Development and preliminary testing of a probe-based duplex real-time PCR assay for the detection of African swine fever virus.

An outbreak of African swine fever (ASF) in China in 2018 caused substantial economic losses to the swine industry. To accurately diagnose clinical infection with ASF virus (ASFV), we developed a TaqMan probe-based duplex real-time PCR that simultaneously detected two discontinuous genes in the virus genome, thereby preventing the inaccurate results obtained with only one reaction. Two sets of ASFV gene-specific primers, along with two fluorescent TaqMan probes were designed to target conserved regions of the B646L and B438L genes. This method had high sensitivity and specificity, with a limit of detection of 10 copies/µL, and it did not cross-react with the genomes of other viral pathogens that affect pigs (i.e., CSFV, PRRSV, PEDV, PRV, PPV and PCV2). Overall, 180 clinical samples from ASFV-infected pig farms were used to compare this method with a commercial kit, which yielded excellent consistency (98.3%). This new diagnostic method should greatly improve the efficiency of ASFV surveillance and reduce economic losses, providing benefits for both animal and public health.

[Correlation between Bone Marrow Microvascular Density, Angiogenesis Factors and Bortezomib Resistance in Multiple Myeloma].

OBJECTIVE: To investigate the correlation between bone marrow microvascular density, angiogenesis factors and bortezomib resistance in multiple myeloma (MM). METHODS: The data of 200 patients with MM treated in our hospital from January 2020 to August 2023 were retrospectively analyzed, and the patients with MM were divided into drug-resistant group(n=68) and non-drug-resistant group(n=132) according to their drug resistance during bortezomib treatment. The univariate and multivariate logistic analysis were used to screen the independent influencing factors of bortezomib resistance in MM patients during treatment. The receiver operating characteristic (ROC) curve and clinical decision curve (DCA) were used to evaluate the predictive performance and clinical application value of the risk prediction model, the consistency between the actual incidence rate and the predicted incidence rate was judged by validating the calibration chart, and the goodness-of-fit of the model was judged by H-L test. RESULTS: 68 of the 200 MM patients developed resistance and poor clinical efficacy during bortezomib treatment, and the clinical resistance rate of bortezomib was 34.0%. The results of multivariate analysis showed that high bone marrow microvessel density (MVD) and high bone marrow supernatant VEGF, HGF, and bFGF expression levels were independent risk factors for bortezomib resistance in MM patients (P < 0.05). The area under the ROC curve (AUC) of the model jointly constructed by bone marrow MVD, serum VEGF, HGF, bFGF and TNF-α levels was 0.924, and its sensitivity and specificity were 92.6% and 78.8%, which were higher than those of the bone marrow MVD model (AUC=0.743) and the vasogenesis factor model (AUC=0.878). The calibration curve of the joint prediction model was close to the standard curve, indicating that the model is more consistent. The results of H-L goodness -of - fit test showed χ2=14.748， P =0.164, the joint prediction model had a good fit. The DCA curve showed that the clinical net benefit of intervention in the range of 0.0~1.0 was greater than that of full intervention and no intervention. CONCLUSION: The prediction model based on bone marrow MVD and vasogenesis factors (VEGF, HGF, bFGF) in MM patients has higher clinical evaluation performance and predictive value.

Selection, proliferation and differentiation of bone marrow-derived liver stem cells with a culture system containing cholestatic serum in vitro.

AIM: To explore the feasibility of direct separation, selective proliferation and differentiation of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Whole bone marrow cells of rats cultured in routine medium were replaced with conditioning selection media containing 20 mL/L, 50 mL/L, 70 mL/L, and 100 mL/L cholestatic sera, respectively, after they attached to the plates. The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures. Then the selected BDLSC were induced to proliferate and differentiate with the addition of hepatocyte growth factor (HGF). The morphology and phenotypic markers of BDLSC were characterized using immunohistochemistry, RT-PCR and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: Bone marrow cells formed fibroblast-like but not hepatocyte-like colonies in the presence of 20 mL/L cholestatic serum. In 70 mL/L cholestatic serum, BDLSC colonies could be selected but could not maintain good growth status. In 100 mL/L cholestatic serum, all of the bone marrow cells were unable to survive. A 50 mL/L cholestatic serum was the optimal concentration for the selection of BDLSC at which BDLSC could survive while the other populations of the bone marrow cells could not. The selected BDLSC proliferated and differentiated after HGF was added. Hepatocyte-like colony-forming units (H-CFU) then were formed. H-CFU expressed markers of embryonic hepatocytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors (HNF-1alpha and HNF-3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selected medium containing cholestatic serum can select BDLSC from whole bone marrow cells. It will be a new way to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system.

AIM: To explore the feasibility of passage of bone-marrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. METHODS: Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. RESULTS: The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units (H-CFUs) were formed. H-CFUs expressed markers of embryonic hepatocytes (alpha-fetoprotein, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors 1alpha and -3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Modified technique of pancreaticojejunal anastomosis with invagination following pancreaticoduodenectomy: a cohort study.

BACKGROUND: Leakage from pancreatic anastomoses remains the single most important morbidity after pancreaticoduodenectomy and contributes to prolonged hospitalization and mortality. This observational cohort study reported the surgical outcome of a modified invagination technique of pancreaticojejunostomy after pancreaticoduodenectomy. METHODS: Between December 2001 and December 2007, a total of 52 consecutive patients underwent elective pancreaticoduodenectomy for benign or malignant pathologies of the pancreas or the periampullary region in a tertiary referral center. All patients underwent our modified invagination technique of pancreaticojejunostomy regardless of the characteristics of the pancreatic stump. Data were collected prospectively. RESULTS: The mean hospital stay was 12.6 +/- 3.2 days. The incidence of overall surgical complications was 9.6%. No patient developed pancreatic fistula. One patient (1.9%) died of respiratory failure on postoperative day 7. CONCLUSIONS: We reported our pancreaticojejunostomy anastomosis technique with a pancreatic fistula rate of 0% and low intra-abdominal complication rate. The favorable results of this technique warrant further investigation in large prospective cohort studies and prospective randomized controlled studies.