Is thyroid function associated with polycystic ovary syndrome? A bidirectional Mendelian randomization study.

OBJECTIVE: Some observational studies have suggested the association between thyroid function and polycystic ovary syndrome (PCOS). However, it remains to be determined whether these associations are causal or not. The aim of this study was to investigate the underlying causal association between different thyroid function status and PCOS. METHODS: Bidirectional Mendelian randomization (MR) analysis was conducted to explore the impact of different thyroid function statuses on PCOS. The study included 10,074 individuals with PCOS and 103,164 controls for the primary analysis, with validation analysis repeated in the FinnGen R9 and EstBB PCOS cohorts. Female-specific thyroid function GWAS data were obtained from European population, including Hyperthyroidism (22,383 cases and 54,288 controls) and Hypothyroidism (27,383 cases and 54,288 controls) from the UK Biobank, and TSH (54,288 cases and 72,167 controls) and FT4 (49,269 cases and 72,167 controls) within the reference range from the ThyroidOmics Consortium. Inverse variance weighting (IVW) was chosen as the principal method, and sensitivity analysis was conducted to test for the presence of horizontal pleiotropy or heterogeneity. RESULTS: The IVW analysis indicated nominal significance between normal TSH levels and PCOS after adjusted for age and BMI [OR (95% CI) = 0.78(0.62,0.97), P = 0.029], suggesting that maintaining normal TSH levels might act as a protective factor against the pathogenesis of PCOS. Besides, in order to increase the statistical power, we pooled PCOS GWAS above together by meta-analysis and found PCOS contributed to the occurrence of hyperthyroidism [OR(95%CI) = 1.37(0.73,2.57), P = 0.012]. However, no causal relationship was found after Bonferroni correction (P-value < 0.0031). CONCLUSION: Although the MR analysis didn't indicate genetic causal association between thyroid function and PCOS after Bonferroni correction. Further efforts are needed to interpret the potential causal relationship between thyroid function and PCOS in different age and BMI subgroup.

High concentration of estradiol has a negative correlation with free thyroxine during the second trimester of pregnancy.

Objective: To explore the relationship between estradiol (E2) and thyroid function during the second trimester of pregnancy and the effect of E2 on sodium iodide transporter (NIS) expression in cultured thyroid cells. Materials and methods: We analyzed relationships between E2 and thyroid function in 196 pregnant women during the second trimester. Multiple linear regression analysis was performed between E2 and thyroid function. The human thyroid Nthy-ori3-1 cells were cultured in different E2 concentrations, and the mRNA levels of NIS, estrogen receptor (ER)-α, and ER-ß were measured by quantitative real-time PCR. Their protein levels were assessed by western blot. Results: E2 was positively correlated with thyroid-stimulating hormone (TSH) and negatively correlated with free thyroxine (FT4) (P < 0.05). When we corrected for age, BMI, alanine aminotransferase, and serum creatinine, E2 was still negatively correlated with FT4 (P < 0.5) during the second trimester. In Nthy-ori3-1 cells treated with 10 nM E2, NIS and ER-ß mRNA levels were significantly reduced, while ER-α mRNA level was not altered (P > 0.5). Moreover, 10 nM E2 significantly decreased protein levels of ER-ß, phosphorylated versions of protein kinase A (p-PKA), phosphorylated versions of cAMP response element-binding protein (p-CREB), and NIS, while treatment with the ER-ß inhibitor restored the expression of p-PKA, p-CREB, and NIS (P < 0.05). Conclusion: High concentration of E2 has a negative correlation with FT4. High concentration of E2 can inhibit the NIS expression through the ER-ß-mediated pathway, which may cause thyroid hormone fluctuations during pregnancy.

Serum Trimethylamine N-oxide and the Diversity of the Intestinal Microbial Flora in Type 2 Diabetes Complicated by Diabetic Kidney Disease.

