Next-generation sequencing technology for diagnosis and efficacy evaluation of a patient with visceral leishmaniasis: A case report.

BACKGROUND: Visceral leishmaniasis (VL) is a parasitic disease caused by Leishmania and transmitted by infected sand flies. VL has a low incidence in China, and its clinical presentation is complex and atypical. This disease is easily misdiagnosed and can become life-threatening within a short period of time. Therefore, early, rapid and accurate diagnosis and treatment of the disease are essential. CASE SUMMARY: A 25-year-old male patient presented with the clinical manifestations of irregular fever, hepatosplenomegaly, increased polyclonal globulin, and pancytopenia. The first bone marrow puncture biopsy did not provide a clear diagnosis. In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis, splenectomy was performed, and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy. Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing (mNGS) technology detection, the patient was finally diagnosed with VL. After treatment with liposomal amphotericin B, the body temperature quickly returned to normal and the hemocytes recovered gradually. Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected. CONCLUSION: As a fast and accurate detection method, mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.

[Effects of Dasatinib on the Expansion, Subsets, Receptor Expression and Cytotoxic Function of NK Cells in Vitro].

OBJECTIVE: To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. RESULTS: The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. CONCLUSION: The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

NK cell predicts the severity of acute graft-versus-host disease in patients after allogeneic stem cell transplantation using antithymocyte globulin (ATG) in pretreatment scheme.

BACKGROUND: Graft-versus-host disease (GVHD) is one of the most complex complications after allogeneic stem cell transplantation. Current standard of grading system is based on clinical symptoms in skin, liver and intestinal. However, it's difficult to differ GVHD and its extent just by clinical manifestation. Here we retrospectively analyzed cell immune function in patients implemented allogeneic stem cell transplantation in Ningbo first Hospital from Jan 2013 to Jan 2018. RESULTS: the data are collected from 51 patients (mean age was 42; 45.1% women). The average NK cell percentage was 39.31% in severe GVHD (Grade III-IV), was 16.98% in mild GVHD (GradeI-II), while was 21.15% in No GVHD group. The statistical analysis showed difference among each grade. Further analysis was performed in Antithymocyte globulin (ATG) treated group and control group. We showed NK Cell percentage was sharply different in ATG treated group: 47.34% in severe GVHD, 11.98% in mild GVHD group, while 18.3% in no GVHD group. However, in control group, the average percentage of NK cells was 23.27% in severe GVHD, was 23.22%in mild GVHD group, while was 21.13% in no GVHD group. CONCLUSION: The data supports that ATG can prevent GVHD by increasing NK cell percentage. The percentage of NK cell seemed to be a useful probe to evaluate the severity of GVHD in allogeneic stem cell transplantation patients using ATG in pretreatment.

Comparison of therapeutic efficacy and complications between autologous stem cell transplantation and chemotherapy for large B-cell lymphoma: a meta-analysis.

OBJECTIVE: To contrast the clinical effects and complications for the treatment of large B-cell lymphoma between autologous stem cell transplantation and chemotherapy. METHODS: Multiple databases were searched to identify relevant studies. Articles that met the inclusion criteria were included. RESULTS: A total of 11 studies including 795 patients were involved, which eventually met the eligibility criteria. 385 and 410 samples were included in ASCT group and chemotherapy group respectively. The heterogeneity test suggested that clinical efficacy (RR = 1.29, 95% CI [1.17, 1.42], P < 0.00001; P for heterogeneity = 0.0002, I2 = 72%), side effects (RR = 1.72, 95% CI [1.15, 2.58], P = 0.009; P for Heterogeneity < 0.0001, I² = 85%) and overall 5-year survival rate (RR = 1.16, 95% CI [1.05, 1.29], P = 0.004; P for Heterogeneity = 0.008, I² = 58%) were significantly different between ASCT group and chemotherapy group, while non-disease 5-year survival rate (RR = 1.24, 95% CI [1.04, 1.48], P = 0.02; P for Heterogeneity = 0.22, I² = 24%) appears insignificantly. CONCLUSIONS: This study presents a comprehensive summary and critical assessment of the current evidence for the treatment of large B-cell lymphoma. The evidence shows that the clinical efficacy of ASCT was better than that of chemotherapy to some extent, but ASCT might lead to more possibility of complications.

[Optimization of flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma by response surface methodology].

Response surface methodology was used to optimize and obtain the optimal flash-type extraction technology of alisol B 23-acetate from Alismatis Rhizoma. With the extraction rate of alisol B 23-acetate as an indicator, single-factor test was used to investigate the effect of ethanol volume fraction, liquid-solid ratio, extraction times and extracting time on the extraction rate of alisol B 23-acetate.The results were combined with Box-Benhnken design and response surface analysis to optimize the technology parameters for extraction process of Alismatis Rhizoma and obtain the optimal flash-type extraction technology under the following conditions： ethanol volume fraction 80%, liquid-solid ratio 12∶1, extraction 4 times, 114 s/time. Flash-type extraction technology of alisol B 23-acetate by response surface methodology is stable, time-saving, efficient, and with the advantages of room temperature extraction and no component damage, so it can be used for massive production.

