CD4(+) T cells from food allergy model are resistant to TCR-dependent apoptotic induction.

BACKGROUND: CD4(+) T cell polarization plays a critical role in the pathogenesis of allergy. How to modulate the skewed CD4(+) T cell polarization is less clear. The specific immunotherapy (SIT) is the only specific remedy for the treatment of allergic diseases; the therapeutic effect is to be improved. OBJECTIVES: This study aims to investigate the role of interleukin (IL)-18 in enhancing the therapeutic effect of SIT. METHODS: A peanut allergy mouse model was developed and treated with SIT or/and IL-18. CD4(+) T cell apoptosis was assessed by flow cytometry. The expression of Fas ligand (FasL) was observed by quantitative real time RT-PCR and Western blotting. Interferon-γ in the culture medium was determined by enzyme-linked immunosorbent assay. The fasL gene promoter methylation in CD4(+) T cells was assessed by methylation specific PCR. RESULTS: The results showed that lower levels of IL-18 were detected in allergic mice; administration of IL-18 significantly enhanced the therapeutic effect of SIT on suppressing the allergic inflammation in the mouse intestine. In the cell culture studies, IL-18 increased the TCR-dependent CD4(+) T cell apoptosis, the expression of FasL in CD4(+) T cells, the production of Interferon-γ and the demethylation of the FasL promoter in CD4(+) T cells. CONCLUSIONS: Administration of IL-18 enhances the effect of SIT on suppressing allergic inflammation in the mouse intestine via enhancing the TCR-dependent CD4(+) T cell apoptosis.

Exploration of the effect of probiotics supplementation on intestinal microbiota of food allergic mice.

Environmental factor-induced alterations in intestinal microbiota have been demonstrated to be associated with increasing prevalence of food allergy. However, it is not clear to what extent oral administration of probiotics can affect gut microbiota composition, thus inhibiting food allergy development. Using ovalbumin (OVA)-sensitized murine model, it was demonstrated that probiotics ameliorated allergic symptoms, including reducing OVA specific-IgE, and -IgG1 levels in the serum, Th2 cytokines release in spleen, and occurrence of diarrhea. Moreover, 16S rRNA analysis showed that the probiotics-mediated protection was conferred by an enrichment of Coprococcus and Rikenella. The present study supports the theory that probiotics can treat food allergy by modulating specific genera of the gut microbiota.

Kinetic changes of intestinal microbiota in the course of intestinal sensitization.

Food allergy (FA) is an adverse immune response to certain innocent food. It is estimated about 2% to 6% of the general population suffer from FA. Symptoms of a food allergic reaction may involve the gastrointestinal tract or/and other organs. The gut microbiota plays a critical role in diet-induced health problems. Whether the changes in the composition of the intestinal microbiota regulate allergic responses to food remains poorly understood. Thus, we created an FA animal model, sequenced the V4-V5 regions of 16S rRNA genes to characterize the genera abundance of gut microbiota. The results showed that mice under FA condition showed different gut bacterial structures. Diverse distribution of the bacterial species was identified between FA and control groups. FA altered the components of intestinal Microbiota in mice. The dysbiosis of the gut metagenome correlated with the development of the FA.

The Pathogenesis of Rheumatoid Arthritis is Associated with Milk or Egg Allergy.

BACKGROUND: Rheumatoid arthritis (RA) is a very complicated autoimmune disease with apparent synovial hyperplasia and cartilage and bone destruction. AIMS: In the present study, we aimed to determine whether the pathogenesis of RA correlates with food allergy and which allergen(s) are relevant. MATERIALS AND METHODS: We used type-II collagen (CII) to induce arthritis (collagen-induced arthritis, CIA) model in Wistar rats, and the development of arthritis was evaluated accordingly by scoring system. Proinflammatory cytokine levels in plasma were measured by enzyme-linked immunosorbent assay (ELISA), and concentrations of circulating immune complexes (CICs) were analyzed by C1q solid phase method. Furthermore, food-specific immunoglobulin G (IgG) and immunoglobulin E (IgE) levels were determined in the CIA model. RESULTS: In the CIA model, we found that levels of tumor necrosis factor-alpha (TNF-a), interleukin (IL)-1, IL-6, and IL-17, as well as CICs, were elevated significantly. Moreover, concentrations of milk- or egg-specific IgG and IgE were enhanced strikingly in CIA rats. CONCLUSION: The results suggest that pathogenesis of RA correlates closely to increased egg- or milk-specific antibodies.

Herbal Formula-3 inhibits food allergy in rats by stabilizing mast cells through modulating calcium mobilization.