BACKGROUND: Trimethylamine N-oxide (TMAO) serves as a metabolite of intestinal bacteria as well as a urotoxin influencing the prognosis of chronic kidney disease (CKD), which has become a research hotspot in the field of kidney disease. This study preliminarily explored the alternations of the microbial flora and serum TMAO in patients with type 2 diabetes mellitus (T2DM) complicated with diabetic kidney disease (DKD). METHODS: Seventeen T2DM patients at the Affiliated Hospital of Zunyi Medical University between September 2018 and February 2019 were included. Among these patients, 8 patients had T2DM complicated with DKD. Eight healthy volunteers constituted the control group. Fresh stool was collected for Illumina sequencing. Based on the sequencing outcomes, the flora diversity and species differences were analyzed. Serum TMAO, cystatin C, urinary albumin/urine creatinine ratios (ACRs), and routine biochemical outcomes were also compared. RESULTS: The DKD group exhibited a significantly higher TMAO level than the remaining groups. The high-TMAO group had a significantly increased ACR level compared with the low-TMAO group. TMAO positively correlated with the ACR. Compared with the control group, the DKD group exhibited a decreased flora diversity. At the genus level, both the T2DM group and the DKD group showed decreased numbers of Alloprevotella and Megasphaera compared with the control group. The difference in Megasphaera between the DKD group and the control group was significant. CONCLUSIONS: The alternation of the intestinal microbial flora may participate in the development of DKD, and TMAO and chronic inflammation might be important factors for DKD development.

Decreased Monocyte Count Is Associated With Gestational Diabetes Mellitus Development, Macrosomia, and Inflammation.

CONTEXT: The immune system plays a central role in the pathophysiology of gestational diabetes mellitus (GDM). Monocytes, the main innate immune cells, are especially important in the maintenance of a normal pregnancy. OBJECTIVE: Here, we investigated the potential effect of monocytes in GDM. METHODS: Monocyte count was monitored throughout pregnancy in 214 women with GDM and 926 women without in a case-control and cohort study. Circulating levels of inflammatory cytokines, placenta-derived macrophages, and their products were measured. RESULTS: Throughout pregnancy, monocyte count was significantly decreased in women with GDM, and was closely associated with glucose level, insulin resistance, and newborn weight. First-trimester monocyte count outperformed that of the second and third trimester as a risk factor and diagnostic predictor of GDM and macrosomia both in the case-control and cohort study. In addition, our cohort study showed that as first-trimester monocyte count decreased, GDM and macrosomia incidence, glucose level, and newborn weight increased in a stepwise manner. Risk of GDM started to decrease rapidly when first-trimester monocyte count exceeded 0.48 × 109/L. Notably, CD206 and interleukin 10 (IL-10) were significantly lower, whereas CD80, CD86, tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) were higher both in GDM placental tissue and peripheral blood. First-trimester monocyte count was positively related to IL-10 and CD206, but negatively related to CD80, CD86, TNF-α, and IL-6. CONCLUSION: Decreased monocyte count throughout pregnancy was closely associated with the development of GDM, macrosomia, and the chronic inflammatory state of GDM. First-trimester monocyte count has great potential as an early diagnostic marker of GDM.

Monocyte to high-density lipoprotein cholesterol ratio as an independent risk factor for papillary thyroid carcinoma.

BACKGROUND: Papillary thyroid carcinoma (PTC) is considered to be an inflammatory disease. This study aimed to investigate the association of monocyte to high-density lipoprotein cholesterol ratio (MHR) with PTC. METHODS: Clinical parameters from 300 patients with PTC and 552 patients with benign thyroid nodule were compared. Serum renal function and liver enzymes, fasting plasma glucose, lipid profile, and blood cell count were measured. RESULTS: Patients with PTC had a higher MONO (p < 0.001) and MHR (p < 0.001). There was a step-wise increase in the prevalence of PTC (p = 0.003) with the tertile of MHR. Logistic regression analysis revealed that MHR could be considered an independent risk factor (p < 0.001) in the case-control study and the cohort study. Pearson correlation analysis and simple linear regression analysis indicated that MHR was positively associated with neutrophil (NEU) and lymphocyte (LYM) count as well as neutrophil-to-lymphocyte ratio (NLR). Area under the curve (AUC) was 0.711. The optimal cutoff of MHR was 0.33 × 109 /mmol. CONCLUSION: This study identifies novel evidence that patients with PTC have a higher MHR. MHR is an independent risk factor for PTC. These findings support the application of MHR to predict, diagnose, and evaluate the occurrence of PTC.

Alterations in CD8+ Tregs, CD56+ Natural Killer Cells and IL-10 Are Associated With Invasiveness of Nonfunctioning Pituitary Adenomas (NFPAs).

