ABSTRACT
Objective:To investigate the molecular mechanism of transcytosis of Leptospira interrogans ( L. interrogans) across vascular endothelial cells. Methods:Transwell assay was performed to observe the ability of L. interrogans strain Lai across the monolayer of human vascular endothelial cells (HUVEC). Transmission electron microscopy and laser confocal microscopy were used to detect the endocytic vesicles containing L. interrogans strain Lai in HUVEC. The leptospiral endocytic pathway was determined by endocytic inhibition test. Laser confocal microscopy was also used detect the co-localization of L. interrogans with lysosomal marker LAMP1 in HUVEC. The exocytosis of L. interrogans from HUVEC was detected using Petroff-Hausser counting chamber and darkfield microscopy. Results:L. interrogans strain Lai could rapidly transmigrate through HUVEC monolayers and be internalized into HUVEC by PI3K-microfilament-dependent endocytosis to form leptospiral endocytic vesicles. The internalized L. interrogans did not co-localize with LAMP1, indicating the leptospiral endocytic vesicles did not fuse with lysosomes. The exocytosis of internalized L. interrogans was through FAK-microfilament/microtubule pathway. Conclusions:L. interrogans strain Lai could transmigrate through HUVEC by transcytosis to diffuse in vivo and cause disease aggravation.
ABSTRACT
Objective To analyze the role of paroxysmal nocturnal hemoglobinuria (PNH) clones in children with acquired aplastic anemia (AA). Methods The relationship between the existence of PNH clones and clinical features in children with AA was retrospectively analyzed. The influence of PNH clones on the efficacy of combined immunosuppressive therapy (IST) of anti-thymocyte globuline (ATG) and cyclosporine A (CSA) was also observed. In addition, multiple factor analysis was used to analyze the main factors affecting the efficacy of AA. Results One hundred and forty-eight children with AA were enrolled, including 74 cases (50%) of granulocyte PNH clones positive, 68 cases (45.9%) of monocyte PNH clones positive, and 93 cases (62.8%) of total PNH clones (granulocytes and / or monocytes) positive. In 49 children having both granulocytes and monocytes PNH clones, the clone size of monocytes and granulocytes was 0.7% (0.4%-1.5%) and 0.2% (0.1%-0.7%), respectively, and the difference was significant (P<0.001) and there was a significantly positive correlation between them (r=0.65, P<0.001). According to the different PNH positive clones (monocytes, granulocytes, total), children were divide into three groups. And there were no differences in gender, age, concurrent infection, white blood cell count, hemoglobin concentration, platelet count, neutrophil absolute count, reticulocyte percentage in different PNH clones positive and negative groups (P>0.05). The group with monocytes PNH clones positive had a positive effect on the efficacy of IST (P=0.02). Multiple factor logistic regression analysis showed that the concentrations of hemoglobin and the positive PNH clones of monocytes were the main factors affecting the efficacy (P<0.05). Conclusions The high concentration of hemoglobin and the positive PNH clones of monocytes contribute the better effect of IST in children with AA.
ABSTRACT
Objective To establish a flow cytometric measurement of detecting minimal residual disease(MRD) according to the leukemia-associated immunophenotypes in children with acute lymphoblastic leukemia(ALL) and to explore the significance of MRD detection in ALL children for a individualized treatment. Methods A variety of four-color fluorescent antibody combinations were used to investigate the children's normal bone marrow. The normal bone marrow pattern at two-parameter plots was established to identify the residual tumor cells, seventy-five bone marrow samples from newly diagnosed ALL children were analyzed with four-color cytometry to determined the optimal combinations which can clearly distinguish the tumor cells from normal cells. The bone marrow samples were monitored with the combination panel in 60 patients at the end of induction therapy and follow-up treatment. Cytomorphology test, PCR amplification of 29 fusion genes as well as IgG and TCR gene rearrangements were performed simultaneously. Results Sixty-nine cases (92.0%) could be identified for effective antibody combinations to monitor MRD by four-color cytometry. Fusion genes or IgG and T cell receptor (TCR) gene rearrangements can be detected in 21 cases (28.0%) to monitor MRD by PCR. No MRD can be detected in 25 bone marrow samples at the end of induction therapy and follow-up treatment. Four-color cytometry could detect as low as 0.021%-4.130% residual leukemia cells. Conclusion MRD can be monitored by flow cytometry which is faster than PCR, and the sensitivity is superior to morphology method.