Fra-2/AP-1 regulates melanoma cell metastasis by downregulating Fam212b.

Metastatic melanoma remains a challenging disease. Understanding the molecular mechanisms how melanoma becomes metastatic is therefore of interest. Herein we show that downregulation of the AP-1 transcription factor member Fra-2 in melanoma cells is associated with an aggressive melanoma phenotype in vitro and in vivo. In vitro, Fra-2 knockdown in melanoma cells promoted cell migration and invasion associated with increased Snail-1, Twist-1/2, and matrix metalloproteinase-2 (MMP-2) expression. In vivo, Fra-2 knockdown in a melanoma cell line led to increased metastasis into the lungs and liver. The increased metastatic potential of Fra-2 knockdown melanoma cells was likely due to an accelerated cell cycle transition and increased tissue angiogenesis. Using Fra-2 knockdown cell lines microarray analysis, we identified the protein Fam212b (family with sequence similarity 212 member B) as a downstream target of Fra-2. By additional knockdown of Fam212b in Fra-2 mutant cells, we mitigated the cell migration, invasion, and cell cycle transition phenotype induced by Fra-2 knockdown. Furthermore, Fam212b overexpression enhanced ß-catenin pathway. Finally, Fam212b expression is correlated with increased melanoma metastasis and poor clinical outcomes in human patients. In summary, these findings reveal the Fra-2-Fam212b axis as a new pathway of melanoma metastasis, which can be in the future used as potential marker of the metastatic properties of melanoma.

Effect of stromal cell-derived factor-1/CXCR4 axis in neural stem cell transplantation for Parkinson's disease.

Previous studies have shown that neural stem cell transplantation has the potential to treat Parkinson's disease, but its specific mechanism of action is still unclear. Stromal cell-derived factor-1 and its receptor, chemokine receptor 4 (CXCR4), are important regulators of cell migration. We speculated that the CXCR4/stromal cell-derived factor 1 axis may be involved in the therapeutic effect of neural stem cell transplantation in the treatment of Parkinson's disease. A Parkinson's disease rat model was injected with 6-hydroxydopamine via the right ascending nigrostriatal dopaminergic pathway, and then treated with 5 µL of neural stem cell suspension (1.5 × 104/L) in the right substantia nigra. Rats were intraperitoneally injected once daily for 3 days with 1.25 mL/kg of the CXCR4 antagonist AMD3100 to observe changes after neural stem cell transplantation. Parkinson-like behavior in rats was detected using apomorphine-induced rotation. Immunofluorescence staining was used to determine the immunoreactivity of tyrosine hydroxylase, CXCR4, and stromal cell-derived factor-1 in the brain. Using quantitative real-time polymerase chain reaction, the mRNA expression of stromal cell-derived factor-1 and CXCR4 in the right substantia nigra were measured. In addition, western blot assays were performed to analyze the protein expression of stromal cell-derived factor-1 and CXCR4. Our results demonstrated that neural stem cell transplantation noticeably reduced apomorphine-induced rotation, increased the mRNA and protein expression of stromal cell-derived factor-1 and CXCR4 in the right substantia nigra, and enhanced the immunoreactivity of tyrosine hydroxylase, CXCR4, and stromal cell-derived factor-1 in the brain. Injection of AMD3100 inhibited the aforementioned effects. These findings suggest that the stromal cell-derived factor-1/CXCR4 axis may play a significant role in the therapeutic effect of neural stem cell transplantation in a rat model of Parkinson's disease. This study was approved by the Animal Care and Use Committee of Kunming Medical University, China (approval No. SYXKK2015-0002) on April 1, 2014.

Transplantation of Nurr1-overexpressing neural stem cells and microglia for treating parkinsonian rats.