Invasive nonfunctioning pituitary adenomas (NFPAs) grow rapidly and the mechanisms are unclear. Among many complex mechanisms, the role of immunity in the development of NFPAs has not been fully explored. Here, we analyzed the clinical features 146 NFPA patients who underwent trans-sphenoidal surgery or craniotomy and examined the effects of immune tolerance in invasiveness of NFPA patients using fluorescence-activated cell sorting and immunohistochemical methods. We found patients with invasive NFPAs had more visual deficits and defective fields, higher tumor size, and lower white blood cell count compared with patients with noninvasive NFPAs. Additionally, compared with patients with noninvasive NFPAs, patients with invasive NFPAs had conspicuously lower CD3-CD56+ natural killer (NK) cells and significantly higher levels of CD3+CD8+CD28-T cells (CD8+ Tregs) and interleukin-10 (IL-10) in peripheral blood. Moreover, patients with invasive NFPAs had lower infiltrated CD56+ cells, less infiltrated CD28+ cells, and significantly greater IL-10 expression. These results demonstrated that low CD56+ cells infiltration and CD28+ cells infiltration, as well as high IL-10 expression in pituitary tumor tissues, were related with increased invasiveness of NFPAs. Levels of CD3-CD56+ NK cells, CD8+ Tregs and IL-10 in the peripheral blood could be feasible diagnostic markers for invasive NFPAs.

IRON DEFICIENCY, A RISK FACTOR FOR THYROID AUTOIMMUNITY DURING SECOND TRIMESTER OF PREGNANCY IN CHINA.

Objective: Previous studies have reported an association between iron deficiency (ID) and increased thyroid peroxidase antibody (TPO-Ab) during early pregnancy. The objective of this study was to explore the relationship between ID and thyroid dysfunction, as well as thyroid autoantibodies, during the second trimester of pregnancy. Methods: A total of 1,592 pregnant women (13 to 28 weeks gestation) were enrolled in this cross-sectional study. According to serum ferritin (SF) concentrations, they were divided into ID (SF <20 µg/L) or non-ID (SF ≥20 µg/L) groups. Logistic regression analysis was used to evaluate the association between ID and subclinical hypothyroidism (thyroid-stimulating hormone [TSH] >4.0 mIU/L and free thyroxine [FT4] within the reference range) and thyroid autoimmunity. Results: The prevalence of ID was 23.43% (373/1,592). Compared with the non-ID group, the ID group had lower FT4 levels (13.94 pmol/L [8.91 to 29.82 pmol/L] versus 14.63 pmol/L [8.22 to 47.24 pmol/L]; P<.001]) and higher TSH levels (1.85 mIU/L [0.01 to 7.84 mIU/L] versus 1.69 mIU/L [0.01 to 10.2 mIU/L]; P<.05). Logistic regression analysis confirmed ID as a risk factor for increased thyroglobulin antibody (TG-Ab) (odds ratio 1.974; 95% confidence interval 1.065, 3.657; P<.05), but not for subclinical hypothyroidism or increased TPO-Ab. Conclusion: ID is associated with increased TG-Ab during the second trimester of pregnancy. Abbreviations: BMI = body mass index; CV = coefficient of variation; FT4 = free thyroxine; Hb = hemoglobin; ID = iron deficiency; IDA = iron deficiency anemia; SF = serum ferritin; T3 = triiodothyronine; T4 = thyroxine; TAI = thyroid autoimmunity; TG = thyroglobulin; TG-Ab = thyroglobulin antibody; TPO = thyroid peroxidase; TPO-Ab = thyroid peroxidase antibody; TSH = thyroid-stimulating hormone.

Association of High Prolactin Level on Postoperative Day 1 and Tumor Invasion with Female Gonadal Dysfunction After Trans-Sphenoidal Surgery of Pituitary Adenomas.

