Natural Killer Cell Receptor+ T‐Lymphocytes in Normal and Helicobacter pylori‐Infected Human Gastric Mucosa

Background: Helicobacter pylori infection is associated with development of chronic inflammation and infiltration of immune cells into the gastric mucosa. As unconventional T‐lymphocytes expressing natural killer cell receptors are considered to play central roles in the immune response against infection, a study investigating their frequencies in normal and H. pylori‐infected gastric mucosa was undertaken. Materials and Methods: Flow cytometry was used to quantify T‐cells expressing the natural killer cell markers CD161, CD56, and CD94 in freshly isolated lymphocytes from the epithelial and lamina propria layers of gastric mucosa. Thirteen H. pylori‐positive and 24 H. pylori‐negative individuals were studied. Results: CD94⁺ T‐cells were the most abundant (up to 40%) natural killer receptor‐positive T‐cell population in epithelial and lamina propria layers of H. pylori‐negative gastric mucosa. CD161⁺ T‐cells accounted for about one‐third of all T‐cells in both compartments, but the lowest proportion were of CD56⁺ T‐cells. Compared with H. pylori‐negative mucosa, in H. pylori‐infected mucosa the numbers of CD161⁺ T‐cells were significantly greater (p = .04) in the epithelium, whereas the numbers of CD56⁺ T‐cells were lower (p = .01) in the lamina propria. A minor population (< 2%) of T‐cells in both mucosal layers of H. pylori‐negative subjects were natural killer T‐cells, and whose proportions were not significantly different (p > .05) to those in H. pylori‐infected individuals. Conclusions: The predominance, heterogeneity, and distribution of natural killer cell receptor‐positive T‐cells at different locations within the gastric mucosa reflects a potential functional role during H. pylori infection and warrants further investigation.     

Untreated and enzyme‐modified bovine whey products reduce association of Salmonella Typhimurium,Escherichia coli O157:H7 and Cronobacter malonaticus (formerly Enterobacter sakazakii) to CaCo‐2 cells

Aims: Adhesion of a micro‐organism to a cell surface is often considered to be the first step in pathogenesis. Inhibiting this process may have therapeutic effects in vivo. This study investigates the inhibitory effects of various bovine whey products on the association of Salm. Typhimurium, E. coli O157:H7 and C. malonaticus (formerly Enterobacter sakazakii) to the human CaCo‐2 cell line. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus was also examined. Methods and Results: Infection assays were performed by incubating pathogenic acteria with CaCo‐2 cells in the presence of untreated (UT) or enzyme‐modified (EM) whey products. Associated micro‐organisms were directly quantified by plate counts. Invasion of CaCo‐2 cells by Salm. Typhimurium and C. malonaticus in the presence/absence of test materials was also quantified using gentamicin protection assays. At a concentration of 40 mg ml^?1, some UT whey products reduced association and invasion, but this effect was enhanced following hydrolysis with porcine pancreatic lipase. Conclusions: Both UT and EM sweet whey protein concentrates (WPCs) were found to be particularly effective inhibitors of association and invasion. All EM whey products significantly (P < 0·05) inhibited invasion of C. malonaticus into epithelial cells, causing a 2‐log reduction in the quantity of these micro‐organisms internalized. Significance and Impact of the Study: The present study suggests that whey products can inhibit association to and invasion of CaCo‐2 cells by selected micro‐organisms and may be useful in the treatment and/or prevention of foodborne infections.     

Probiotic‐mediated competition,exclusion and displacement in biofilm formation by food‐borne pathogens

The objective of this study was to examine the inhibitory effect of probiotic strains on pathogenic biofilm formation in terms of competition, exclusion and displacement. Probiotic strains (Lactobacillus acidophilus KACC 12419, Lact. casei KACC 12413, Lact. paracasei KACC 12427 and Lact. rhamnosus KACC 11953) and pathogens (Salmonella Typhimurium KCCM 40253 and Listeria monocytogenes KACC 12671) were used to evaluate the auto‐aggregation, hydrophobicity and biofilm formation inhibition. The highest auto‐aggregation abilities were observed in Lact. rhamnosus (17·5%), Lact. casei (17·2%) and Lact. acidophilus (15·1%). Salm. Typhimurium had the highest affinity to xylene, showing the hydrophobicity of 53·7%. The numbers of L. monocytogenes biofilm cells during the competition, exclusion and displacement assays were effectively reduced by more than 3 log when co‐cultured with Lact. paracasei and Lact. rhamnosus. The results suggest that probiotic strains can be used as alternative way to effectively reduce the biofilm formation in pathogenic bacteria through competition, exclusion and displacement.