BACKGROUND: Neural stem cells (NSCs) transplantation is considered a promising treatment for Parkinson's disease. But most NSCs are differentiated into glial cells rather than neurons, and only a few of them survive after transplantation due to the inflammatory environment. METHODS: In this study, neural stem cells (NSCs) and microglial cells both forced with the Nurr1 gene were transplanted into the striatum of the rat model of PD. The results were evaluated through reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunofluorescence analysis. RESULTS: The behavioral abnormalities of PD rats were improved by combined transplantation of NSCs and microglia, both forced with Nurr1. The number of tyrosine hydroxylase+ cells in the striatum of PD rats increased, and the number of Iba1+ cells decreased compared with the other groups. Moreover, the dopamine neurons differentiated from grafted NSCs could still be detected in the striatum of PD rats after 5 months. CONCLUSIONS: The results suggested that transplantation of Nurr1-overexpressing NSCs and microglia could improve the inhospitable host brain environments, which will be  a new potential strategy for the cell replacement therapy in PD.

MiR-142-3p functions as a tumor suppressor by targeting RAC1/PAK1 pathway in breast cancer.

MicroRNA-142-3p (miR-142-3p) was previously investigated in various cancers, whereas, it's role in breast cancer (BC) remains far from understood. In this study, we found that miR-142-3p was markedly decreased both in cell lines and BC tumor tissues. Elevated miR-142-3p expression suppressed growth and metastasis of BC cell lines via gain-of-function assay in vitro and in vivo. Mechanistically, miR-142-3p could regulate the ras-related C3 botulinum toxin substrate 1 (RAC1) expression in protein level, which simultaneously suppressed the epithelial-to-mesenchymal transition related protein levels and the activity of PAK1 phosphorylation, respectively. In addition, rescue experiments revealed RAC1 overexpression could reverse tumor-suppressive role of miR-142-3p. Our results showed miR-142-3p could function as a tumor suppressor via targeting RAC1/PAK1 pathway in BC, suggesting a potent therapeutic target for BC treatment.

Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation.

The crosstalk between bone marrow adipocytes and tumor cells may play a critical role in the process of bone metastasis. A variety of methods are available for studying the significant crosstalk; however, a two-dimensional transwell system for coculture remains a classic, reliable, and easy way for this crosstalk study. Here, we present a detailed protocol that shows the coculture of bone marrow adipocytes and melanoma cells. Nevertheless, such a coculture system could not only contribute to the study of cell signal transductions of cancer cells induced by bone marrow adipocytes, but also to the future mechanistic study of bone metastasis which may reveal new therapeutic targets for bone metastasis.

microRNA-485-5p Functions as a Tumor Suppressor in Colorectal Cancer Cells by Targeting CD147.

Colorectal cancer(CRC) is a prevalent malignancy in the world. There is growing evidence that microRNAs (miRNAs) as crucial modulator are in connection with many tumor-related diseases including CRC. Though miR-485-5p has been reported as an anti-oncogene in certain cancers, it remains unclear in CRC. In this research, we found that miR-485-5p was at lower level expression in CRC tissues and cell lines compared to the paired paracancerous tissues and the normal colon epithelial cell line FHC, correspondingly. Furthermore, Experimental up-regulation miR-485-5p in DLD-1 and SW480 cells with mimic could inhibit the ability of proliferation, migration, invasion of CRC cell lines and facilitate cells apoptosis. Also, we confirmed that CD147 existed typically negative regulation by miR-485-5p through binding a conserved sequence specifically within the CD147 3'-untranslated region (3'UTR) and reintroduction of CD147 could rescue the phenotypic changes caused by miR-485-5p. The findings provide evidence to demonstrate the role of miR-485-5p/CD147 interaction in CRC and indicate that miR-485-5p might be exploited therapeutically in CRC.

Nurr1 promotes neurogenesis of dopaminergic neuron and represses inflammatory factors in the transwell coculture system of neural stem cells and microglia.

INTRODUCTION: Neural stem cells (NSCs) are the most promising cells for cell replacement therapy for Parkinson's disease (PD). However, a majority of the transplanted NSCs differentiated into glial cells, thereby limiting the clinical application. Previous studies indicated that chronic neuroinflammation plays a vital role in the degeneration of midbrain DA (mDA) neurons, which suggested the developing potential of therapies for PD by targeting the inflammatory processes. Thus, Nurr1 (nuclear receptor-related factor 1), a transcription factor, has been referred to play a pivotal role in both the differentiation of dopaminergic neurons in embryonic stages and the maintenance of the dopaminergic phenotype throughout life. AIM: This study investigated the effect of Nurr1 on neuroinflammation and differentiation of NSCs cocultured with primary microglia in the transwell coculture system. RESULTS: The results showed that Nurr1 exerted anti-inflammatory effects and promoted the differentiation of NSCs into dopaminergic neurons. CONCLUSIONS: The results suggested that Nurr1 protects dopaminergic neurons from neuroinflammation insults by limiting the production of neurotoxic mediators by microglia and maintain the survival of transplanted NSCs. These phenomena provided a new theoretical and experimental foundation for the transplantation of Nurr1-overexpressed NSCs as a potential treatment of PD.