BACKGROUND The aim of this study was to evaluate the risk factors of gonadal dysfunction among Chinese women of reproductive age with pituitary adenomas (PAs) after trans-sphenoidal surgery. MATERIAL AND METHODS We retrospectively evaluated 317 women (16-44 years old) who underwent gonadal function and hormone testing before and after trans-sphenoidal surgery for PAs during 2003-2012. Gonadal function was assessed on the basis of menstrual status. RESULTS Three women were excluded because of pre-existing gynecological diseases. Before trans-sphenoidal surgery, 34 (10.7%) women were eugonadal and 283 (89.3%) women had gonadal dysfunction. After trans-sphenoidal surgery, 130/189 (68.7%) women with follow-up menstruation data were eugonadal, and 59/189 (31.2%) women exhibited gonadal dysfunction. In addition, 67.4% women of reproductive age with PAs and gonadal dysfunction were restored by trans-sphenoidal surgery (P<0.01). Postoperative gonadal dysfunction was independently associated with high prolactin level at day 1 after trans-sphenoidal surgery (odds ratio (OR)=1.024; 95% confidence interval (CI)=1.005-1.043; P=0.012) and tumor invasion (OR=5.752; 95%CI=1.618-20.447; P<0.01). Based on the receiver operating characteristic (ROC) curve, prediction of gonadal dysfunction in women of reproductive age after trans-sphenoidal surgery for PAs using prolactin >46.82 µg/L on postoperative day 1 had sensitivity of 88%, specificity of 95%, positive predictive value of 98%, and negative predictive value of 76%, and an area under the ROC curve of 0.701. CONCLUSIONS Gonadal dysfunction is very common in Chinese women of reproductive age with PAs and can be effectively restored by trans-sphenoidal surgery. Prolactin >46.82 µg/L at 1 day after trans-sphenoidal surgery and tumor invasion can predict postoperative gonadal dysfunction in these patients.

Evaluation of serum ferritin and thyroid function in the second trimester of pregnancy.

Ferritin is a universal intracellular protein that acts as an iron carrier. Several studies have indicated that iron deficiency affects thyroid function in non-pregnant women. Our objective was to assess the relationship between serum ferritin levels and thyroid function in pregnant women during the second trimester. Pregnant women with sufficient iodine intake and normal antithyroid antibodies during the second trimester were recruited from the obstetric outpatient department of the Fifth People's Hospital of Fudan University. Serum ferritin (SF) levels, thyroid function, anti-thyroid antibodies and vitamin B12 were determined by electrochemiluminescence immunoassay kit. Maternal serum iron (Fe), unsaturated iron binding capacity (UIBC), hemoglobin (Hb), creatinine (Cr), fasting blood glucose (FBG), and alanine aminotransferase (ALT) were also evaluated. Stepwise regressions performed to evaluate the associations between SF and other maternal parameters. In the second trimester, 11.4% pregnant women had a SF concentration less than 12 µg/L, and 7.6% pregnant women were anemic. SF levels were negatively correlated with serum TSH levels (r = -0.219, p < 0.05), and positively correlated with FT4 levels (r = 0.203, p < 0.05). Linear regression analysis showed only SF, age, week of gestation were significant predictors of regression with TSH as the dependent variable (ß: -0.007, -0.059, and 0.118 respectively; all p < 0.05). However consistent relation between the SF levels and FT4 was not observed in stepwise linear regression. Maternal iron status is a determinant of TSH concentrations during pregnancy in pregnant women during the second trimester.

STAT6 deficiency ameliorates Graves' disease severity by suppressing thyroid epithelial cell hyperplasia.

Signal transducer and activator of transcription 6 (STAT6) is involved in epithelial cell growth. However, little is known regarding the STAT6 phosphorylation status in Graves' disease (GD) and its role in thyroid epithelial cells (TECs). In this study, we found that STAT6 phosphorylation (p-STAT6) was significantly increased in TECs from both GD patients and experimental autoimmune Graves' disease mice and that STAT6 deficiency ameliorated GD symptoms. Autocrine IL-4 signalling in TECs activated the phosphorylation of STAT6 via IL-4 R engagement, and the downstream targets of STAT6 were Bcl-xL and cyclin D1. Thus, the IL-4-STAT6-Bcl-xL/cyclin D1 pathway is crucial for TEC hyperplasia, which aggravates GD. More importantly, in vitro and in vivo experiments demonstrated that STAT6 phosphorylation inhibited by AS1517499 decreased TEC hyperplasia, thereby reducing serum T3 and T4 and ameliorating GD. Thus, our study reveals that in addition to the traditional pathogenesis of GD, in which autoantibody TRAb stimulates thyroid-stimulating hormone receptors and consequently produces T3, T4, TRAb could also trigger TECs producing IL-4, and IL-4 then acts in an autocrine manner to activate p-STAT6 signalling and stimulate unrestricted cell growth, thus aggravating GD. These findings suggest that STAT6 inhibitors could be potent therapeutics for treating GD.

Decrease in proportion of CD19+ CD24(hi) CD27+ B cells and impairment of their suppressive function in Graves' disease.