[Fast measurement method based on near infrared spectroscopy in extraction process of Tianshu capsules].

Based on the near infrared spectroscopy, partial least square (PLS) method was used to respectively develop the quantitative calibration models to fast measure the contents of the total solid and ferulic acid in extraction process of Tianshu capsule extracts. The results showed that in the quantitative model of solid content, the correlation coefficients (R²) of calibration set and cross validation set were 0.967 301 and 0.947 726. The root-mean square error of calibration set (RMSEC) was 0.054 7 and root-mean square error of cross validation set (RMSECV) was 0.069 8. Besides, in the quantitative model of ferulic acid, the correlation coefficients (R²) of calibration set and cross validation set were 0.986 879 and 0.962 243. RMSEC was 1.402 6 and RMSECV was 2.400 2. When the established models were applied to on-line monitoring, the correlation coefficients of predicted results and measured values for total solid content and ferulic acid were 0.993 3 and 0.991 6; root-mean square error of predicted value (RMSEP) was 0.039 3 and 1.669 3 respectively; mean relative deviation of predicted value (RSEP) was 3.49% and 3.58%. The results indicated that the established models can be used to fast measure the contents of the total solid and ferulic acid in extraction process of Tianshu capsule extracts.

[Quantitative and qualitative evaluation on tablets of Ginkgo biloba leaves using fingerprint and LC-MS analysis].

A reasonable method for the quality control of tablets of Ginkgo biloba leaves was established in this paper. The total flavonol glycosides and terpene lactones of G. biloba tablets were quantified by HPLC. Totally, 16 batches of the commercially available tablets of G. biloba leaves were determined. Among of them, 2 batches were unqualified in the content of total flavonol glycosides, and 3 batches were unqualified in the content of terpene lactones. A validated HPLC fingerprint method was established to evaluate the commercially available tablets of G. biloba leaves with the assistance of LC-MS. Sixteen batches showed the similarity of 0.763-0.989. There were 31 fingerprint chromatogram peaks were identified as flavonoids compositions by LC-MS. This provides a research idea for the quality control of tablets of G. biloba leaves.

The general transition metal (Tro) and Zn2+ (Znu) transporters in Treponema pallidum: analysis of metal specificities and expression profiles.

Acquisition of transition metals is central to the struggle between a bacterial pathogen and its mammalian host. Previous studies demonstrated that Treponema pallidum encodes a cluster-9 (C9) ABC transporter (troABCD) whose solute-binding protein component (TroA) ligands Zn(2+) and Mn(2+) with essentially equal affinities. Bioinformatic analysis revealed that T. pallidum encodes an additional C9 transporter (tp0034-36) orthologous to Zn(2+)-uptake (Znu) systems in other bacteria; the binding protein component, ZnuA, contains a His-rich tract characteristic of C9 Zn(2+)-binding proteins. Metal analysis and metal-reconstitution studies demonstrated that ZnuA is a Zn(2+)-binding protein; parallel studies confirmed that TroA binds Zn(2+), Mn(2+) and Fe. Circular dichroism showed that ZnuA, but not TroA, undergoes conformational changes in the presence of Zn(2+). Using isothermal titration calorimetry (ITC), we demonstrated that TroA binds Zn(2+) and Mn(2+) with affinities approximately 100-fold greater than those previously reported. ITC analysis revealed that ZnuA contains multiple Zn(2+)-binding sites, two of which are high-affinity and presumed to be located within the binding pocket and His-rich loop. Quantitative reverse transcription polymerase chain reaction of tro and znu transcripts combined with immunoblot analysis of TroA and ZnuA confirmed that both transporters are simultaneously expressed in T. pallidum and that TroA is expressed at much greater levels than ZnuA. Collectively, our findings indicate that T. pallidum procures transition metals via the concerted utilization of its general metal (Tro) and Zn(2+) (Znu) transporters. Sequestration of periplasmic Zn(2+) by ZnuA may free up TroA binding capacity for the importation of Fe and Mn(2+).

[Influence of chronic epilepsy on spatial memory retrieval in rats].

OBJECTIVE: To investigate the influence of chronic epilepsy on spatial memory retrieval in rats, and to evaluate the effects of TAK-147, an acetylcholinesterase inhibitor, and histidine, the precursor of histamine, on the amnesia induced by epilepsy. METHODS: After successfully trained in the 8-arm (4-arm baited) radial maze, the rats were ip injected with a subconvulsive dose of pentylenetetrazole (PTZ) every 48 h until fully kindled. Memory retrieval was tested at the same maze. RESULT: Impairment of memory retrieval was in a steady state 1 to 18 days after fully kindled, the ability of memory retrieval returned to the control level 31 days after fully kindled. TAK-147 showed an ameliorating effect on memory impairment induced by epilepsy, including reference and working memory in a dose-dependent manner. Histidine only ameliorated reference but not working memory. CONCLUSION: PTZ-kindled seizure impair spatial memory retrieval, which it might be due to a decrease of brain acetylcholine and histamine induced by epilepsy.