The Predictive and Prognostic Role of Stromal Tumor-infiltrating Lymphocytes in HER2-positive Breast Cancer with Trastuzumab-based Treatment: a Meta-analysis and Systematic Review.

Background Tumor-infiltrating lymphocytes (TILs) are white blood cells that have left the bloodstream and migrated into a tumor, involving in the prognosis of breast cancer (BC) patients. Published studies reported the value of TILs in patients with HER2-positive receiving trastuzumab-based treatment. However, the results obtained remain controversial. Here, we conducted this study to explore the predictive and prognostic role of TILs for HER2-positive BC patients receiving trastuzumab therapies. Method To identify the related published studies, a comprehensive literature search dating up to July 2017 was performed in the databases of PubMed, PMC, Web of Science and China National Knowledge Infrastructure (CNKI) according to predefined selection criteria. The pathologic complete response (pCR) and survival outcome of patients were measured by odds ratio (OR) and hazard ratio (HR) with corresponding 95% confidence interval (95% CI), respectively. The association between TILs and trastuzumab benefit was analyzed by using STATA version 11.0. Result Eleven eligible studies comprising 3228 patients were identified in the present study. The pooled results showed that high level of TILs was associated with a significantly improved pCR rate (OR = 1.32; 95% CI = 1.10-1.60) and longer survival (HR = 0.97; 95% CI = 0.96-0.99), particularly in the subgroups of retrospective study design and 10% INC cut-off value. Moreover, stratified analysis revealed that elevated TILs was a predictor of higher pCR rate in the Asian population and improved survival in the subgroups of Caucasian population and multivariate analysis. Conclusion This meta-analysis indicated that the level of stromal TILs was an independent predictive and prognostic marker for better outcome in HER2-positive BC patients receiving trastuzumab-based treatment. High level of TILs was significantly associated with trastuzumab benefit.

Association between SNPs in Long Non-coding RNAs and the Risk of Female Breast Cancer in a Chinese Population.

Long non-coding RNAs (LncRNAs) have been reported to be involved in tumorigenesis and tumor progression. Single nucleotide polymorphisms (SNPs) in the lncRNAs also play a vital role in carcinogenesis. The aim of this study was to assess the relationships between the four selected tagSNPs (rs944289, rs3787016, rs1456315, rs7463708) in the lncRNAs and the risk of female breast cancer in a Chinese population. A case-control study was carried out involving in a total of 439 breast cancer patients and 439 age-matched healthy controls. The genotyping was performed with Sequenom MassARRAY and the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2) in tumor tissues was measured by the immunohistochemistry (IHC) assay. We found that rs3787016 TT genotype (adjusted odds ratio (OR) = 1.62, 95% confidence interval (CI) = 1.09-2.41, P = 0.018) was associated with an increased risk of female breast cancer, especially among the patients with premenopausal status (adjusted OR = 2.55, 95% CI = 1.30-4.97, P = 0.006). Moreover, a statistically significant increased risk of the rs3787016 TT genotype was observed among the patients with advanced tumor stage (Ⅲ and Ⅳ), poor histological grade (G3-G4), positive lymph node involvement, positive expression of ER and PR and negative expression of HER-2; rs7463708 GT and GT/GG genotype were associated with decreased risk of breast cancer in the subgroup of patients with postmenopausal status (GT versus (vs.) TT: adjusted OR = 0.67, 95% CI = 0.46-0.99, P = 0.043; GT/GG vs. TT: adjusted OR = 0.68, 95% CI = 0.47-0.98, P = 0.041) and tumor late-stage (GT vs. TT: adjusted OR = 0.65, 95% CI = 0.43-0.97, P = 0.037; GT/GG vs. TT: adjusted OR = 0.65, 95% CI = 0.44-0.96, P = 0.029). In short, rs3787016 TT genotype was associated with increased breast cancer risk and clinicopathologic features of the tumor, especially among premenopausal women.

