance autophagy and also suppress inflammatory IL-1ß expression in Salmonella-infected IECs via membrane ATG16L1 protein expression. Probiotics may enhance autophagic clearance of Salmonella infection and modulate inflammatory responses to protect the hosts. Hence, we can assume that probiotics could treat infectious and autoimmune diseases through mechanisms involving ATG16L1.

The different effects of probiotics treatment on Salmonella-induced interleukin-8 response in intestinal epithelia cells via PI3K/Akt and NOD2 expression.

Salmonella spp. remains a major public health problem for the whole world. Intestinal epithelial cells serve as an essential component of the innate mucosal immune system to defend against Salmonella infection. A substantial amount of evidence has accumulated that probiotics can regulate interleukin 8 (IL-8) involved in innate immunity. However, the exact effect of probiotics on epithelial IL-8 response to Salmonella infection is not well understood. Therefore, we investigated the action of probiotics on Salmonella-infected Caco-2 cells and its novel mechanisms. Two probiotic strains were examined for Salmonella-induced IL-8 responses and regulating proteins using Caco-2 cell cultures. We demonstrated probiotic, either Lactobacillus rhamnosus GG or Bifidobacterium animalis subsp. lactis DSM10140, administered before Salmonella infection conferred significantly suppressive effect on Salmonella-induced IL-8 responses in Caco-2 cells, either in secreted protein or mRNA, via the PI3K/Akt signal pathway while probiotic administered after infection enhanced Salmonella-induced IL-8 responses via nucleotide-binding oligomerisation domain-containing protein 2 expression in membrane. These findings suggest that the different regulation of probiotics on Salmonella-induced IL-8 responses in Caco-2 cells according to the administered timing supports a rationale for the therapeutic use of probiotics in the treatment of Salmonella colitis and inflammatory bowel disease. This can explain the reported controversial effect of probiotics on these diseases.

Study on the clinical value of alprostadil combined with α-lipoic acid in treatment of type 2 diabetes mellitus patients with erectile dysfunction.

OBJECTIVE: We investigated the clinical value of alprostadil combined with the α-lipoic acid in treating type 2 diabetes mellitus with erectile dysfunction (DMED). PATIENTS AND METHODS: We selected a total of 76 cases of patients who were admitted to endocrinology department of our hospital from June 2014 to June 2015 and diagnosed as DMED, and the average age was (46.7 ± 7.2) years old, average course of diabetes mellitus was (6.2 ± 2.8) years and average body mass index was (25.4 ± 1.3) kg/m2. 40 cases were randomly divided in the observation group while 36 cases were divided in the control group. They received blood glucose control therapy. The patients in the observation group received 60 mg alprostadil hydrochloride and 600 mg α-lipoic acid added into 250 mL normal saline, intravenous drip once per day for 2 weeks. The patients in the control group took tadalafil 5 mg orally, once per night for 2 weeks as a course of treatment. There were no cases of loss. RESULTS: The effective rate of treatment in observation group is significantly higher than that in the control group (95.0% vs. 80.5%, p < 0.05). The score of IIEF-5, EHGS and the FMD value of brachial artery of the observation group after the operation were significantly higher than that of the control group (p < 0.05). The adverse reaction rate in the observation group was lower than that in the control group (7.5% vs. 13.9%, p < 0.05). CONCLUSIONS: Alprostadil combined with α-lipoic acid can improve DMED patients' vascular endothelial function and erection hardness to treat erectile dysfunction with less adverse effects and better safety.

A cluster of Mycobacterium massiliense keratitis in foundry workers.

Three consecutive workers from the same foundry had Mycobacterium massiliense keratitis. The strains isolated from each patient were identical. This is the first report of a non-surgery-related outbreak of non-tuberculous mycobacterial ocular infection. An investigation revealed that injured cornea with exposure to aerosolized non-tuberculous mycobacteria might account for this outbreak.

1,2,4-Triazolo[4,3-a]quinoxaline-1,4-diones as antiallergic agents.

A series of new 1,4-dihydro-1,2,4-triazolo[4,3-]quinoxaline-1,4-diones has been prepared. These compounds were tested as inhibitors of antigen-induced release of histamine (AIR) in vitro from rat peritoneal mast cells (RMC) and as inhibitors of IgE-mediated rat passive cutaneous anaphylaxis (PCA). Most of this new class of antiallergic agents showed good activity in the RMC and PCA tests. The most potent compound, 2-acetyl-7-chloro-5-n-propyl-1,2,4-triazolo[4,3-a]quinoxaline-1,4-dione (1x), with an I50 value of 0.1 microM, is 30 times more potent than disodium cromoglycate (DSCG) in the RMC assay.

