[Polarization of human acute monocytic leukemia THP-1 cells-derived macprophages induced by Nippostrongylus brasiliensis proteins in vitro].

OBJECTIVE: To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by Nippostrongylus brasiliensis proteins in vitro, so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections. METHODS: The in-vitro culture of N. brasiliensis was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The in-vitro culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the mRNA expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by N. brasiliensis proteins in vitro. RESULTS: Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (χ2 = 3 721.00, P < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups (χ2 = 105.43, P < 0.001). There were significant differences among groups in terms of the mRNA expression of CCL2 (F = 191.95, P < 0.001), TNF-α (F = 129.95, P < 0.001), IL-12b (F = 82.89, P < 0.001), PPARγ (F = 11.30, P < 0.001), IL-10 (F = 9.51, P < 0.001) and Mrc1 genes (F = 12.35, P < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups (χ2 = 24 004.33 and 832.50, P < 0.001). Higher IL-1ß and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group (P < 0.001), and greater TGF-ß1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group (P < 0.05). CONCLUSIONS: Both L3 and L5 proteins of N. brasiliensis may induce the polarization of THP-1-derived macrophages to M2 type in vitro.

P1BS, a conserved motif involved in tolerance to phosphate starvation in soybean.

Available phosphate (Pi) is a major limiting factor for plant growth, development, and productivity. Phosphate starvation response 1 (PHR1) is a binding dimer that binds to an imperfect palindromic sequence. PHR1-binding sequences (GnATATnC) exist in the promoter of Pi starvation-responsive structural genes, indicating an effect occurring downstream in the Pi starvation signaling pathway. These sequences are referred to as PHR1-binding site (P1BS) structures. In this study, the sequences of GmPHR1 and GmSPX1 from Glycine max (L.) Merr. soybean were determined and analyzed. We found that GmPHR1 is an MYB-related transcription factor. In addition, GmSPX1 contained a P1BS structure, which is an important cis-regulatory motif in the phosphate signaling pathway. We found that GmPHR1 can physically interact with GmSPX1 through the cis-element, which may be a major pathway for the GmPHR1-mediated Pi starvation stress response. Thus, the P1BS structure in the Pi starvation signaling pathway is an important cis-regulatory motif that improves the tolerance to low phosphorus conditions in soybean.

Molecular cloning and tissue distribution profiles of the chicken R-spondin1 gene.

Rspo1 belongs to the Rspo family, which is composed of 4 members (Rspo1-4) that share 40 to 60% sequence homology and similar domain organizations, and regulate the WNT signaling pathway via a common mechanism. Rspo1 plays a key role in vertebrate development and is an effective mitogenic factor of gastrointestinal epithelial cells. We report the cloning of chicken Rspo1 and its gene expression distribution among tissues. It contained an open reading frame of 783 bp encoding a protein of 260 amino acids, and its molecular weight was predicted to be 28.80 kDa. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that chicken Rspo1 was highly expressed in the stomach muscle tissue, but was expressed at low levels in the lung, brain, jejunum, cecum, ileum, spleen, pancreas, kidney, and glandular stomach. These results suggest that Rspo1 plays a major role in muscular immune protection.

Investigations into the association between polymorphisms in the interleukin-10 gene and risk of early-onset preeclampsia.

In this case-control study, we assessed the influence of IL-10 -1082A/G and -819T/C on the development of preeclampsia. The IL-10 -1082A/G and -819T/C polymorphisms were assessed by polymerase chain reaction-restriction fragment length polymorphism. The genotype distributions of the IL-10 -1082A/G and -819T/C polymorphisms in the control subjects were in conformance with Hardy-Weinberg equilibrium (HWE; P = 0.46 and 0.17). Unconditional logistic regression analyses revealed that individuals carrying the CC genotype of IL-10 -819T/C were associated with an increased risk of preeclampsia compared to the TT genotype. The odds ratio (95% confidence interval) for the CC genotype of IL-10 -819T/C was 1.71 (1.07-3.27) compared to the TT genotype. In conclusion, the results of our study indicated that the IL-10 -819T/C polymorphism was associated with an increased risk of preeclampsia in a Chinese population.

Independent and joint effects of the IL-6 and IL-10 gene polymorphisms in pulmonary tuberculosis among the Chinese Han population.

We investigated the association between interleukin (IL)-6 and IL-10 gene polymorphisms and the susceptibility to pulmonary tuberculosis (PTB). DNA samples were obtained from 191 Han Chinese patients with PTB and 191 healthy control subjects. IL-6 (-572, -174, -597) and IL-10 (-1082, -819) polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism. The IL-6 -572 C/C and IL-10 -819 T/T genotypes were observed less frequently in the case group than in the control group, with crude odds ratios of 0.591 [95% confidence interval (CI) = 0.381-0.917] and 0.401 (95%CI = 0.257-0.627), respectively. A significant association remained after adjusting for environmental factors in multivariate logistic analysis. The homozygote genotypes of IL-6 -572 and IL-10 -819 had an adjusted OR of 0.565 (95%CI = 0.356-0.898) and 0.341 (95%CI = 0.210-0.553), respectively. These results indicate that the mutant heterozygote IL-10 -1082 A/ G+G/G genotype and the homozygote IL-10 -819 T/T genotype have a combined effect on PTB. These results suggest that the IL-6 -572 C/C and IL-10 -819 T/T genotype polymorphisms are protective factors against PTB.

Structural analysis of a 4414-bp element in Drosophila melanogaster.

We cloned a 4414-bp element from a mutant of Drosophila melanogaster. Its insertion site was 18,929,626 bp. Analysis of the nucleotide and amino acid sequences demonstrated that the element is homologous to Pifo_I, first obtained from D. yabuka, which belongs to the gypsy/Ty3 subfamily. We also obtained a 3754-bp length element from a wild-type fly by PCR, with a pair of primers designed from the conserved region of the 4414-bp length element. The two elements included a pair of long terminal repeats and part of the GAG and ENV proteins, but the POL protein was completely lost. This element is found in the subgenus of D. melanogaster, but it is a degenerate type of Pifo_I and is not infective. Also, a 714-bp region structured in 5.0 tandem repeats of 143 bp each was found in the 5'UTR of the degenerate element; these could interact with transcription factor CF2. Phylogenetic analysis and alignment of amino acids indicated that the Pifo_I element was closer to the ZAM retrotransposon, which gave us some clues to their functional similarity. Based on these data, we propose that there is a relationship between the degenerate element and the mutant phenotype, which would provide a foundation for further research.