This study reports on three patients with Shwachman-Diamond syndrome (SDS) who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the First Affiliated Hospital of Zhejiang University School of Medicine. Based on relevant literature, the clinical manifestations and genetic mutation characteristics of SDS were summarized, and the efficacy and timing of allo HSCT for such patients were explored. Three SDS patients were all male, with transplant ages of 32, 33, and 32 years old, respectively. All three patients were diagnosed in childhood. Case 1 presented with anemia as the initial clinical manifestation, which gradually progressed to a decrease in whole blood cells; Case 2 and 3 both present with a decrease in whole blood cells as the initial clinical manifestation. Case 1 and 3 have intellectual disabilities, while case 3 presents with pancreatic steatosis and chronic pancreatitis. All three patients have short stature. Three patients all detected heterozygous mutations in the SBDS: c.258+2T>C splice site. The family members of the three patients have no clinical manifestations of SDS. All three patients were treated with a reduced dose pre-treatment regimen (Fludarabine+Busulfan+Me-CCNU+Rabbit Anti-human Thymocyte Globulin). Case 1 and case 2 underwent haploid hematopoietic stem cell transplantation, while case 3 underwent unrelated donor hematopoietic stem cell transplantation. Case 1 was diagnosed with myelodysplastic syndrome transforming into acute myeloid leukemia before transplantation, but experienced early recurrence and death after transplantation; Case 2 is secondary implantation failure, dependent on platelet transfusion; Case 3 was removed from medication maintenance treatment after transplantation, and blood routine monitoring was normal.

Objective This study aims to construct and identify the chimeric antigen receptor NK92 (CAR-NK92) cells targeting NKG2D ligand (NKG2DL) (secreting IL-15Ra-IL-15) and verify the killing activity of NKG2D CAR-NK92 cells against multiple myeloma cells. Methods The extracellular segment of NKG2D was employed to connect 4-1BB and CD3Z, as well as IL-15Ra-IL-15 sequence to obtain a CAR expression framework. The lentivirus was packaged and transduced into NK92 cells to obtain NKG2D CAR-NK92 cells. The proliferation of NKG2D CAR-NK92 cells was detected by CCK-8 assay, IL-15Ra secretion was detected by ELISA and killing efficiency was detected by lactate dehydrogenase (LDH) assay. The molecular markers of NKp30, NKp44, NKp46, the ratio of apoptotic cell population, CD107a, and the secretion level of granzyme B and perforin were detected using flow cytometry. In addition, the cytotoxic mechanism of NKG2D CAR-NK92 cells on the tumor was verified by measuring the degranulation ability. Moreover, after NKG2D antibody inhibited effector cells and histamine inhibited tumor cells, LDH assay was utilized to detect the effect on cell-killing efficiency. Finally, the multiple myeloma tumor xenograft model was constructed to verify its anti-tumor activity in vivo. Results Lentiviral transduction significantly increased NKG2D expression in NK92 cells. Compared with NK92 cells, the proliferation ability of NKG2D CAR-NK92 cells was weaker. The early apoptotic cell population of NKG2D CAR-NK92 cells was less, and NKG2D CAR-NK92 cells had stronger cytotoxicity to multiple myeloma cells. Additionally, IL-15Ra secretion could be detected in its culture supernatant. NKp44 protein expression in NKG2D CAR-NK92 cells was clearly increased, demonstrating an enhanced activation level. Inhibition test revealed that the cytotoxicity of CAR-NK92 cells to MHC-I chain-related protein A (MICA) and MICB-positive tumor cells was more dependent on the interaction between NKG2D CAR and NKG2DL. After stimulating NKG2D CAR-NK92 cells with tumor cells, granzyme B and perforin expression increased, and NK cells obviously upregulated CD107α. Furthermore, multiple myeloma tumor xenograft model revealed that the tumors of mice treated with NKG2D CAR-NK92 cells were significantly reduced, and the cell therapy did not sensibly affect the weight of the mice. Conclusion A type of CAR-NK92 cell targeting NKG2DL (secreting IL-15Ra-IL-15) is successfully constructed, indicating the effective killing of multiple myeloid cells.
Humans ; Mice ; Animals ; Receptors, Chimeric Antigen/genetics* ; Interleukin-15 ; NK Cell Lectin-Like Receptor Subfamily K/metabolism* ; Granzymes ; Cell Line, Tumor ; Multiple Myeloma/therapy* ; Perforin

