[Safety and efficacy of humanized CD19-targeted CAR-T cells in patients with relapsed/refractory acute B cell lymphoblastic leukemia].

Objective: This study aimed to evaluate the safety and efficacy of humanized CD19-targeted chimeric antigen receptor T-cell (CAR-T) in patients with relapsed/refractory acute B cell lymphoblastic leukemia (R/R B-ALL) . Methods: The clinical data of 41 patients with R/R B-ALL treated with humanized CD19-targeted CAR-T cells in the First Affiliated Hospital of Zhejiang University School of Medicine from February 2020 to July 2021 were analyzed. Results: Cytokine release syndrome occurred in all patients, and 63.4% (26/41) were grades 1-2. Immune effector cell-associated neurotoxicity syndrome developed in three patients. On median day 15 (9-47) , the complete remission rate was 95.1% (39/41) , of which 38 patients tested negative for bone marrow minimal residual disease detected by flow cytometry. Among the 39 patients with complete remission, 17 patients did not receive further treatment, and 70.6% (12/17) remained in remission at the end of follow-up, with a progression-free survival of 11.6 months of the two patients with the earliest infusion. Another 17 patients underwent consolidation allogeneic hematopoietic stem cell transplantation (10 cases) or CD22 CAR-T cell sequential therapy (seven cases) after remission, and 76.5% (13/17) of the patients were still in remission at the end of follow-up. The remaining five patients who did not receive consolidation therapy relapsed at a median of 72 (55-115) days after CAR-T cell therapy. Conclusion: In patients with R/R B-ALL, the humanized CD19-targeted CAR-T cells had a high response and manageable toxicity.

A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein.

The QM gene that encodes for the ribosomal protein L10 was firstly identified from human tumour cells as a tumour suppressor. In this study, a QM gene was identified in silkworm Bombyx mori (BmQM) and its immunomodulatory function was explored. BmQM messenger RNA (mRNA) and protein were highly expressed in the silk gland and fat body, and expressed in all stages of silkworm growth. After challenged with four different microorganisms, the expression levels of BmQM mRNA in fat body or haemocytes were significantly upregulated compared with the control. After knock-down of BmQM gene, the expressions of some immune genes (PGRPS6, Gloverin0, Lysozyme and Moricin) were affected, and the transcripts of prophenoloxidase1 and prophenoloxidase2 have different degrees of change. The phenoloxidase activity was significantly reduced when the purified recombinant BmQM protein was injected. Recombinant BmQM protein inhibited systemic melanization and suppressed prophenoloxidase activation stimulated by Micrococcus luteus, but it did not affect phenoloxidase activity. Far-western blotting assays showed that the BmQM protein interacted with silkworm BmJun protein, which negatively regulates AP-1 expression. Our results indicated that BmQM protein could affect some immune gene expression and negatively regulate the prophenoloxidase-activating system, and it may play an important role in regulation of the innate immunity in insects.

[Jinghuaweikang capsules combined with Quadruple therapy in the treatment of Helicobacter pylori infection:a multicenter,randomized,controlled,clinical study].

Objective: To explore the efficacy of Jinghuaweikang capsules combined with Quadruple therapy in the treatment of Helicobacter pylori (H.pylori)infection. Methods: Patients who were infected with H.pylori in 7 centers in Gansu Province were recruited in this prospective simple randomized study. All the patients are divided into four groups randomly: patients in Group A1 were treated with esomeprazole (20 mg, twice a day) + pectin bismuth (200 mg, three times a day) + amoxicillin (1 000 mg, twice a day) + clarithromycin (500 mg, twice a day), while Group A2 with Jinghuaweikang capsules(160 mg, three times a day) based on group A2, Group B1 with esomeprazole (20 mg, twice a day) + bismuth pectin (200 mg, three times a day) + amoxicillin (1 000 mg, twice a day) + furazolidone (100 mg, twice a day) and Group B2 with Jinghuaweikang capsules(160 mg, three times a day) based on group B2. The treatment time was 14 days for all 4 groups. In the course of treatment, abdominal pain, acid reflux, abdominal distension, belching, hiccups were observed at the time before treatment, 14 days and 30 days after treatment and were scored. Finally, all patients received (13)C or (14)C for H.pylori at the time of 30 days after the treatment. Result: A total of 455 patients were included in 7 hospitals from February 2016 to May 2017 in Gansu province, and there were 189 male patients. Group A1 included 129 cases, group A2 96 cases, group B1 112 cases and group B2 118 cases. The eradication rates that accorded with program data analysis (PP) were A1[46.9%(60/128)], A2[63.8%(60/94)], B1[60.7%(68/112)], B2[68.6%(81/118)] (P<0.004). Compared with group A1, the eradication rate of H.pylori in group B1 and group A2 increased (P<0.001, P=0.032), there was no statistical difference between group B2 and group A2, group B1 and group B2 (P=0.208, P=0.461). According to intentional analysis (ITT), the eradication rates of H.pylori in group A1 were 46.5% (60/129),group A2 were 62.5% (60/96),group B1 were 60.7% (68/112),and group B2 were 68.6% (81/118).The radical rate of A2 was higher than A1 (P=0.017), group B2 was not higher than group B1 (P=0.208), and there was no significant difference among the other groups. The symptoms of abdominal pain, abdominal distention, acid reflux, belching and hiccup in the group A2 and group B2 were improved than those in group A1 and group B1 (P<0.05). No serious adverse reactions occurred in all groups. Conclusion: Jinghuaweikang capsules can improve the eradication rate of Helicobacter pylori, and improve the symptoms of patients.