[1,4]Benzoxazine-2,3-diones as antiallergic agents.

The synthesis of a series of [1,4]benzoxazine-2,3-diones and a new class of compounds, benzobisoxazinetetrones, is described. These compounds were evaluated for their effect in the rat mast cell (RMC) test passively sensitized in vitro with rat antiovalbumin serum and for their effect in inhibitory passive cutaneous anaphylaxis (PCA) in the rat. Some of these compounds are of the same potency level as disodium cromglycate in the RMC test and some are effective orally in PCA.

Synthesis of 2-(2,3-dihydro-2-oxo-1,3,4-oxadiazol-5-yl) benzo heterocycles. A novel series of orally active antiallergic agents.

A series of new 2-(2,3-dihydro-2-oxo-1,3,4-oxadiazol-5-yl) benzo heterocycles has been prepared. These compounds were tested as inhibitors of antigen-induced release of histamine (AIR) in vitro from rat peritoneal mast cells (RMC) and as inhibitors of IgE-mediated rat passive cutaneous anaphylaxis in the rat (PCA). Most of this new class of antiallergic agents showed good activity in the RMC assay. The most potent compound, 3-chloro-2-(2,3-dihydro-2-oxo-1,3,4-oxadiazol-5-yl)benzo[b]thiophe ne (6t), with an I50 value of 0.2 microM, is 15 times more potent than disodium cromoglycate (DSCG) in the RMC assay. Many compounds were orally active in the PCA test, and several of these compounds showed higher potency when given in this way to that shown by DSCG when given intraperitoneally.

Novel cyclic compounds as potent phosphodiesterase 4 inhibitors.

The synthesis and biological activity of a novel series of 2, 2-disubstituted indan-1,3-dione-based PDE4 inhibitors are described. This structurally unique class of PDE4 inhibitors is markedly different from the known PDE4 inhibitors such as RP 73401 (2) and CDP 840 (3). Structure-activity relationship (SAR) studies led to the identification of inhibitors with nanomolar potency and oral activity in a murine endotoxemia model for TNF-alpha inhibition. Unlike other classical PDE4 inhibitors, several analogues were found to be nonemetic in a canine emesis model at intravenous doses of up to 3 mg/kg.

Differential effects of a series of hydroxamic acid derivatives on 5-lipoxygenase and cyclooxygenase from neutrophils and 12-lipoxygenase from platelets and their in vivo effects on inflammation and anaphylaxis.

The synthesis of a series of novel substituted hydroxamates has been described along with their profile of inhibitory activity against 5-lipoxygenase, 12-lipoxygenase, and cyclooxygenase enzymes. The structure--activity relationship suggests that future molecules could be designed to specifically inhibit one or more of these enzymes since there were definite differences in structure--activity relationships for these different enzymes. A representative number of these compounds have been tested in vivo and found to possess potent oral activity in a systemic anaphylaxis model mediated by leukotrienes and topical activity in an arachidonic acid induced inflammation model. One of these molecules, compound 20, demonstrated that a leukotriene antagonist pharmacophore can be modified such that it contains both antagonist activity and 5-lipoxygenase inhibitory activity.

The development of a novel series of (quinolin-2-ylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 3. Structural variation of the acidic side chain to give antagonists of enhanced potency.

This paper is the third in a series outlining the development of orally active sulfido peptide leukotriene antagonists containing a (quinolin-2-ylmethoxy)phenyl moiety. In this work the systematic variation of the acid side chain substituents led to dramatic and reproducible changes in the oral activity of these compounds, presumably due to alterations in their pharmacokinetic properties. The most potent compound identified, 5-[4-[4-(quinolin-2-yl-methoxy)phenyl]-3-methylbutyl]tetrazole (32), represents a convergence of good in vitro antagonist activity and a 3-10-fold improvement in oral potency over the current clinical candidate 2. The new findings from these optimization studies are as follows: oxygen substitution in the acid side chain was not necessary for antagonist activity, in vitro and in vivo activity was enhanced by alkyl or phenyl substitution on the gamma-carbon of the acid side chain of para-substituted (quinolin-2-ylmethoxy)phenyl derivatives, and free rotation about the side chain carbon atom adjacent to the (quinolin-2-ylmethoxy)phenyl ring was required for activity. The lead compound of this report (32) is a competitive inhibitor of [3H]LTD4 binding to receptor membrane purified from guinea pig lung (Ki = 12 +/- 3 nM) and of the spasmogenic activity of LTC4, LTD4, and LTE4 in guinea pig lung strip. Dosed orally in guinea pigs, this compound blocks LTD4-induced bronchoconstriction (ED50 0.8 mg/kg) and antigen-induced systemic anaphylaxis (ED50 = 1.2 mg/kg).