Objective: To investigate the epidemiological characteristics of rabies-exposed populations in Anji County, Zhejiang Province from 2017 to 2021, so as to provide insights into rabies control in the county. Methods: All data pertaining to rabies were captured from Monthly Report of Rabies-exposed Populations in Huzhou City and Investigation Form of Multiple Dog Injuries reported by dog injury clinics in Anji County from 2017 to 2021, and the species of animals causing dog injuries, duration, degree and site of exposure, and post-exposure treatment of rabies-exposed populations were descriptively analyzed. Results: Totally 46 186 cases with rabies exposure were reported in dog injury clinics in Anji County from 2017 to 2021, and the rate of exposure appeared a tendency towards a decline year by year (Z=-23.249, P<0.001), with an annual mean exposure rate of 1 739.59/105. The number of cases with exposure to rabies peaked in July and August (10 066 cases, 21.79%). Dogs were predominant animals causing injuries (31 732 cases, 68.70%), and the rate of exposure to dog bites appeared a tendency towards a decline year by year (Z=-35.541, P<0.001). There were 11 350 cases with cat-causing injuries (24.57%), and the rate of exposure to cat bites appeared a tendency towards a rise (Z=14.834, P<0.001). Lower extremity was the main site of exposure (22 364 cases, 48.42%), and the proportions of grade Ⅱ and Ⅲ exposure to rabies were 72.85% and 25.23%, the rates of exposure both appeared a tendency towards a decline (Z=-14.522, P<0.001; Z=-21.820, P<0.001). The proportion of using human rabies immune globulin was 25.72% among populations with grade Ⅲ exposure, which appeared a tendency towards a rise (Z=6.636, P<0.001). Conclusions The rate of exposure to rabies appeared a tendency towards a decline in Anji County from 2017 to 2021. Dogs were predominant animals causing injuries, and the rate of cat bites appeared a tendency towards a rise from 2017 to 2021; however, the proportion of using human rabies immune globulin remains to be improved among populations with grade Ⅲ exposure.

Traditionally, Tripterygium hypoglaucum (Levl.) Hutch (THH) are widely used in Chinese folk to treat rheumatoid arthritis (RA). This study aimed to investigate whether the anti-RA effect of THH is related with the gut microbiota. The main components of prepared THH extract were identified by HPLC-MS. C57BL/6 mice with adjuvant-induced arthritis (AIA) were treated with THH extract by gavage for one month. THH extract significantly alleviated swollen ankle, joint cavity exudation, and articular cartilage destruction in AIA mice. The mRNA and protein levels of inflammatory mediators in muscles and plasma indicated that THH extract attenuated inflammatory responses in the joint by blocking TLR4/MyD88/MAPK signaling pathways. THH extract remarkably restored the dysbiosis of the gut microbiota in AIA mice, featuring the increases of Bifidobacterium, Akkermansia, and Lactobacillus and the decreases of Butyricimonas, Parabacteroides, and Anaeroplasma. Furthermore, the altered bacteria were closely correlated with physiological indices and drove metabolic changes of the intestinal microbiota. In addition, antibiotic-induced pseudo germ-free mice were employed to verify the role of the intestinal flora. Strikingly, THH treatment failed to ameliorate the arthritis symptoms and signaling pathways in pseudo germ-free mice, which validates the indispensable role of the intestinal flora. For the first time, we demonstrated that THH extract protects joint inflammation by manipulating the intestinal flora and regulating the TLR4/MyD88/MAPK signaling pathway. Therefore, THH extract may serve as a microbial modulator to recover RA in clincial practice.ver RA in clincial practice.
Mice ; Animals ; Gastrointestinal Microbiome ; Tripterygium ; Myeloid Differentiation Factor 88/genetics* ; Toll-Like Receptor 4/genetics* ; Mice, Inbred C57BL ; Arthritis, Experimental/drug therapy*