Adipogenic niches for melanoma cell colonization and growth in bone marrow.

Bone marrow (BM) adipocytes are abundant in BM and may be involved in the process of bone metastasis. However, their behaviors in metastatic BM niches during bone metastasis have not been fully explored. In this study, intracardiac transplantation of B16-F10 melanoma cells into immunocompetent C57BL/6 mice was performed. Tibial marrow sections were stained with hematoxylin and eosin, Masson's trichrome, tartrate-resistant acid phosphatase, and fatty acid-binding protein 4 (FABP4) and analyzed using a histomorphometric system. The results showed that the number of BM adipocytes rapidly increased in melanoma metastatic BM niches, which were in direct contact with metastasizing melanoma cells and acted as a tumor stromal population in the BM-melanoma niche. Melanoma cell-derived factors could enhance BM adipogenesis, which promotes melanoma cell proliferation and cell cycle transitions. Moreover, BM adipocytes might aid in the modification of the osteolytic BM microenvironment. These results indicate that an increase in the number of BM adipocytes in a metastatic BM niche may facilitate melanoma cell colonization and growth in BM. BM adipocytes might therefore support the development of bone metastases.

A randomized, controlled trial of efficacy and safety of Anbainuo, a bio-similar etanercept, for moderate to severe rheumatoid arthritis inadequately responding to methotrexate.

The objective of the study was to evaluate the efficacy and safety of etanercept (Anbainuo) treatment in Chinese moderate to severe rheumatoid arthritis (RA) with inadequate response to methotrexate (MTX-IR); 600 patients (360 in phase III-1 and 240 in phase III-2) poorly responding to MTX were enrolled in the study and randomized at a ratio of 2:1 into an Anbainuo treatment or control group. The study was designed as a 12-week double-blind, placebo-controlled period followed by a 12-week open-label study. The primary endpoint was the ACR20 response rate at week 12. Secondary endpoints included the ACR50, ACR70, ACR-N, and safety. At week 12, ACR20 response was observed in 60.9 % of the Anbainuo group-significantly higher than that of the control group (20.6 %). At week 24, the ACR20 response in the Anbainuo group increased to 70.2 %; there was no significant difference compared with that of the control group (61.8 %, P > 0.05). At week 12, the ACR50 and ACR70 responses of the Anbainuo group increased to 25.6 and 6.8 %, compared to 4 and 1 % in the control group (P < 0.001, P = 0.002). The ACR-N was 2.85 ± 6.73 vs. -3.24 ± 8.78 % in the control group (P < 0.001). During the first 12 weeks of treatment, 66 adverse events (AE) were reported in the Anbainuo group (15.6 %) and 21 AEs (10.5 %) occurred in the control group, whereby the rate of the Anbainuo group was slightly higher than the control group (P = 0.042). Severe adverse events (SAEs) occurred in the Anbainuo group (1.3 %) and one (SAE) occurred in the control group (0.5 %) (P = 0.19). Anbainuo displays a rapid onset of efficacy as well as good tolerance and safety in MTX-IR patients having moderate to severe RA.

High fat diet increases melanoma cell growth in the bone marrow by inducing osteopontin and interleukin 6.

The impact of metabolic stress induced by obesity on the bone marrow melanoma niche is largely unknown. Here we employed diet induced obese mice model, where mice received high-fat (HFD) or normal diet (ND) for 6 weeks before challenge with B16F10 melanoma cells. Tumor size, bone loss and osteoclasts numbers were assessed histologically in the tibial bones. For defining the molecular pathway, osteopontin knock-out mice, interleukin 6 neutralizing antibody or Janus kinase 2 inhibition were carried out in the same model. Mechanistic studies such as adipocyte-melanoma co-cultures for defining adipocyte induced changes of tumor cell proliferation and expression profiles were also performed. As results, HFD enhanced melanoma burden in bone by increasing tumor area and osteoclast numbers. This process was associated with higher numbers of bone marrow adipocytes expressing IL-6 in direct vicinity to tumor cells. Inhibition of IL-6 or of downstream JAK2 blocked HFD-induced tumor progression. Furthermore, the phenotypic changes of melanoma cells triggered macrophage and osteoclast accumulation accompanied by increased osteopontin expression. Osteopontin triggered osteoclastogenesis and also exerted a positive feedback loop to tumor cells, which was abrogated in its absence. Metabolic stress by HFD promotes melanoma growth in the bone marrow by an increase in bone marrow adipocytes and IL-6-JAK2-osteopontin mediated activation of tumor cells and osteoclast differentiation.

