Anemia Is Associated with Disease Severity, Hepatic Complications, and Progression of Wilson Disease: A Retrospective Cohort Study.

INTRODUCTION: Anemia is a common manifestation of chronic liver diseases. It is a predictor of severe disease, a high risk of complications, and poor outcomes in various liver diseases. However, it remains unclear whether anemia serves as a similar indicator in patients with Wilson disease (WD). Therefore, this study aimed to investigate the relationship between anemia and severity, hepatic complications, and the progression of WD. METHODS: Medical data were collected retrospectively from January 1, 2016, to December 31, 2020. Univariate and multivariate analyses were carried out to investigate the relationship between anemia and liver-associated disease severity, hepatic complications, and the progression of WD. RESULTS: A total of 288 WD patients (48 with and 240 without anemia) were enrolled in the study. Multivariate linear regression revealed that WD patients with anemia had significantly higher levels of bilirubin, alanine transaminase, prothrombin time, international normalized ratio, type Ⅳ collagen, and hyaluronic acid and significantly lower levels of albumin, total cholesterol, and high-density lipoprotein-cholesterol (all p < 0.05). Multivariate logistic regression showed that anemia was a risk factor for gastric varices and ascites (all p < 0.05). Fully adjusted Cox regression revealed that anemia was an independent risk factor for advanced Child-Pugh classification (p = 0.034). CONCLUSIONS: Anemia was common in WD patients and was associated with greater disease severity, a higher risk of hepatic complications, and a faster progression.

[Clinical Application of Chromosomal Microarray Analysis in Karyotyping with Uncertain Genomic Rearrangement].

OBJECTIVES: To apply chromosomal microarray analysis (CMA) in the diagnosis of karyotyping with uncertain genomic rearrangement. METHODS: We retrospectively reviewed 48 samples (34 samples of amniotic fluid, 14 samples of peripheral blood) of karyotype analyses with uncertain genomic rearrangement in patients admitted to our department from September 2014 to April 2016. The CMA results were compared with those of karyotyping. RESULTS: The 48 samples consisted of 13 samples with marker chromosomes, 19 samples with derivative chromosomes, and 16 samples with balanced translocation. Sixteen cases (33.33%) were detected with abnormalities by CMA. In the 32 samples with marker chromosomes or derivative chromosomes, 16 cases were detected with deletions or duplications (>5 Mb) by CMA, including 1 case 21-trisomy, 2 cases XYY syndrome and 3 cases microdeletion/ microduplication syndromes (22q11 duplication syndrome, Wolf-Hirschhorn syndrome and 15q26 overgrowth syndrome). In the 16 balanced translocation cases, all revealed negative results in CMA. CONCLUSIONS: CMA can confirm the karyotyping with uncertain genomic rearrangement and clarify its clinical significance.

[The use of human papillomavirus genotying kit based on gene chip technology for detecting and typing of human papillomavirus].

OBJECTIVE: To evaluate a new human papillomavirus (HPV) genotyping technique based on gene chip technology (HPG) for HPV genotyping and its clinical efficacy. METHODS: HPV genotyping (HPG) test, hybrid capture II (HC2) test and DNA sequencing assay were performed in 151 patients aged 20-75 years with diagnosis of chronic cervicitis or abnormal vaginal bleeding. The cervical specimens were collected from cervical epithelium. All the cervical samples were analyzed by the HPG test, HC2 test and DNA sequencing. The clinical efficacy of the HPG test was analyzed. RESULTS: The consistent rate between HPG test and HC2 test was 87.42% (kappa = 0.75, P < 0.05). When DNA sequencing assay was regarding as the final test result, the sensitivity and specificity of HPG test for high risk HPV were 100% and 96.49%, respectively. The consistent rate between HPG test and direct DNA sequencing was 98.70% (kappa = 0.97, P < 0.05). The most common six HPV genotypes detected by HPG test were HPV 16 (13.25%), 58 (11.92%), 52 (11.92%), 31 (6.62%) 39 (5.96%), 33 (5.96%) in descending order of frequency. The incidence of multiple-types infection detected by HPG test was 23.84%. CONCLUSION: HPG test is a rapid and accurate test for HPV genotyping which could detect 29 types of HPV infection at one time. It is suitable for cervical HPV infection screening in clinic.

Type 3 innate lymphoid cells as an indicator of renal dysfunction and serum uric acid in hyperuricemia.