[The effect of WT1 expression on the prognosis of allogeneic hematopoietic stem cell transplantation in acute leukemia].

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) , χ(2)=4.086, P=0.043; 53.8% (7/13) vs 87.5% (56/64) , χ(2)=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.

Molecular cloning, expression, and characterization of E2F transcription factor 4 from Antheraea pernyi.

The E2F transcription factor family is distributed widely in eukaryotes and has been well studied among mammals. In the present study, the E2F transcription factor 4 (E2F4) gene was isolated from fat bodies of Antheraea pernyi and sequenced. E2F4 comprised a 795 bp open reading frame encoding a deduced amino acid sequence of 264 amino acid residues. The recombinant protein was expressed in Escherichia coli (Transetta DE3), and anti-E2F4 antibodies were prepared. The deduced amino acid sequence displayed significant homology to an E2F4-like protein from Bombyx mori L. Quantitative real-time polymerase chain reaction analysis revealed that E2F4 expression was highest in the integument, followed by the fat body, silk glands, and haemocytes. The expression of E2F4 was upregulated in larvae challenged by bacterial (Escherichia coli, Micrococcus luteus), viral (nuclear polyhedrosis virus), and fungal (Beauveria bassiana) pathogens. These observations indicated that E2F4 is an inducible protein in the immune response of A. pernyi and probably in other insects.

Characterization and functional analysis of serpin-1 like gene from oak silkworm Antheraea pernyi.

Serpins are a broadly distributed family of proteases found in various organisms that play an important role in regulating the immune response. Here, we identified a serpin-1 gene from Antheraea pernyi that encodes a 279 amino acid protein with a molecular weight of 30.8 kDa. We expressed the recombinant Ap-serpin-1 protein in Escherichia coli and used the purified protein to prepare rabbit anti-Ap-serpin-1 polyclonal antibodies. We calculated the enzyme-linked immunosorbent assay titer of the antibody as 1:128000. Quantitative real-time polymerase chain reaction analysis revealed that Ap-serpin-1 was expressed in all examined tissues, including hemolymph, malpighian tubules, midgut, silk gland, integument and the fat body; the highest Ap-serpin-1 expression levels was detected in the fat body. We next investigated the expression patterns of Ap-serpin-1 in both fat body and hemolymph samples, following treatment with E. coli, Beauveria bassiana, Micrococcus luteus and nuclear polyhedrosis virus (NPV). We reported that NPV and M. luteus significantly enhanced Ap-serpin-1 expression in the fat body. While, in the hemolymph samples, treatment with B. bassiana and M. luteus was shown to upregulate Ap-serpin-1 expression at 24 h induction. Altogether, our results suggest that Ap-serpin-1 is involved in the innate immunity of A. pernyi.

Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.

PTEN inactivation by germline/somatic c.950_953delTACT mutation in patients with Lhermitte-Duclos disease manifesting progressive phenotypes.

Lhermitte-Duclos disease (LDD), a neurological manifestation of Cowden syndrome (CS), is a rare and benign cerebellar disorder, featured by dysplastic cerebellar ganglion cells which replace granular and Purkinje cells. Phosphatase and Tensin Homolog (PTEN) is confirmed as the susceptibility gene for CS which represents the most complex features and is not easily recognizable. We reported two index patients with LDD diagnosed either in an isolated form or coexist with CS. These two patients displayed progressive though comparable phenotypes and were found to carry an identical PTEN c.950_953delTACT mutation in either germline or somatic sources of DNA, respectively. Negative or moderate expression levels of PTEN were validated by immunohistochemistry in the corresponding patients' affected tissues. This study has revealed a novel pathogenicity locus to LDD/CS as a candidate for early molecular diagnosis. In addition, the differential PTEN mutation status with corresponding LDD phenotypes suggests a potential correlation between germline or somatic mutation and coexisting LDD/CS or isolated LDD, respectively. Furthermore, our data could lend some reference to the underlying molecular mechanism of LDD pathogenesis in the future.