IL-10-producing B cells (B10 cells) have been shown to play a suppressive role in the peripheral blood of humans, with their numbers and function altered in several autoimmune diseases. However, the role of B10 cells in Graves' disease (GD) remains unknown. In this study, we demonstrated that B10 cells in human peripheral blood belonged to a CD24(hi)CD27(+) B cell subpopulation. The proportion of B10 cells along with the CD19(+)CD24(hi)CD27(+) B cell subset was significantly lower in new-onset patients compared with healthy individuals. In recovered patients, these proportions were restored to levels similar to those seen in healthy individuals. Additionally, we found that CD19(+)CD24(hi)CD27(+) B cells from healthy individuals inhibited proliferation and TNF-α production of CD4(+) T cells via an IL-10-independent pathway. They also inhibited IFN-γ production by CD4(+) T cells, through an IL-10-dependent pathway. In contrast, their suppressive function on CD4(+) T cell proliferation and cytokine production was impaired in new-onset and recovered patients compared with healthy individuals. Our study provides evidence that CD19(+)CD24(hi)CD27(+) B cells may possess the capacity to downregulate immune responses, partially by production of IL-10 in human peripheral blood. Impairment of their immunosuppressive function may contribute to GD pathogenesis and relapse.

Galactosylated chitosan/5-fluorouracil nanoparticles inhibit mouse hepatic cancer growth and its side effects.

AIM: To observe the curative effect of galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticles in liver caner mice and its side effects. METHODS: The GC/5-FU nanoparticle is a nanomaterial made by coupling GC and 5-FU. The release experiment was performed in vitro. The orthotropic liver cancer mouse models were established and divided into control, GC, 5-FU and GC/5-FU groups. Mice in the control and GC group received an intravenous injection of 200 µL saline and GC, respectively. Mice in the 5-FU and GC/5-FU groups received 200 µL (containing 0.371 mg 5-FU) 5-FU and GC/5-FU, respectively. The tumor weight and survival time were observed. The cell cycle and apoptosis in tumor tissues were monitored by flow cytometry. The expression of p53, Bax, Bcl-2, caspase-3 and poly adenosine 50-diphosphate-ribose polymerase 1 (PARP-1) was detected by immunohistochemistry, reverse transcription-polymerase chain reaction and Western blot. The serum blood biochemical parameters and cytotoxic activity of natural killer (NK) cell and cytotoxicity T lymphocyte (CTL) were measured. RESULTS: The GC/5-FU nanoparticle is a sustained release system. The drug loading was 6.12% ± 1.36%, the encapsulation efficiency was 81.82% ± 5.32%, and the Zeta potential was 10.34 ± 1.43 mV. The tumor weight in the GC/5-FU group (0.4361 ± 0.1153 g vs 1.5801 ± 0.2821 g, P < 0.001) and the 5-FU (0.7932 ± 0.1283 g vs 1.5801 ± 0.2821 g, P < 0.001) was significantly lower than that in the control group; GC/5-FU treatment can significantly lower the tumor weight (0.4361 ± 0.1153 g vs 0.7932 ± 0.1283 g, P < 0.001), and the longest median survival time was seen in the GC/5-FU group, compared with the control (12 d vs 30 d, P < 0.001), GC (13 d vs 30 d, P < 0.001) and 5-FU groups (17 d vs 30 d, P < 0.001). Flow cytometry revealed that compared with the control, GC/5-FU caused a higher rate of G0-G1 arrest (52.79% ± 13.42% vs 23.92% ± 9.09%, P = 0.014 ) and apoptosis (2.55% ± 1.10% vs 11.13% ± 11.73%, P < 0.001) in hepatic cancer cells. Analysis of the apoptosis pathways showed that GC/5-FU upregulated the expression of p53 at both the protein and the mRNA levels, which in turn lowered the ratio of Bcl-2/Bax expression; this led to the release of cytochrome C into the cytosol from the mitochondria and the subsequent activation of caspase-3. Upregulation of caspase-3 expression decreased the PARP-1 at both the mRNA and the protein levels, which contributed to apoptosis. 5-FU increased the levels of aspartate aminotransferase and alanine aminotransferase, and decreased the numbers of platelet, white blood cell and lymphocyte and cytotoxic activities of CTL and NK cells, however, there were no such side effects in the GC/5-FU group. CONCLUSION: GC/5-FU nanoparticles can significantly inhibit the growth of liver cancer in mice via the p53 apoptosis pathway, and relieve the side effects and immunosuppression of 5-FU.