Objective To investigate the risk factors for spontaneous bacterial peritonitis (SBP) in patients with cirrhotic ascites, and to establish a new model for predicting the development of SBP. Methods A total of 215 patients who were diagnosed with cirrhotic ascites in Hebei General Hospital from September 2016 to September 2020 were enrolled, and according to the presence or absence of SBP, they were divided into SBP group with 55 patients and non-SBP group with 160 patients. Related clinical data were collected and albumin-bilirubin (ALBI) score, Model for End-Stage Liver Disease (MELD) score, MELD combined with serum sodium concentration (MELD-Na) score, and Child-Pugh score were calculated. The t -test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups; a multivariate logistic regression analysis was used to screen out independent risk factors, and the receiver operating characteristic (ROC) curve was plotted to evaluate the performance of ALBI score, procalcitonin (PCT), polymorphonuclear neutrophil (PMN) count in ascites, and the ALBI-PMN-PCT combined model in the diagnosis of SBP. Results Compared with the SBP group, the non-SBP group had a significantly higher concentration of Na + ( Z =-3.414, P =0.001) and significantly lower total bilirubin ( Z =-2.720, P =0.007), creatinine ( Z =-1.994, P =0.046), urea nitrogen ( Z =-2.440, P =0.015), C-reactive protein ( Z =-9.137, P < 0.001), PCT ( Z =-8.096, P < 0.001), prothrombin time ( Z =-1.969, P =0.049), international normalized ratio ( Z =-2.073, P =0.038), PMN ( Z =-8.292, P < 0.001), MELD score ( Z =-2.736, P =0.006), MELD-Na score ( Z =-3.188, P =0.001), Child-Pugh score ( Z =-3.419, P =0.001), and ALBI score ( t =-5.010, P < 0.001), and there were also significant differences between the two groups in the presence or absence of gastrointestinal bleeding or hepatic encephalopathy ( χ 2 =16.551 and 8.142, P < 0.001 and P =0.004). The multivariate logistic regression analysis showed that ALBI score (odds ratio [ OR ]=3.460, 95% confidence interval [ CI ]: 1.296-9.240, P =0.013), PMN ( OR =1.012, 95% CI : 1.007-1.017, P < 0.001), and PCT ( OR =6.019, 95% CI : 2.821-12.843, P < 0.001) were independent risk factors for SBP in patients with cirrhotic ascites. The ROC curve showed that ALBI, PCT, PMN, and ALBI-PMN-PCT had areas under the ROC curve of 0.711, 0.866, 0.875, and 0.934, respectively, in the diagnosis of SBP, with sensitivities of 50.91%, 73.36%, 72.73%, and 89.09%, respectively, and specificities of 86.87%, 81.25%, 100.00%, and 91.87%, respectively. The patients with ALBI-PMN-PCT > 0.272 had an increased risk of developing SBP. Conclusion The ALBI-PMN-PCT combined model has a high value in predicting the onset of SBP in patients with cirrhotic ascites.

Objective:To observe the curative effects of berberine in rats with high-fat diet induced non-alcoholic fatty liver and to further explore its possible mechanism.Methods:Twenty-six Sprague-Dawley rats (120-160 g) were randomly divided into 3 groups: control group ( n = 8), model group ( n = 10) and treatment group ( n = 8). Rats in the control group were fed with regular diet, and the model group and the treatment group were fed a high-fat diet. At the 12th week, two rats in the in the model group were sacrificed to verify whether model was successful established. Subsequently, treatment group rats were given a gavage of berberine at a dose of 150 mg·kg -1·d -1 for 4 weeks, and the control and the model group rats were given the same dose of normal saline. Rats were sacrificed at week 16th. HE staining was used to observe the changes in the intestinal mucosa of rats. Sudan black B staining was used to observe the fatty changes in liver. Immunohistochemical staining was used to observe the expression level of occludin protein in the intestinal epithelium. A real-time 16S rDNA PCR method was used to measure the number of escherichia coli, bacteroides and faecalibacterium prausnitzii in the feces of rats. Results:Model group had a higher serum levels of endotoxin (0.288 ± 0.045) and tumor necrosis factor (TNF)-α (1.07 ± 0.11) than the control group (0.192 ± 0.049, 0.94 ± 0.07) ( P < 0.05). Berberine intervention had significantly reduced endotoxin (0.213 ± 0.025) and TNF-α level (0.93 ± 0.07) ( P < 0.05). The expression level of occludin protein was significantly lower in the intestinal mucosa of model group than that of control group (0.166 ± 0.014), and berberine had promoted the expression of occludin protein in intestinal mucosa (0.055 ± 0.009), but the difference was not statistically significant ( P > 0.05). At the same time, compared with the model group (7.29 ± 0.47), the number of bacteroidetes in the control group (9.49 ± 0.59) was decreased, while the number of bacteroidetes in the treatment group was increased (9.77 ± 0.87). The number of escherichia coli (6.92 ± 0.77) and faecalibacterium prausnitzii (8.70 ± 0.62) in the model group were increased than control group (5.42 ± 0.63, 9.49 ± 0.59), while the number of escherichia coli (6.34 ± 0.71) and faecalibacterium prausnitzii (9.77 ± 0.87) ( P < 0.05) was reduced with the intervention of berberine. Conclusion:Berberine could effectively protect the intestinal barrier function in rats with NAFLD and the possible mechanism of action behind it may be the regulation of intestinal flora.