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene receptor antagonists. 1. Initial structure-activity relationships.

This series of reports describes the development of orally active, highly potent, specific antagonists of the peptidoleukotrienes containing a (2-quinolinylmethoxy)phenyl moiety. Described in this first report are the structure-activity relationships that led to a more than a 20-fold improvement of the potency and selectivity of the initial chemical lead (RG 5901). From this series of compounds, RG 7152 (16) was identified and selected for further evaluation in the clinic as an antiasthmatic agent. Compound 16 competitively inhibits [3H]LTD4 binding to membranes from guinea pig lung (Ki = 38 +/- 6 nM) and the spasmogenic activity of LTC4, LTD4, and LTE4 in parenchymal lung strips from guinea pigs. Unlike the original lead (RG 5901), compound 16 does not inhibit 5-lipoxygenase from guinea pig PMNs. Following oral administration to guinea pigs, 16 blocks LTD4-induced dermal permeability (ED50 = 6.9 mg/kg), LTD4-induced bronchoconstriction (ED50 = 1.1 mg/kg), antigen-induced bronchoconstriction (ED50 = 2.5 mg/kg), and anaphylactic-induced mortality (ED50 = 16 mg/kg). These studies on structure-activity relationships indicate that there is a requirement for an acidic function and the presence of the (2-quinolinylmethoxy)phenyl moiety in a specific geometric arrangement.

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene D4 receptor antagonists. 2. Effects of an additional phenyl ring on receptor affinity.

This series of reports describe the development of orally active, highly potent, specific antagonists of the peptidoleukotrienes containing a (2-quinolinylmethoxy)phenyl moiety. The compounds reported in this paper contain an additional phenyl ring, which has significantly improved the receptor affinity. The effect of changes in the linkage between the two phenyl rings as well as the orientation of the acidic functional group on biological activity are discussed. Many of these compounds have high affinity to the sulfidopeptide leukotriene D4 receptors with Ki values ranging between 2 and 20 nM and are orally active. Compound 27 [RG 12525, 5-[[2-[[4-(2-quinolinylmethoxy)phenoxy]- methyl]phenyl]methyl]-1H-tetrazole] represents the best combination of in vitro and in vivo biological activity in this series and has been selected for further evaluation in clinical studies of asthma.

Development of a novel series of (2-quinolinylmethoxy)phenyl-containing compounds as high-affinity leukotriene D4 receptor antagonists. 4. Addition of chromone moiety enhances leukotriene D4 receptor binding affinity.

The combination of the benzopyran-4-one ring, a moiety found in the prototype leukotriene antagonist, FPL 55,712, with the (2-quinolinylmethoxy)phenyl group led to a significant increase in leukotriene receptor binding affinity. This modification resulted in a 10,000-fold improvement in binding affinity compared to FPL 55,712. Compound 7 (RG 12553), with a Ki value of 0.1 nM, has higher affinity than the natural agonist LTD4 and is one of the most potent LTD4 antagonists reported. The structure-activity relationships of this series of potent leukotriene antagonists are discussed.

A novel series of [2-[methyl(2-phenethyl)amino]-2-oxoethyl] benzene-containing leukotriene B4 antagonists: initial structure-activity relationships.

This report describes the synthesis of a new class of LTB4 receptor antagonists containing [2-[methyl(2-phenethyl)amino]-2-oxoethyl]benzene as a key binding domain for interaction with high-affinity LTB4 receptors. In addition to this binding domain, two other structural features, an acid function and a lipophilic group, are also required by these compounds for high binding affinity. Our studies indicate that maximal binding affinity in this series is controlled by the spatial relationship of these groups relative to one another. The structure-activity relationships are discussed. The most potent compound in this chemical series, (E)-5-[2-[methyl(2-phenethyl)-amino]-2-oxoethyl]-2-(benzyloxy)cinn amic acid (32), has an IC50 of 2 nM in a guinea pig spleen cell membrane assay. In the whole-cell human neutrophils binding assay, (Z)-5-[2-[methyl-(2-phenethyl)amino]-2-oxoethyl]-2-(benzyloxy)cinn amic acid (30) was the most potent compound with an IC50 of 50 nM.