Relation of Age, Sex and Bone Mineral Density to Serum 25-Hydroxyvitamin D Levels in Chinese Women and Men.

OBJECTIVE: To investigate the relation of circulating 25-hydroxyvitamin D (25[OH]D) levels to age, sex, and bone mineral density (BMD) in adults living in Guangzhou Province. METHODS: This cross-sectional study comprised 188 women and 122 men aged 17-88 years who were randomly sampled among community-dwelling Guangzhou residents. BMD at the lumbar spine and femoral neck was measured by dual energy X-ray absorptiometry, and serum concentrations of 25(OH)D, parathyroid hormone (PTH), procollagen I N-terminal peptide, and beta C-telopeptide of collagen were assayed by electrochemiluminescence immunoassay. Serum 25(OH)D concentrations were divided into four subgroups: severe deficiency (<10 ng/mL), deficiency (10-20 ng/mL), insufficiency (20-30 ng/mL), and sufficiency (≥30 ng/mL). RESULTS: The mean age of participants was 47.39 ± 19.32 years. Serum 25(OH)D levels were significantly lower in women than men (25.35 ± 6.59 ng/mL vs 27.25 ± 7.94 ng/mL, P < 0.05). The prevalence of 25(OH)D severe deficiency (<10 ng/mL) was 1.6% in men, zero in women; 25(OH)D deficiency (10-20 ng/mL) was 22.9% in women and 20.5% in men; and 25(OH)D insufficiency (20-30 ng/mL) was 73.4% in women and 65.6% in men. An inverse relationship between serum 25(OH)D levels and age (r = -0.249, P < 0.01) was observed in men, but no correlation was found in women (r = 0.130, P > 0.05). Serum 25(OH)D levels were positively associated with lumbar spine and femoral neck BMD (r = 0.382, P < 0.01; r = 0.384, P < 0.01, respectively) in elderly women and (r = 0.332, P < 0.05; r = 0.260, P < 0.05, respectively) and in young men. When adjustments were made for age, correlations between serum 25(OH)D levels and lumbar spine and femoral neck BMD persisted (r = 0.325, P < 0.05; r = 0.323, P < 0.05, respectively) in elderly women. However, age-adjusted serum 25(OH)D levels were positively correlated with BMD at lumbar spine (r = 0.278, P < 0.05) but not at femoral neck (r = 0.165, P > 0.05) in young men. No association between unadjusted or age-adjusted serum 25(OH)D levels and lumbar spine and femoral neck BMD was found in young and middle-aged women and in middle-aged and elderly men. Neither serum PTH levels nor bone turnover markers were related to unadjusted and age-adjusted serum 25(OH)D levels in our participants. CONCLUSION: More than two-third of participants residing in Guangzhou had vitamin D insufficiency. Serum 25(OH)D level is an important biomarker for BMD in elderly women and young men.

Sclerostin inhibition reverses systemic, periarticular and local bone loss in arthritis.

OBJECTIVE: To test whether inhibition of sclerostin by a targeted monoclonal antibody (Scl-Ab) protects from bone and cartilage damage in inflammatory arthritis. Sclerostin is a potent inhibitor of bone formation and may be responsible for the low level of bone repair in patients with rheumatoid arthritis. METHODS: Human tumour necrosis factor transgenic mice (hTNFtg mice) developing inflammatory arthritis and local and bone loss were administered either vehicle, anti-TNF antibody, Scl-Ab, or a combination of both agents. Inflammation, systemic and periarticular bone loss, bone erosion and cartilage damage were evaluated at baseline (week 8) and after 3 weeks of treatment by clinical assessment, micro-CT and histology. RESULTS: Scl-Ab did not affect joint swelling or synovitis. Systemic bone loss in the spine and periarticular bone loss in the proximal tibia were completely blocked and partially reversed by inhibition of sclerostin but not by inhibition of TNF. Moreover, Scl-Ab completely arrested the progression of bone erosion in hTNFtg mice and in combination with TNF inhibition even led to significant regression of cortical bone erosions. Protective effects of Scl-Ab were also observed for the articular cartilage. CONCLUSIONS: These data suggest that sclerostin inhibition is a powerful tool to enhance bone repair in inflammatory arthritis.