BACKGROUND: Type 3 innate lymphoid cells (ILC3s) are a newly identified group of innate immune cells that participate in the progression of several metabolic diseases by secreting interleukin (IL)-17 and IL-22. These cytokines are associated with hyperuricemia (HUA) severity and development; however, the relationship between ILC3s and HUA remains unclear. OBJECTIVES: To determine the characteristics of circulating ILC3s in patients with HUA. MATERIAL AND METHODS: Type 3 innate lymphoid cells and their subsets were detected using flow cytometry in peripheral blood mononuclear cells (PBMCs) of 80 HUA patients and 30 healthy controls (HC). Plasma levels of IL-17A and IL-22 were measured with enzyme-linked immunosorbent assay (ELISA). Clinical data of enrolled subjects were collected from electronic medical records. RESULTS: In patients with HUA, the frequency of circulating ILC3s was elevated and positively correlated with levels of serum uric acid and serum creatinine (Scr). Although there was no significant difference in the plasma concentration of IL-17A between the patients with HUA and healthy controls, positive correlations between plasma IL-17A and the concentration of serum uric acid and frequency of circulating ILC3s were observed in the patients with HUA. CONCLUSIONS: In patients with HUA, positive correlations were detected between circulating ILC3 levels, plasma IL-17A and serum uric acid. Therefore, ILC3s and IL-17A may be useful indicators of disease severity, and are potential new therapeutic targets in HUA.

Reduced type 3 innate lymphoid cells related to worsening kidney function in renal dysfunction.

Intestinal mucosa barrier injury and immunity imbalance contribute to chronic kidney disease (CKD) progression. Type 3 innate lymphoid cells (ILC3s) are essential for normal intestinal homeostasis. Nevertheless, the relationship between ILC3s and CKD remains largely unknown. The aim of this study was to investigate the relationship linking ILC3s to clinical indicators among patients with renal dysfunction. The levels of circulating ILC3s and dendritic cells, as well as their subsets, in patients with renal dysfunction and healthy controls were determined through flow cytometry. The levels of human plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) were measured using enzyme-linked immunosorbent assay. Renal function was evaluated by measuring the estimated glomerular filtration rate (eGFR), as well as the levels of serum creatinine, blood urea nitrogen (BUN), and uric acid. The results revealed that the proportion of peripheral ILC3s was significantly decreased in patients with renal dysfunction. This reduction was positively associated with the levels of eGFR, and inversely associated with the levels of BUN and uric acid. Similarly, the percentage of circulating C-C motif chemokine receptor 6-positive (CCR6 +) ILC3s was also obviously reduced, and demonstrated positive and negative associations with the levels of eGFR and BUN, respectively. Furthermore, the levels of CCR6 + ILC3s correlated positively with those of GM-CSF, as well as type 1 conventional dendritic cells (cDC1s), which also decreased in parallel with kidney function. Thus, the reduction of ILC3s, particularly CCR6 + ILC3s, was related to worsening kidney function in patients with renal dysfunction. This effect may delay renal function impairment by regulating cDC1s via the secretion of GM-CSF, indicating that CCR6 + ILC3s may serve as efficient biomarkers for evaluating kidney function.

Fresh Washed Microbiota Transplantation Alters Gut Microbiota Metabolites to Ameliorate Sleeping Disorder Symptom of Autistic Children.

Accumulating studies have raised concerns about gut dysbiosis associating autism spectrum disorder (ASD) and its related symptoms. However, the effect of gut microbiota modification on the Chinese ASD population and its underlying mechanism were still elusive. Herein, we enrolled 24 ASD children to perform the first course of fresh washed microbiota transplantation (WMT), 18 patients decided to participate the second course, 13 of which stayed to participate the third course, and there were 8 patients at the fourth course. Then we evaluated the effects of fresh WMT on these patients and their related symptoms. Our results found that the sleeping disorder symptom was positively interrelated to ASD, fresh WMT significantly alleviated ASD and its sleeping disorder and constipation symptoms. In addition, WMT stably and continuously downregulated Bacteroides/Flavonifractor/Parasutterella while upregulated Prevotella_9 to decrease toxic metabolic production and improve detoxification by regulating glycolysis/myo-inositol/D-glucuronide/D-glucarate degradation, L-1,2-propanediol degradation, fatty acid ß-oxidation. Thus, our results suggested that fresh WMT moderated gut microbiome to improve the behavioral and sleeping disorder symptoms of ASD via decrease toxic metabolic production and improve detoxification. Which thus provides a promising gut ecological strategy for ASD children and its related symptoms treatments.

[Establishment of a cisplatin-resistant human cervical cancer cell line].

OBJECTIVE: To establish a cisplatin (cDDP)-resistant human cervical cancer cell line named SiHa/cDDP and researched its biological characteristics. METHODS: The development of cDDP resistance in SiHa cell line was induced by continuously stepwise exposure of the cells to cDDP. Cell growth curve, doubling time and resistance index (RI) were evaluated by MTT analysis. The expression of P-gp, GST-pi, Topo I were assessed with immunocytochemical method. RESULTS: SiHa/cDDP cell line was successfully established which had stable growth, subculture, frozen reservation and resuscitation in the concentration of 2 microg/ml cDDP, doubling time was (45.82 +/- 3.69) h and RI to cDDP was 16.131. It also showed different degrees of cross-resistance to several anticancer drugs. As compared to parental SiHa, SiHa/cDDP had over-expression in P-gp and GST-pi (P < 0.01), but Topo I showed no statistically significant differences. CONCLUSION: We successfully established the cDDP-resistant human cervical cancer cell line SiHa/cDDP, which may provide ideal experimental model for research of human cervical carcinoma.

[Isolation and biological characterization of human amniotic fluid-derived stem cells].

