Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.     

Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells

Activation of T lymphocytes requires protein kinase C theta (PKC-theta) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-theta on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.     

A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

GABA_A receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.     

Development of 2,4-diaminopyrimidine derivatives as novel SNSR4 antagonists

2,4-Diaminopyrimidines derivatives were developed as a novel class of SNSR4 antagonists. Structure activity relationship of the diamino pyrimidine core was explored and a tool compound suitable for target validation was identified.     

Evaluation of (R)-(-)-α-methoxy phenyl acetic acid as a chiral shift reagent for resolution and determination of R and S enantiomers of modafinil in bulk drugs and formulations by 1H NMR spectroscopy

(R)-(-)-α-Methoxy phenyl acetic acid, (S)-(-)-1,1'-(2-naphthol), and (R)-(+)-α-methoxy-α-trifluoromethyl phenyl acetic acid were evaluated as chiral shift reagents (CSRs) for (1)H NMR spectroscopic resolution and determination of R and S enantiomers of modafinil (MDL) in bulk drugs and formulations. Effects of the nature of CSR and the weight ratio of substrate to shift reagent on enantiomeric discrimination were investigated. Intramolecular and intermolecular hydrogen bonding interactions between the drug and the CSR seem to be the driving force for desired resolution. A mechanism was proposed to explain the interactions between (R, S)-enantiomers of MDL and (R)-(-)-α-methoxy phenyl acetic acid. The method was validated and applied successfully to determine the enantiomeric purity of MDL in tablet formulations.     

Stabilized beta-catenin potentiates Fas-mediated T cell apoptosis

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) T cells. Using transgenic mice expressing a stabilized beta-catenin (beta-cat(Tg)), we show here that beta-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, beta-cat(Tg) mice exhibited accelerated deletion of CD4(+)Vbeta8(+) T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from beta-cat(Tg) mice than that from wild type mice. Additionally, T cells from beta-cat(Tg) mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, beta-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the beta-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.     

Effect on growth and reproduction of hormone immersed and masculinized fighting fish Betta splendens

To produce all-male progenies in the fighting fish, Betta splendens, six groups of fry were subjected to discrete immersion treatment at different 17alpha-methyltestosterone (MT) doses (viz. 100, 200, 500, 700, 900, and 1,000 microg/l) for a constant duration (3 hr/day) and frequency (second, fifth, and eighth day after hatching). The treatment at 900 microg/l led to 98% masculinization and 71% survival at sexual maturity. Treated groups, which showed significant deviation from the 1:1 sex ratio, were classified into two different series: S1 and S2. The groups that showed nearly cent-percent masculinization were classified as S1, and the other groups were classified as S2. The S1 males showed remarkably slower growth and attained 3.5 cm total length compared to 6.0 cm attained by a normal male. The S2 males attained 5.4 cm total length. Apart from these morphological defects, both S1 and S2 males suffered functional (decreased sperm count and sperm motility) and behavioral defects (incomplete embracing during mating) in their reproductive ability, leading to approximately 50% and 30% reduction in fecundity per mating, respectively. The cumulative fecundity loss suffered by the S1 male during its active reproductive phase is discussed. When normal and sex-reversed males were presented, a female preferred the former. Progeny testing of the sex-reversed males showed the occurrence of 12.75% males, indicating the possible role of autosomal genes in the sex determination mechanism of this species. Discrete immersion treatment at optimal/super-optimal doses ensured not only a higher percentage of masculinization, but also a higher frequency of homogametic males (XX).     

The role of the charged residues of the GP2 helical regions in Ebola entry

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.     

Growth enhancement and food conversion efficiency of transgenic fish Labeo rohita

Three family lines of fast growing transgenic rohu Labeo rohita (rohu) were generated by electroporated-sperm-mediated transfer of the vectors harboring CMV promoter or grass carp beta-actin promoter fused to endogenous rohu GH (rGH) cDNA. The gene transfer efficiency was 25%. The transgenic rohu (family line 1) with CMV promoter showed a growth enhancement of four times normal size, whereas those (family lines 2 and 3) generated with beta-actin promoter grew 4.5 and 5.8 times faster than their respective control siblings. Southern analysis confirmed the transgene extrachromosomal (Te) persistence until the 60th week in family 1. The individuals of family lines 2 and 3, however, showed integration (Ti), as well as persistence as extrachromosomal copies (Te) until the age of 30 weeks. Mosaicism of the transgene was shown at the levels of its presence and expression. The ectopic expression of rGH mRNA was confirmed by RT-PCR. Feeding experiments revealed that the transgenic rohu ate food at a lower rate but grew more efficiently than their control siblings.