Significance and Impact of the Study

This study provides new insight into biofilm control strategy based on probiotic approach. Probiotic strains effectively inhibited the biofilm formation of Listeria monocytogenes through the mechanisms of competition, exclusion and displacement. These findings contribute to better understand the probiotic‐mediated competition, exclusion and displacement in biofilm formation by pathogens.     

Establishment and characterization of a novel ‘double‐hit’ follicular lymphoma cell line,FL‐SJC

About 5 per cent of follicular lymphoma (FL) cases are double‐hit (DH) lymphomas. Double‐hit follicular lymphoma (DHFL) cell lines can improve our understanding and drug development on FL. But there are only few DHFL cell lines. Here, we established a new MYC/BCL2 DHFL cell line, FL‐SJC. The cells were obtained from the hydrothorax of a patient with MYC/BCL2 DHFL and cultured for 140 passages in vitro. FL‐SJC cells demonstrated CD19⁺⁺, CD20⁺, CD22⁺⁺, HLA‐DR⁺, CD10⁺, CD38⁺, Lambda⁺ CD23^‐, CD5^‐ and Kappa^‐. The chromosome karyotypic analysis confirmed the co‐existence of t(8;22)(q24;q11) and t(14;18)(q32;q21), as well as additional abnormalities involving chromosomes 2 and 3. Fluorescence in situ hybridization analysis (FISH) showed IGH/BCL2 fusion gene and the MYC rearrangement. In addition, the FL‐SJC cells displayed KMT2D/MLL2 and CREBBP gene mutations. After subcutaneous inoculation of FL‐SJC cells, the SCID mice developed solid tumour masses within 6‐8 weeks. FL‐SJC cells were proven to be free of Epstein‐Barr (EB) virus infection and be multidrug‐resistant. In a conclusion, the FL‐SJC cell line has been identified as a novel MYC/BCL2 double‐hit follicular lymphoma that can be used as a potentially available tool for the clinical and basic research, together with the drug development for MYC/BCL2 DHFL.     

The proteins (12 and 15 kDa) isolated from heat‐killed Lactobacillus plantarum L67 induces apoptosis in HT‐29 cells

A number of scientific studies have revealed that Lactobacillus strains have beneficial bioactivities in the gastrointestinal tract. In this study, the production of intracellular reactive oxygen species (ROS) and the amounts of intracellular calcium, protein kinase C activity, cytochrome c, Bid, Bcl‐2, Bax and the apoptosis‐mediated proteins [caspase‐8, caspase‐3 and poly ADP ribose polymerase (PARP)] were evaluated to understand the induction of programmed cell death in HT‐29 cells by Lactobacillus plantarum L67. The results obtained from this study indicated that the relative intensities of the apoptotic‐related factors (intracellular ROS and intracellular calcium) and of apoptotic signals (Bax and t‐Bid) increased with increasing concentrations of the membrane proteins isolated from heat‐killed L. plantarum L67, whereas the relative intensities of cytochrome c, Bcl‐2, caspase‐8, caspase‐3 and PARP decreased. This study determines whether proteins (12 and 15 kDa) isolated from heat‐killed L. plantarum L67 induce programmed cell death in HT‐29 cells. Proteins isolated from L. plantarum L67 can stimulate the apoptotic signals and then consequently induce programmed cell death in HT‐29 cells. The results in this study suggest that the proteins isolated from L. plantarum L67 could be used as an antitumoural agent in probiotics and as a component of supplements or health foods. Copyright © 2015 John Wiley & Sons, Ltd.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Genomewide identification and analysis of heat‐shock proteins 70/110 to reveal their potential functions in Chinese soft‐shelled turtle Pelodiscus sinensis

Heat‐shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft‐shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved “heat shock protein 70” domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue‐specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B‐L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B‐L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.     