OBJECTIVE: To establish in vitro culture procedure for human amniotic fluid-derived CD117 positive stem cells, and to identify the characteristics of CD117 positive stem cells. METHODS: 86 amniotic fluid samples (10 mL of each) were obtained by second-trimester amniocentesis. Isolation of amniotic fluid-derived stem cells expressing CD117 antigen was performed via magnetic cell sorting using the CD117 MicroBead Kit. The karyotype of CD117 positive stem cells was analysed through repeated freezing. Adipogenic differentiation of these CD117 positive stem cells was displayed by Oil Red O staining. Osteogeneic differentiation of these CD117 positive stem cells was confirmed by Alizarin Red staining. RESULTS: The CD117 positive stem cells were successfully isolated and cultured from 61 samples, with all showing normal karyotype. Product analysis of specific staining confirmed that under specific culture mediums, these cells could be successfully induced to differentiate into adipocytes and osteocytes. CONCLUSION: Based on this study, we estimate that isolating CD117 positive stem cells from second-trimester amniotic fluid obtained by amniocentesis has a success rate of 70.93%. These cells maintain morphological and genetic stability in vitro. Human amniotic fluid-derived CD117 positive stem cells have the ability to differentiate in vitro into adipocytes and osteocytes under specific culture mediums and may be applied in cell transplantation and regenerative medicine.

Two novel variants of the PHEX gene in patients with X­linked dominant hypophosphatemic rickets and prenatal diagnosis for fetuses in these families.

X­linked hypophosphatemic rickets (XLHR; OMIM 307800) is an X­linked dominant disorder caused by mutations in the phosphate­regulating neutral endopeptidase homolog X­linked (PHEX) gene, which is located at Xp22.11. In the present study, two novel variants of the PHEX gene were identified in two unrelated families with XLHR by directly sequencing all 22 exon regions and intron/exon boundaries of the PHEX gene. One missense variant, NM_000444.5: c.1721T>A, was identified in exon 17 of the PHEX gene in Family 1, which led to an amino acid change in the p.Ile574Lys protein. The other splicing variant identified was NM_000444.5: c.591A>G, in exon 5 in Family 2, resulting in a deletion of 77 bp in the 3' site of exon 5 during splicing, which was verified by direct cDNA sequencing of the PHEX gene. According to the results of reverse transcription­quantitative polymerase chain reaction analysis, the affected male with the splicing variant c.591A>G showed normal gene expression of PHEX, whereas the affected female exhibited low gene expression, compared with normal females. Based on these findings, prenatal diagnoses were made for the fetuses with a family history of XLHR using the backup amniotic fluid samples. One fetus without the missense variant was confirmed to be a healthy girl in a follow­up visit 1 month following birth.

miR-34 increases in vitro PANC-1 cell sensitivity to gemcitabine via targeting Slug/PUMA.

miR-34 was deregulated in tumor tissues compared with corresponding noncancerous tissue samples. Furthermore, miR-34 may contribute to cancer-stromal interaction associated with cancer progression. However, whether miR-34 could decrease chemoresistance of cancer cells to chemotherapeutic agent remains unclear. In our study, we examined whether overexpression of miR-34 could sensitize gemcitabine -mediated apoptosis in human pancreatic cancer PANC-1 cells. We found that miR-34 markedly induced gemcitabine -mediated apoptosis in PANC-1 cells. miR-34 induced down-regulation of Slug expression and upregulation of p53 up-regulated modulator of apoptosis (PUMA) expression. The over-expression of Slug or downregulation of PUMA by Slug cDNA or PUMA siRNA transfection markedly blocked miR-34-induced gemcitabine sensitization. Furthermore, miR-34 induced PUMA expression by downregulation of Slug. Taken together, our study demonstrates that miR-34 enhances sensitization against gemcitabine-mediated apoptosis through the down-regulation of Slug expression, and up-regulation of Slug-dependent PUMA expression.

[Effects of ovariectomy on fracture healing in female rats].

OBJECTIVE: To observe the effects of ovariectomy on fracture healing in female rats. METHODS: Thirty-six 6-month-old female SD rats were divided into OVX group and SHAM group. The osteoporotic model was established by ovariectomy. The right femur was broken by operation at 8 weeks later. The rats were killed in batches at the end of 2, 4, 6, 8 weeks postoperatively for study. The examinations included the bone roentgenography, the histological appearance of bone calli and the biomechanical properties of right femur. RESULTS: The density of calli was decreased obviously in OVX group. The line of fracture existed in OVX group while the line of fracture was unclear or disappeared in SHAM. In the OVX group, there were cartilages at the early callus formation stage, and there were small-sized osteoblasts and increased number of osteoclasts on the surface of osseous trabecula. The osseous trabecula became thinner and disrupted obviously in OVX group, and it became massive, thicker and closer gradually 8 weeks after fracture in SHAM group. The area of osseous trabecula in the SHAM group was bigger than that in the OVX group. CONCLUSION: After ovariectomy, the absorption of bone prevails over osteogenesis, the osteoporotic changes occur, and the fracture healing is poor in quality.