A multicenter, randomized, double-blind clinical trial of combination therapy with Anbainuo, a novel recombinant human TNFRII:Fc fusion protein, plus methotrexate versus methotrexate alone or Anbainuo alone in Chinese patients with moderate to severe rheumatoid arthritis.

This study aims to evaluate the clinical and radiological efficacy as well as safety profiles of Anbainuo, a recombinant human TNFRII:Fc fusion protein, combined with methotrexate (MTX) versus MTX alone or Anbainuo alone in the treatment of Chinese patients with moderate to severe rheumatoid arthritis (RA). In this 24-week, multicenter, double-blind, active comparator-controlled study, 396 RA patients were randomized into combination therapy group (Anbainuo plus MTX), Anbainuo group, or MTX group. Clinical response was assessed using the American College of Rheumatology (ACR)-N, ACR20, ACR50, ACR70, and van der Heijde modification of Sharp score, among which ACR-N and ACR20 were defined as primary major endpoints. After 24 weeks of treatment, the ACR-N in the combination therapy group (12.79 ± 9.24 %) was significantly higher than that in Anbainuo group (9.56 ± 11.16 %) and in MTX group (5.08 ± 11.1 %) (p = 0.00 and p = 0.00, respectively). Patients in Anbainuo group had significantly higher ACR-N than those in MTX group (p = 0.02). More patients in the combination therapy group (53.6 %) achieved ACR50 improvement response than those in the MTX group (30.8 %). ACR70 of combination therapy group (27.7 %) was significantly higher than that of Anbainuo group (15.8 %) and MTX group (7.70 %), with no significant difference between Anbainuo group and MTX group. DAS28-ESR in the combination therapy group was significantly reduced compared to either monotherapy groups. Moreover, DAS28-ESR was significantly lower in Anbainou group than in MTX group. The combination therapy group also showed significantly less radiographic progression than the MTX group (p = 0.03). The total adverse events (AE) in the combination group (40.9 %) was significantly higher than those in the MTX group (28.8 %) (p < 0.05). Anbainuo combined with MTX therapy can effectively control the disease activity and radiographic progression of RA, while the incidence of AE also increased compared to either Anbainuo or MTX.

Values of three different preoperative regimens in comprehensive treatment for young patients with stage Ib2 cervical cancer.

OBJECTIVE: To compare the clinical efficacy of concurrent chemoradiotherapy, neoadjuvant chemotherapy, and intracavity brachytherapy in comprehensive treatment for young patients with stage Ib2 cervical cancer. METHODS: One hundred and twelve young patients with stage Ib2 cervical cancer were enrolled retrospectively in our hospital from January 2003 to June 2005. They were categorized into three groups according to preoperative regimens, including the concurrent chemoradiotherapy group (Group 1, n=38), the neoadjuvant chemotherapy (Group 2, n=49), and the intracavity brachytherapy group (Group 3, n=25). Radical hysterectomy was performed following these regimens. Chemotherapy and radiotherapy were given according to pelvic lymph node metastasis, deep cervical stromal invasion, intravascular cancer emboli, histological grading, vaginal stump and positive surgical margin. RESULTS: The cancer disappearance and superficial muscle invasion rates were statistically significantly better in the concurrent chemoradiotherapy group than in the other two groups (P<0.01). No statistically significant difference was noted in the deep muscle invasion rate, surgical time and intraoperative blood loss among three groups, but significantly more postoperative complications occurred in the concurrent chemoradiotherapy group. The 2-year pelvic recurrence was statistically significantly lower in the concurrent chemoradiotherapy group compared to other two groups, while the 5-year survival was higher. CONCLUSION: Concurrent chemoradiotherapy is efficacious for young patients with stage Ib2 cervical cancer.