5-Fluorouracil nanoparticles inhibit hepatocellular carcinoma via activation of the p53 pathway in the orthotopic transplant mouse model.

Biodegradable polymer nanoparticle drug delivery systems provide targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and fewer side effects. These drug delivery systems are widely used for delivering cytotoxic agents. In the present study, we synthesized GC/5-FU nanoparticles by combining galactosylated chitosan (GC) material with 5-FU, and tested its effect on liver cancer in vitro and in vivo. The in vitro anti-cancer effects of this sustained release system were both dose- and time-dependent, and demonstrated higher cytotoxicity against hepatic cancer cells than against other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in hepatic cancer tissues. GC/5-FU significantly inhibited tumor growth in an orthotropic liver cancer mouse model, resulting in a significant reduction in tumor weight and increased survival time in comparison to 5-FU alone. Flow cytometry and TUNEL assays in hepatic cancer cells showed that GC/5-FU was associated with higher rates of G0-G1 arrest and apoptosis than 5-FU. Analysis of apoptosis pathways indicated that GC/5-FU upregulates p53 expression at both protein and mRNA levels. This in turn lowers Bcl-2/Bax expression resulting in mitochondrial release of cytochrome C into the cytosol with subsequent caspase-3 activation. Upregulation of caspase-3 expression decreased poly ADP-ribose polymerase 1 (PARP-1) at mRNA and protein levels, further promoting apoptosis. These findings indicate that sustained release of GC/5-FU nanoparticles are more effective at targeting hepatic cancer cells than 5-FU monotherapy in the mouse orthotropic liver cancer mouse model.

Preliminary pharmacology of galactosylated chitosan/5-fluorouracil nanoparticles and its inhibition of hepatocellular carcinoma in mice.

Biodegradable polymer nanoparticle drug delivery systems are characterized by targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability and lowered side effects; these drug delivery systems are widely used for delivery of cytotoxic agents. The galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticle is a nanomaterial made by coupling GC, a polymer known to have the advantages described above, and 5-FU. The GC/5-FU nanoparticle is a sustained release system, it was showed that the peak time, half-life time, mean residence time (MRT) and area of under curve (AUC) of GC/5-FU were longer or more than those of the 5-FU group, but the maximum concentration (Cmax) was lower. The distribution of GC/5-FU in vivo revealed the greatest accumulation in the hepatic cancer tissues, and the hepatic cell was the target of the nanoparticles. Toxicology research showed that the toxicity of GC-5-FU was lower than that of 5-FU in mice. In vivo experiments showed that GC/5-FU can significantly inhibit tumor growth in an orthotropic liver cancer mouse model. GC/5-FU treatment can significantly lower the tumor weight and increase the survival time of mice when compared with 5-FU treatment alone. Flow cytometry and the TUNEL assay revealed that compared with 5-FU, GC/5-FU caused higher rates of G 0-G 1 arrest and apoptosis in hepatic cancer cells.

Synthesis of galactosylated chitosan/5-fluorouracil nanoparticles and its characteristics, in vitro and in vivo release studies.

Biodegradable polymer nanoparticle drug delivery systems are characterized by targeted drug delivery, improved pharmacokinetic and biodistribution, enhanced drug stability, and lowered side effects; these drug delivery systems are widely used for delivery of cytotoxic agents. The galactosylated chitosan (GC)/5-fluorouracil (5-FU) nanoparticle is a nanomaterial made by coupling GC, a polymer known to have the advantages described above, and 5-FU. We found that when 5-FU and GC were mixed at the mass ratio of 10:1, the nanoparticle reached a maximum encapsulation efficiency of 81.82% ± 5.32%, with a drug loading of 6.12% ± 1.36%, a particle size of 35.19 ± 9.50 nm, and a Zeta potential of +10.34 ± 1.43 mV. The GC/5-FU nanoparticle is a sustained release system, whose anticancer effects were shown to be dose and time dependent, with a higher cytotoxicity to hepatic cancer than to other cell types. The distribution of GC/5-FU in vivo revealed the greatest accumulation in the hepatic cancer tissues, with an 8.69-, 23.35-, 79.96-, and 85.15-fold increase when compared to normal liver tissue, kidney, heart and blood, respectively, suggesting that the hepatic cell was the target of the nanoparticles. In vivo experiments showed that GC/5-FU can significantly inhibit tumor growth in an orthotropic liver cancer mouse model. GC/5-FU treatment can significantly lower the tumor weight and increase the survival time of mice when compared to 5-FU treatment alone. Flow cytometry revealed that compared to 5-FU, GC/5-FU caused higher rates of G0-G1 arrest and apoptosis in hepatic cancer cells.

Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo.

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.

[Construction of recombinant plasmid pIRES-GM-CSF-IL-21 and its therapeutic effect on orthotopically transplanted liver cancer in mice].

OBJECTIVE: To construct a recombinant plasmid pIRES-GM-CSF-IL-21, and to investigate its antitumor effect on tumors in the mice. METHODS: Fifty Bal b/c mice were included in this study. Cultured hepatoma H22 cells were inoculated in the left lobe of the liver to develop orthotopically transplanted liver tumor models. The mice with orthotopically transplanted liver tumor were randomly divided into 5 groups (n = 10): (1) Each mouse received injection of recombinant plasmid pIRES-GM-CSF-IL-21; (2) Received injection of plasmid pIRES-GM-CSF; (3) pIRES-IL-21; (4) Received injection of ampty plasmid pIRES (H22/neo group); (5) Received injection of PBS (H22 group) via the tail vein, respectively. Therefore, the anti-tumor effect was induced by both GM-CSF and IL-21, or by either of them alone. The serum levels of IFN-γ and IL-2 were detected by ELISA, and the cytotoxicity of spleen NK and CTL cells were tested by MTT colorimetry. RESULTS: Comparing with the H22 and H22/Neo groups, the tumor weight in the mice of H22/GM-CSF group was (0.603 ± 0.223) g, H22/IL21-treated group (0.583 ± 0.290) g and H22/GM-CSF-IL21-treated group (0.303 ± 0.323) g, significantly lower than that in the H22 group [(1.591 ± 0.280) g] and H22/Neo group [(1.489 ± 0.155) g]. Among them the tumor growth was most significantly inhibited in the H22/GM-CSF-IL-21 group (0.303 ± 0.323) g, compared with that of H22 and H22/neo groups (P < 0.01). But there was no significant difference between the tumor weights of the H22/GM-CSF group and H22/IL-21 group, and between the tumor weights of the H22 and H22/Neo groups (P > 0.05). The levels of IFN-γ and IL-2 in peripheral blood of mouse models treated with H22/GM-CSF-IL-21 were significantly increased than that in the H22/GM-CSF group and H22/IL-21 group (all P < 0.01), but significantly decreased in the H22group and H22/Neo group (P < 0.01). The anti-tumor activity of splenic NK cells and CTLs in the H22/GM-CSF-IL21 group was significantly enhanced (P < 0.01), compared with the significantly decreased in the H22 and H22/Neo groups. CONCLUSIONS: Our results demonstrate apparent antitumor effect of the plasmid pIRES-GM-CSF-IL-21 on the orthotopically transplanted liver tumor in mice. The combination of both pIRES-GM-CSF and IL-21 is more effective than that of pIRES/IL21 or pIRES/GM-CSF treatment alone. In addition, the plasmid pIRES-GM-CSF-IL-21 can also promote the secretion of IFN-γ and IL-2 in vivo, and enhance the cytotoxic activity of splenic NK and CTLs against the transplanted liver tumor.

Mutation analysis in a Chinese family with multiple endocrine neoplasia type 1.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome which is caused by germline mutations of the tumor suppressor gene MEN1. This study aimed to identify mutations in a Chinese pedigree with MEN1. METHODS: A large Chinese family with MEN1 was collected. All of the coded regions and their adjacent sequences of the MEN1 gene were amplified and sequenced. RESULTS: In this family, a heterozygous cytosine insertion in exon 10 (c.1546_1547insC) inducing a frame shift mutation of MEN1 was found in the proband and the other two suffering members of his family. This mutation was linked to a novel single nucleotide polymorphism (SNP) in intron 3 (IVS3 + 18C > T). CONCLUSIONS: The mutation in exon 10 of MEN1 gene might induce development of parathyroid hyperplasia and pituitary adenoma and cosegregate with MEN1 syndrome. The significance of the new found IVS3 + 18C > T of MEN1 needs a further investigation.