Porphyromonas gingivalis induces the production of interleukin‐31 by human mast cells,resulting in dysfunction of the gingival epithelial barrier

Interleukin (IL)‐31 is important for innate immunity in mucosal tissues and skin, and increased IL‐31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL‐31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL‐31 expression in the gingival tissues of wild‐type mice but not in those of mast cell‐deficient mice. The P. gingivalis‐induced IL‐31 production by human mast cells occurred through the activation of the JNK and NF‐κB signalling pathways and was dependent on the P. gingivalis lysine‐specific protease gingipain‐K. P. gingivalis infection induced IL‐31 receptor α and oncostatin M receptor β expression in human gingival epithelial cells. Notably, the P. gingivalis‐induced IL‐31 production by mast cells led to the downregulation of claudin‐1, a tight junction molecule, in gingival epithelial cells, resulting in an IL‐31‐dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL‐31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

Permeabilized Escherichia coli Whole Cells Containing Co‐Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo‐Inositol from myo‐Inositol

Scyllo‐inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor‐balance biotransformation for the production of SI from myo‐inositol (MI) by thermophilic myo‐inositol 2‐dehydrogenase (IDH) and scyllo‐inositol 2‐dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co‐expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole‐cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L^?1 of SI is produced from 250 g L^?1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors’ limited knowledge. This study provides a promising method for the large‐scale industrial production of SI.     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

UHPLC/HR‐ESI‐MS/MS Profiling of Phenolics from Tunisian Lycium arabicum Boiss. Antioxidant and Anti‐lipase Activities’ Evaluation

This study was performed in the aim to evaluate nine different extracts from Tunisian Lycium arabicum for their total phenolic and total flavonoid contents, phytochemical analyses as well as their antioxidant and anti‐lipase activities. The in vitro antioxidant property was investigated using three complementary methods (DPPH, ferric reducing antioxidant power (FRAP), and β‐carotene‐linoleic acid bleaching assays) while anti‐lipase activity was evaluated using 4‐methylumbelliferyl oleate method. From all of the tested extracts the most potent found to be the polar MeOH extracts especially those of stems and leaves. In order to investigate the chemical composition of these extracts and possible correlation of their constituents with the observed activities, an UHPLC/HR‐ESI‐MS/MS analysis was performed. Several compounds belonging to different chemical classes were tentatively identified such as rutin and kampferol rutinoside, the major constituents of the leaves, and N‐caffeoyltyramine, lyciumide A, N‐dihydrocaffeoyltyramine as well as fatty acids: trihydroxyoctadecadienoic acid and hydroxyoctadecadienoic acid isomers were detected abundantly in the stems. These results showed that the MeOH extracts of stems and leaves of L. arabicum can be considered as a potential source of biological active compounds.     

Inhibition of yes‐associated protein down‐regulates PD‐L1 (CD274) expression in human malignant pleural mesothelioma

Although tumour PD‐L1 (CD274) expression had been used as a predictive biomarker in checkpoint immunotherapy targeting the PD1/PD‐L1 axis in various cancers, the regulation of PD‐L1 (CD274) expression is unclear. Yes‐associated protein (YAP), an important oncogenic protein in Hippo signalling pathway, reportedly promotes cancer development. We investigated whether inhibition of YAP down‐regulates PD‐L1 (CD274) in human malignant pleural mesothelioma (MPM). Western blotting showed that 2 human MPM cell lines (H2052 and 211H) had increased PD‐L1 protein expression compared to H290, MS‐1 and H28 cells. In H2052 and 211H cells, PD‐L1 mRNA expression was significantly increased compared to other MPM cell lines; YAP knockdown by small interfering RNA decreased PD‐L1 protein and mRNA expression. Forced overexpression of the YAP gene increased PD‐L1 protein expression in H2452 cells. Chromatin immunoprecipitation (ChIP) assay showed the precipitation of PD‐L1 enhancer region encompassing 2 putative YAP‐TEAD‐binding sites in H2052 cells. We found that, in human MPM tissue microarray samples, YAP and PD‐L1 concurrently expressed in immunohistochemistry stain (n = 70, P < .05, chi‐square). We conclude that PD‐L1 is correlated with YAP expression, and inhibition of YAP down‐regulates PD‐L1 expression in human MPM. Further study of how YAP regulates PD‐L1 in MPM is warranted.     