[Cloning and expression of the gene encoding rat IgE-dependent histamine-releasing factor and effect study on the recombinant protein].

AIM: To clone and express the gene encoding rat IgE-dependent histamine-releasing factor (rHRF) and to study the effect of recombinant rHRF to induce histamine release from rat sensitized mast cells. METHODS: The complete coding region of rHRF was cloned and expressed in BL21 cells. The soluble recombinant rHRF was purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately stimulated by recombinant rHRF at different concentration. The released histamine was measured by the OPT spectrofluorometric procedure. RESULTS: The cloned DNA fragment showed 97% nucleotide sequence identity and 95% deduced amino acid sequence identity with the mRNA of rat IgE-dependent histamine-releasing factor (or translationally controlled tumor protein) in GenBank. The recombinant rHRF showed a relative molecular mass of 24 000 in SDS-PAGE. The purified rHRF which was independent of the allergen induced histamine releasing from the sensitized mast cells in a dose-dependent manner. CONCLUSION: The recombinant rHRF, which showed the effect of inducing histamine release from sensitized mast cells, would play an important role in the allergen diseases.

Modulatory effects of 1,25-dihydroxyvitamin D3 on human B cell differentiation.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) can modulate immune responses, but whether it directly affects B cell function is unknown. Patients with systemic lupus erythematosus, especially those with antinuclear Abs and increased disease activity, had decreased 1,25(OH)(2)D(3) levels, suggesting that vitamin D might play a role in regulating autoantibody production. To address this, we examined the effects of 1,25(OH)(2)D(3) on B cell responses and found that it inhibited the ongoing proliferation of activated B cells and induced their apoptosis, whereas initial cell division was unimpeded. The generation of plasma cells and postswitch memory B cells was significantly inhibited by 1,25(OH)(2)D(3), although the up-regulation of genetic programs involved in B cell differentiation was only modestly affected. B cells expressed mRNAs for proteins involved in vitamin D activity, including 1 alpha-hydroxylase, 24-hydroxylase, and the vitamin D receptor, each of which was regulated by 1,25(OH)(2)D(3) and/or activation. Importantly, 1,25(OH)(2)D(3) up-regulated the expression of p27, but not of p18 and p21, which may be important in regulating the proliferation of activated B cells and their subsequent differentiation. These results indicate that 1,25(OH)(2)D(3) may play an important role in the maintenance of B cell homeostasis and that the correction of vitamin D deficiency may be useful in the treatment of B cell-mediated autoimmune disorders.

[Parasite-origin IgE-dependent histamine-releasing factors in inducing histamine release from sensitized mast cells].

OBJECTIVE: To obtain the recombinant IgE-dependent histamine-releasing factors of Schistosoma japonicum and Clonorchis sinensis (rSjHRF and rCsHRF) and to study the effect of recombinant HRFs to induce histamine release from sensitized rat mast cells. METHODS: The complete coding regions of SjHRF and CsHRF were cloned separately, and the recombinant plasmids were respectively transformed and expressed in BL21 cells. The soluble recombinant rSjHRF and rCsHRF were purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately incubated with rSjHRF and rCsHRF and the released histamine was measured by the OPT spectrofluorometric procedure. The dose-dependent curves and the kinetics of histamine release induced by rSjHRF and rCsHRF were prepared. RESULTS: The recombinant plasmids pET-30-rSjHRF and pET-30-rCsHRF were constructed successfully and the purified soluble recombinant proteins rSjHRF and rCsHRF were obtained by affinity chromatography. rSjHRF and rCsHRF induced histamine release from sensitized mast cells in a dose-dependent manner. At the concentration of 150 mg/L, the average rate of histamine release from sensitized mast cells induced by rSjHRF and rCsHRF were 49.78% and 32.63%, respectively. Histamine release increased with prolonged reaction time and the maximal release occurred at 35 min. CONCLUSION: The recombinant parasite-originated IgE-dependent HRFs show an effect of inducing histamine release from sensitized mast cells, suggesting that this protein would play a role in type I hypersensitivity in hosts with parasitic infections.