Objective: To study the effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute leukemia (AL) and its significance as molecular marker to dynamically monitor minimal residual disease (MRD) . Methods: Retrospectively analyzed those AL patients who underwent allo-HSCT in the First Hospital Affiliated to Zhejiang University School of Medicine during Jan 2016 to Dec 2017, a total number of 314 cases, 163 males and 151 females, median age was 30 (9-64) years old. Comparing the difference of WT1 expression at diagnosed, pre-HSCT and after HSCT. Using the receiver operating characteristic (ROC) curve to determine the WT1 threshold at different time so as to predict relapse. The threshold of WT1 expression before transplantation was 1.010%, within 3 months after HSCT was 0.079% and 6 months after HSCT was 0.375%. According to these thresholds, WT1 positive patients were divided into low expression groups and high expression groups. Analyzed the relationship between overall survival (OS) , disease-free survival (DFS) , cumulative incidence of relapse (CIR) and WT1 expression. Results: The OS and DFS of high expression group pre-HSCT were lower than low expression group [69.2% (9/13) vs 89.1% (57/64) , χ²=4.086, P=0.043; 53.8% (7/13) vs 87.5% (56/64) , χ²=9.766, P=0.002], CIR was higher than low expression group [30.8% (4/13) vs 7.8% (5/64) , P=0.017]. There was no significant difference of OS and DFS between high expression and low expression group of 3 months after HSCT (P=0.558, P=0.269) . The OS and DFS of high expression group of 6 months after transplantation were both lower than low expression group (P=0.049, P=0.035) . Multivariate analysis showed that WT1>0.375% when 6 months after transplantation was the only independent prognostic factor for shorter DFS (P=0.022) . There was no statistically significant difference in CIR between the high-expression group and the low-expression group 3 months after transplantation and 6 months after transplantation (P=0.114, P=0.306) . Conclusion High expression of WT1 before and after HSCT was an adverse prognosis factor. It is of clinical practical value to use WT1 as a transplant recommendation index for patients with acute leukemia and as a marker to monitor MRD dynamically.

Objective To investigate the effects of curcumin on myosin light chain kinase(MLCK)in the intestinal mucosa of rats with nonalcoholic fatty liver disease(NAFLD). Methods 35 SD rats were randomly di-vided into the normal control group ,the high fat group and the curcumin group. The high fat group ,curcumin group were given high fat diet for 16 weeks. After 8 weeks of high fat feeding in the curcumin group ,the rats were gavaged with 200 mg/(kg·d)curcumin for 8 weeks. The levels of ALT,AST,LPS and DAO in blood and expres-sion of MLCK in the intestinal mucosa were detected. The changes of liver pathology and tight junction(TJ)of the intestinal mucosa were observed. Results Compared with the control group,the blood levels of ALT,AST,LPS and DAO in the high fat group were obviously increased(P<0.05);Compared with the high fat group,the blood levels of ALT、AST、LPS and DAO in the curcumin group were obviously decreased (P < 0.05). In the high fat group ,hepatocellular steatosis was obvious ,while in the curcumin group hepatocellular steatosis was decreased. TJ was disrupted in the high fat group ,and the intercellular space was larger in the TJ group than the control group (P<0.05). The intercellular space was narrower in the curcumin group than the high fat group(P<0.05). The expression of MLCK in the high fat group was significant higher than that in the control group(P<0.05). The pos-itive staining in the curcumin group was significant lower than that in the high fat group (P < 0.05). Conclusion Curcumin can ameliorate hepatic steatosis by downregulating expression of MLCK in the intestinal mucosa of rats with NAFLD,improving TJ structure of the intestinal mucosa.

Objective To compare the clinic significance of four clinical scoring systems in evaluating prognosis of acute pancreatitis: bedside index for severity in acute pancreatitis(BISAP), acute physiology and chronic health evaluation (APACHEⅡ), Ranson’s scoring system, computed tomography severity index (CTSI) in AP. Methods Patients visited our clinic with AP (n=114) in recent 2 years were retrospectively analyzed. BISAP and APACHEⅡscores were obtained at 24 hours after admission; Ranson ’s score was obtained at 48 hours after admission and CTSI are obtained was obtained at 72 hours after admission. Results of four scoring system were compared under different causes and different severity of the dis?ease. Correlation between BISAP score and the other three scores were analyzed and the predicative value of all four scoring systems for severity of AP and death were also compared. Results The mean values of four scoring systems show no signifi?cant difference in AP patients with different etiology (P>0.05). The BISAP score is positively correlated with APACHE-Ⅱ, Ranson ’s score and CTSI score (P<0.01). The four scoring systems all present good predictive value on the severity of AP and death (P<0.01). Conclusion The four scoring systems can all be applied to grading and prognosis for AP of various causes. BISAP is a simple, prompt, economical scoring system in clinical practice.