Electrophilic [N—F]+ catalysed asymmetric allylation of (E)‐N,1‐diphenylmethanimine

New efficient catalysts based on electrophilic N‐fluoro quaternary ammonium salts are reported for catalytic allylation of (E)‐N,1‐diphenylmethanimine. The chiral version of the catalyst based on cinchonidine (F‐CD‐BF₄) shows high catalytic activity with approximately 94% ee and TOF (>800 h^?1). The F‐CD‐BF₄ is prepared from cinchonidine and Selectfluor by one‐step transfer fluorination.     

A novel kinase function of a nucleoside‐diphosphate‐kinase homologue in Porphyromonas gingivalis is critical in subversion of host cell apoptosis by targeting heat‐shock protein 27

We have previously shown that a homologue of a conserved nucleoside‐diphosphate‐kinase (Ndk) family of multifunctional enzymes and secreted molecule in Porphyromonas gingivalis can modulate select host molecular pathways including downregulation of reactive‐oxygen‐species generation to promote bacterial survival in human gingival epithelial cells (GECs). In this study, we describe a novel kinase function for bacterial effector, P. gingivalis‐Ndk, in abrogating epithelial cell death by phosphorylating heat‐shock protein 27 (HSP27) in GECs. Infection by P. gingivalis was recently suggested to increase phosphorylation of HSP27 in cancer‐epithelial cells; however, the mechanism and biological significance of antiapoptotic phospho‐HSP27 during infection has never been characterised. Interestingly, using glutathione S‐transferase‐rNdk pull‐down analysed by mass spectrometry, we identified HSP27 in GECs as a strong binder of P. gingivalis‐Ndk and further verified using confocal microscopy and ELISA. Therefore, we hypothesised P. gingivalis‐Ndk can phosphorylate HSP27 for inhibition of apoptosis in GECs. We further employed P. gingivalis‐Ndk protein constructs and an isogenic P. gingivalis‐ndk‐deficient‐mutant strain for functional examination. P. gingivalis‐infected GECs displayed significantly increased phospho‐HSP27 compared with ndk‐deficient‐strain during 24 hr infection. Phospho‐HSP27 was significantly increased by transfection of GFP‐tagged‐Ndk into uninfected‐GECs, and in vitro phosphorylation assays revealed direct phosphorylation of HSP27 at serines 78 and 82 by P. gingivalis‐Ndk. Depletion of HSP27 via siRNA significantly reversed resistance against staurosporine‐mediated‐apoptosis during infection. Transfection of recombinant P. gingivalis‐Ndk protein into GECs substantially decreased staurosporine‐induced‐apoptosis. Finally, ndk‐deficient‐mutant strain was unable to inhibit staurosporine‐induced Cytochrome C release/Caspase‐9 activation. Thus, we show for the first time the phosphorylation of HSP27 by a bacterial effector—P. gingivalis‐Ndk—and a novel function of Ndks that is directly involved in inhibition of host cell apoptosis and the subsequent bacterial survival.     

Enhanced resistance in Theobroma cacao against oomycete and fungal pathogens by secretion of phosphatidylinositol‐3‐phosphate‐binding proteins

The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol‐3‐phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P‐binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P‐binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P‐binding site, or by a secreted PI4P‐binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P‐binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P‐binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.     