Structure-activity relationships study of two series of leukotriene B4 antagonists: novel indolyl and naphthyl compounds substituted with a 2-[methyl(2-phenethyl)amino]-2-oxoethyl side chain.

N-Methyl-N-phenethylphenylacetamide has been reported to be a key binding domain to LTB4 receptors. Here we describe the synthesis and structure-activity relationship (SAR) studies of two new series of LTB4 receptor antagonists in which the phenyl ring of this receptor binding domain is replaced with indole and naphthalene, respectively. Results of these studies indicate that, in addition to the 2-[methyl(2-phenethyl)amino]-2-oxoethyl moiety, the presence of an acid group and a lipophilic side chain, as well as the spatial relationship of these three functions, is crucial for high binding affinity with LTB4 receptors. Our SAR studies also reveal that an arenecarboxylic acid, or an enoic acid in which the carboxyl group is conjugated with the central ring, is the preferred polar group. The lipophilic side chain of the naphthyl series was found to tolerate minor variations, ranging from a phenylmethoxy group to phenyl and alkyloxy groups. The most active compounds are 2-ethyl-3-[1-[2-[methyl(2-phenethyl) amino]-2-oxoethyl]-5-(phenylmethoxy)indol-3-yl]propenoic+ ++ acid (4g) of the indolyl series and 4-[2-[methyl(2-phenethyl)amino]-2-oxoethyl]-8-(phenylmethoxy )-2-naphthalenecarboxylic acid (2a) or the naphthyl series, with IC50 of 8 and 4.7 nM respectively, in the receptor binding assay using intact human neutrophils.

The significance of functioning gallbladder visualization on hepatobiliary scintigraphy in infants with persistent jaundice.

UNLABELLED: The purpose of this study was to determine whether gallbladder visualization can help exclude biliary atresia in hepatobiliary scintigraphic studies of infants with persistent jaundice. METHODS: One hundred fifty-two infants with persistent jaundice (49 patients with a final diagnosis of biliary atresia and 103 with biliary patency) were studied using both hepatobiliary scintigraphy and abdominal sonography. Food was withheld for 4 h before the examination, and the infants were fed nothing but glucose until 6 h after the initial injection of (99m)Tc-disofenin or until the gallbladder was seen. If the gallbladder was seen, the infants were fed milk, and imaging was continued to observe gallbladder contractility. RESULTS: In none of the 49 patients with biliary atresia could the gallbladder be seen with hepatobiliary scintigraphy, but abdominal sonography revealed 9 normal-sized gallbladders. Of the 103 patients with biliary patency, hepatobiliary scintigraphy detected the gallbladder more frequently (74%, 76/103) than did abdominal sonography (63%, 65/103). All visualized gallbladders contracted after the infants were fed milk. If we include visualization of both the gallbladder and bowel radioactivity as criteria, the specificity of biliary atresia on hepatobiliary scintigraphy increases to 86% (89/103). CONCLUSION: Gallbladders were usually visible on hepatobiliary scintigraphy of fasting patients with biliary patency. A functioning gallbladder, with or without visualization of bowel radioactivity, indicated biliary patency.

Distribution of morphinan and benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somninferum.

The opium poppy Papaver somniferum L. produces the antimicrobial benzo[c]phenanthridine alkaloid sanguinarine and the narcotic analgesic morphinan alkaloid morphine. Transcripts of three genes of alkaloid biosynthesis in P. somniferum in developing seedlings, mature plants and plant cell suspension culture were monitored for temporal/spatial or for methyl jasmonate-induced accumulation by RNA gel blot analysis. These genes encoded (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B1) that is common to morphine and sanguinarine biosynthesis, the berberine bridge enzyme (BBE) that lies on the pathway to sanguinarine, and codeinone reductase (COR) the penultimate enzyme of morphine biosynthesis. In developing P. somniferum seedlings, the morphine precursor thebaine was present throughout the first twenty days of germination. In contrast, sanguinarine was present in detectable quantities only after day five after germination and continued to increase at least until day twenty. Accumulation of cyp80b1, bbe1 and cor1 gene transcripts paralleled these differences. In the mature poppy plant, cyp80b1, bbe1 and cor1 gene transcripts were detected in the root, the stem, the leaf lamina and the leaf mid rib. Only cyp80b1 and cor1, however, were found in the flower bud and the capsule. Consistent with the fact that sanguinarine accumulation, but not that of morphine, can be induced in opium poppy cell suspension culture by addition of methyl jasmonate to the culture medium, cyp80b1 and bbe1, but not cor1 transcript accumulated in response to elicitor treatment.