BACKGROUND: The prevalence of food allergy has increased dramatically during the last three decades, but currently there was no effective therapy except avoidance of allergen. This study aimed to investigate the effect of a modified Chinese herbal formula, Formula-3, on mast cell degranulation and the anti-allergic activity in both animal and cell models. METHODS: With OVA-sensitized food allergic model in Brown-Norway rats, we checked tissue injury in the small intestines by H&E staining. The Th2 cytokine levels and IgE production in serum or supernatant of the intestinal mucosa homogenates were analyzed by ELISA. Meanwhile, rat peritoneal mast cell activation and degranulation were examined by Toluidine Blue Stain and the release of histamine was measured. Furthermore, the regulation of Formula-3 on Ca(2+) mobilization was investigated by probing intracellular Ca(2+) with fluo-4 fluorescence. The direct effect of Formula-3 on mast cell stabilization was also studied in RBL-2H3 cell line. RESULTS: In vivo Formula-3 administration significantly reduced tissue damage in the small intestines of rat and suppressed Th2 cytokine secretion and IgE production. We demonstrated that Formula-3 treatment significantly suppressed FcεR1-mediated mast cell degranulation no matter in OVA-challenged allergic rats or IgE-sensitized RBL-2H3 cell line. Furthermore, Formula-3 significantly decreased Ca(2+) influx through store-operated calcium channels (SOCs) evoked by dinitrophenyl-BSA or thapsigargin in mast cells. CONCLUSION: Taken together, our data indicate that Formula-3 stabilizes mast cells by suppressing FcεR1-induced Ca(2+) mobilization mainly through inhibiting Ca(2+) entry via SOCs, thus exerting a protective effect against OVA-sensitized food allergy.

Polydatin attenuated food allergy via store-operated calcium channels in mast cell.

AIM: To investigate the effect of polydatin (PD), a resveratrol glucoside, on mast cell degranulation and anti-allergic activity. METHODS: After the rats were orally sensitized with ovalbumin (OVA) for 48 d and underwent PD treatment for 4 d, all the rats were stimulated by 100 mg/mL OVA for 24 h and then sacrificed for the following experiments. The small intestines from all the groups were prepared for morphology examination by hematoxylin and eosin staining. We also used a smooth muscle organ bath to evaluate the motility of the small intestines. The OVA-specific immunoglobulin E (IgE) production and interleukin-4 (IL-4) levels in serum or supernatant of intestinal mucosa homogenates were analyzed by enzyme-linked immunosorbent assay (ELISA). Using toluidine blue stain, the activation and degranulation of isolated rat peritoneal mast cells (RPMCs) were analyzed. Release of histamine from RPMCs was measured by ELISA, and regulation of PD on intracellular Ca(2+) mobilization was investigated by probing intracellular Ca(2+) with fluo-4 fluorescent dye, with the signal recorded and analyzed. RESULTS: We found that intragastric treatment with PD significantly reduced loss of mucosal barrier integrity in the small intestine. However, OVA-sensitization caused significant hyperactivity in the small intestine of allergic rats, which was attenuated by PD administration by 42% (1.26 ± 0.13 g vs OVA 2.18 ± 0.21 g, P < 0.01). PD therapy also inhibited IgE production (3.95 ± 0.53 ng/mL vs OVA 4.53 ± 0.52 ng/mL, P < 0.05) by suppressing the secretion of Th2-type cytokine, IL-4, by 34% (38.58 ± 4.41 pg/mL vs OVA 58.15 ± 6.24 pg/mL, P < 0.01). The ratio of degranulated mast cells, as indicated by vehicles (at least five) around the cells, dramatically increased in the OVA group by 5.5 fold (63.50% ± 15.51% vs phosphate-buffered saline 11.15% ± 8.26%, P < 0.001) and fell by 65% after PD treatment (21.95% ± 4.37% vs OVA 63.50% ± 15.51%, P < 0.001). PD mediated attenuation of mast cell degranulation was further confirmed by decreased histamine levels in both serum (5.98 ± 0.17 vs OVA 6.67 ± 0.12, P < 0.05) and intestinal mucosa homogenates (5.83 ± 0.91 vs OVA 7.35 ± 0.97, P < 0.05). Furthermore, we demonstrated that administration with PD significantly decreased mast cell degranulation due to reduced Ca(2+) influx through store-operated calcium channels (SOCs) (2.35 ± 0.39 vs OVA 3.51 ± 0.38, P < 0.01). CONCLUSION: Taken together, our data indicate that PD stabilizes mast cells by suppressing intracellular Ca(2+) mobilization, mainly through inhibiting Ca(2+) entry via SOCs, thus exerting a protective role against OVA-sensitized food allergy.