Immunolocalization and expression of small‐conductance calcium‐activated potassium channels in human myometrium

Small‐conductance calcium‐activated potassium (SK3) channels have been detected in human myometrium and we have previously shown a functional role of SK channels in human myometrium in vitro. The aims of this study were to identify the precise localization of SK3 channels and to quantify SK3 mRNA expression in myometrium from pregnant and non‐pregnant women. Myometrial biopsies were obtained from pregnant (n = 11) and non‐pregnant (n = 11) women. The expression of SK3 channels was assessed using immunohistochemistry and SK3 mRNA was determined by qRT‐PCR. In non‐pregnant myometrium SK3 immunoreactivity was observed in CD34 positive (CD34⁺) interstitial Cajal‐like cells (ICLC), now called telocytes. Although CD34⁺ cells were also present in pregnant myometrium, they lacked SK3 immunoreactivity. Furthermore, the immunohistochemical results showed that SK3 expression in vascular endothelium was similar between the two groups. CD117 immunoreactivity was only detected in small round cells that resemble mast cells. Compared to non‐pregnant myometrium we found significantly less SK3 mRNA in pregnant myometrium. We demonstrate that SK3 channels are localized solely in CD34⁺ cells and not in smooth muscle cells, and that the molecular expression of SK3 channels is higher in non‐pregnant compared to pregnant myometrium. On the basis of our previous study and the present findings, we propose that SK3 activators reduce contractility in human myometrium by modulating telocyte function. This is the first report to provide evidence for a possible role of SK3 channels in human uterine telocytes.     

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25‐residue innate immunity peptide released by pepsin from the N‐terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR‐NH₂) retains antimicrobial activity and is thought to constitute the “antimicrobial active‐site” (Tomita, Acta Paediatr Jpn. 1994; 36 : 585–91). Here we report on N‐acylation of 1‐Me‐Trp⁵‐LfB6, Cn‐RRWQ[1‐Me‐W]R‐NH₂, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp⁵) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn‐RRWQ[1‐Me‐W ]R‐NH₂ (deuterated 1‐Me‐Trp⁵ underlined), were monitored by solid state ³¹P NMR and ²H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn‐RRWQ[&1‐Me‐W ]R‐NH₂ peptides added at a 1:25 peptide to lipid ratio. ²H‐NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The ²H NMR quadrupolar splittings suggest that the 1‐Me‐Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. ³¹P‐NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0‐RRWQ[1‐Me‐W ]R‐NH₂, resulted in a 3–4 fold increase in antimicrobial activity against E. coli. N‐acylation with a C12 fatty acid enhanced activity almost 90 fold. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Expression of PD‐1/LAG‐3 and cytokine production by CD4+ T cells during infection with Plasmodium parasites

CD4⁺ T cells play critical roles in protection against the blood stage of malarial infection; however, their uncontrolled activation can be harmful to the host. In this study, in which rodent models of Plasmodium parasites were used, the expression of inhibitory receptors on activated CD4⁺ T cells and their cytokine production was compared with their expression in a bacterial and another protozoan infection. CD4⁺ T cells from mice infected with P. yoelii 17XL, P yoelii 17XNL, P. chabaudi, P. vinckei and P. berghei expressed the inhibitory receptors, PD‐1 and LAG‐3, as early as 6 days after infection, whereas those from either Listeria monocytogenes‐ or Leishmania major‐infected mice did not. In response to T‐cell receptor stimulation, CD4⁺ T cells from mice infected with all the pathogens under study produced high concentrations of IFN‐γ. IL‐2 production was reduced in mice infected with Plasmodium species, but not in those infected with Listeria or Leishmania. In vitro blockade of the interaction between PD‐1 and its ligands resulted in increased IFN‐γ production in response to Plasmodium antigens, implying that PD‐1 expressed on activated CD4⁺ T cells actively inhibits T cell immune responses. Studies using Myd88^?/?, Trif^?/? and Irf3^?/? mice showed that induction of these CD4⁺ T cells and their ability to produce cytokines is largely independent of TLR signaling. These studies suggest that expression of the inhibitory receptors PD‐1 and LAG‐3 on CD4⁺ T cells and their reduced IL‐2 production are common characteristic features of Plasmodium infection.