A Krüppel-like factor in Caenorhabditis elegans with essential roles in fat regulation, cell death, and phagocytosis.

We demonstrate that a Caenorhabditis elegans Krüppel-like transcription factor is involved in fat regulation, cell death, and phagocytosis in C. elegans. Suppression of C. elegans klf-1 function by RNA interference (RNAi) results in increased fat storage in the intestine of the RNAi worm that directly or indirectly causes germ cells to die. These dead cells are not engulfed or phagocytosed in the RNAi worm. High-level expression of Ce-klf-1 during larval development, as well as its specific localization in the worm's intestine, supports a direct role for Ce-klf-1 in fat regulation. The C. elegans klf-1 encodes a C(2)H(2) zinc finger protein that is known to act as transcriptional modulator of tissue-specific expression. Members of the Krüppel-like factor (KLF) family play a variety of important roles in vertebrate tissue differentiation. KLFs have recently been implicated in energy and glucose homeostasis through their expression in pancreas, adipose, liver, and muscle tissues. The extensive fat storage and increased cell death in the Ce-klf-1 RNAi worm is important in that it may explain the connection between Ce-klf-1 signaling, cell death, and fat storage. This is the first evidence involving Ce-KLF-1 protein in such functions. In future studies, a thorough analysis of cellular functions of other members of C. elegans Krüppel-like transcription factors together with their interactions and pathway activities with other molecular partners should yield significant insights into mammalian KLF proteins.

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans.

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.

The Caenorhabditis elegans CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization.

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myoepithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes.

CeRh1 (rhr-1) is a dominant Rhesus gene essential for embryonic development and hypodermal function in Caenorhabditis elegans.

Rhesus (Rh) proteins share a conserved 12-transmembrane topology and specify a family of putative CO(2) channels found in diverse species from microbes to human, but their functional essentiality and physiological importance in metazoans is unknown. To address this key issue and analyze Rh-engaged physiologic processes, we sought to explore model organisms with fewer Rh genes yet are tractable to genetic manipulations. In this article, we describe the identification in nematodes of two Rh homologues that are highly conserved and similar to human Rh glycoproteins, and we focus on their characterization in Caenorhabditis elegans. RNA analysis revealed that CeRh1 is abundantly expressed in all developmental stages, with highest levels in adults, whereas CeRh2 shows a differential and much lower expression pattern. In transient expression in human cells, both CeRh1 and CeRh2-GFP fusion proteins were routed to the plasma membrane. Transgenic analysis with GFP or LacZ-fusion reporters showed that CeRh1 is mainly expressed in hypodermal tissue, although it is also in other cell types. Mutagenesis analysis using deletion constructs mapped a minimal promoter region driving CeRh1 gene expression. Although CeRh2 was dispensable, RNA interference with CeRh1 caused a lethal phenotype mainly affecting late stages of C. elegans embryonic development, which could be rescued by the CbRh1 homologue from the worm Caenorhabditis briggsae. Taken together, our data provide direct evidence for the essentiality of the CeRh1 gene in C. elegans, establishing a useful animal model for investigating CO(2) channel function by cross-species complementation.

[Effect of transfection of hEndostatin gene on CNE2 cell xenograft growth in nude mice].

BACKGROUND & OBJECTIVE: Tumor angiogenesis play an important role in growth and metastasis of cancer. Angiogenesis inhibitors induce apoptosis in cancer by inhibiting tumor angiogenesis and have strong inhibiting effect on both growth and metastasis of cancer. This study was designed to explore the effect of transfection of human endostatin gene on nasopharyngeal carcinoma CNE2 cells xenograft growth in nude mice. METHODS: The plasmids (pBlast-hIL-hEndostatin, pBlast-hEndostatin, and pBlast-MCS) were transfected and lipofectin-mediated into the CNE2 cell line. The biological activity of secreted hEndostain from gene-transferred cell lines was determined using MTT method in vitro. Then the transfected CNE2 cells were injected into the nude mice and tumorigenicity of CNE2 was observed in vivo. RESULTS: The supernatant of CNE2 cell transfected with pBlast-hIL-hEndostatin effectively inhibited the growth of endothelial cell (ECV304). The volume and the weight of tumor in pBlast-hIL -hEndostatin transfecting cells group were less than those in control group (P< 0.01). The growth speed of tumor in pBlast-hIL-hEndostatin transfecting cells group was slower than that in control group. CONCLUSION: Transfection of hEndostatin gene could inhibit CNE2 cell growth in nude mice.