Linkage and mapping analyses of the normal marking gene +P in the silkworm (Bombyx mori) using SSR markers.

In the silkworm, Bombyx mori, normal markings are mainly controlled by the +P gene, which is located on the second chromosome. Due to a lack of crossing over in females, reciprocal backcrossed F1 (BC1) progenies were used for linkage analysis and mapping of the +P gene based on an SSR linkage map using silkworm strains P50 and H9, which are normal marking and sex-limited marking, respectively. The +P gene was found to be linked to 3 SSR markers. Using a reciprocal BC1M cross, we constructed a linkage map of 22.5 cM, with +P mapped at 11.3 cM and the nearest SSR marker S0206 at a distance of 3.0 cM. Based on a fine genome map of domesticated silkworms, Kaikoblast analysis showed that the physical distance between the nearest markers (containing the +P gene) is 995 kb. Further analysis showed that BGIBMGA009689, BGIBMGA009688, and BGIBMGA009687 are closer to +P, and that BGIBMGA009689 is closest to +P, with a physical distance of 19.1 kb.

Relationship between TNFA, TNFB and TNFRII gene polymorphisms and outcome after unrelated hematopoietic cell transplantation in a Chinese population.

This study aimed to analyze the association between cytokine gene polymorphisms and outcome following allogeneic hematopoietic SCT (allo-HSCT). A total of 138 unrelated donor/recipient pairs who underwent allo-HSCT from 2001 to 2009 were tested for TNFA-1031 (T>C), -863 (C>A), -857 (C>T), -238 (G>A), TNFB+252 (A>G) and TNFRII codon 196 (T>G) single nucleotide polymorphisms by multiplex SnaPshot analysis. Transplantation involving recipients and/or donors with TNFA-857 C/C genotype or TNFB+252 G allele-positivity resulted in a higher incidence of acute GVHD (aGVHD), which was independent of HLA mismatching. In multivariate analysis, TNFA-857 C/C genotype donors (relative risk (RR)=2.29, P=0.006) and TNFB+252 G allele-positive recipients (RR=1.789, P=0.036) were found to be significantly associated with an increased incidence of aGVHD. TNFA-857 C/C genotype donors (RR=3.748, P=0.002) and TNFB+252 G allele-positive recipients (RR=1.823, P=0.063) were also associated with the development of grades II-IV aGVHD. TNFRII polymorphism in recipients was also related to relapse rate, but no significant associations were found between TNFA, TNFB or TNFRII 196 genotype and cGVHD, relapse or overall survival after transplantation. These results provide the first report of an association between TNFA, TNFB and TNFRII polymorphic features and outcome of allo-HSCT in a Chinese population, and suggest an interaction between TNFA-857 and TNFB+252 genotypes and risk of aGVHD.

A rat model of chronic allograft liver rejection.

INTRODUCTION: The aim of this study was to develop a rat model of chronic irreversible rejection, which is a major causes of late graft loss and retransplantation after orthotopic liver allotransplantation. METHODS: Allogeneic liver transplantation was performed in a rat combination of Dark Agouti (DA) to Brown Norway (BN). Group A was left without treatment, group B received cyclosporine' (CsA; 1 mg/kg/d) and group C, CsA (4 mg/kg/d). Animals were followed for 6 months. Liver tissue was harvested to construct a time course of histological changes after liver transplantation using histopathological and morphometric techniques. We compared the total histological score of rejection activity index and survival rates. RESULTS: In untreated animals, irreversible acute rejection developed, all animals died within 15 days. In the low-dose CsA group, all animals that survived more than 30 days developed moderate to severe manifestations of chronic liver rejection, with graft infiltration, ductular damage or proliferation, obliterative arteriopathy, and liver fibrosis. No apparent histological alterations were observed in group C. Survival analysis showed significant differences between the three groups. CONCLUSIONS: In the rat strain combination of DA --> BN with low-dose immunosuppression, early mild inflammation was followed by the development of chronic rejection.

Self-guided robotic camera control for laparoscopic surgery compared with human camera control.

BACKGROUND: In laparoscopic surgery, the surgeon no longer has direct visual control of the operation area, and a camera assistant who maneuvers the laparoscope is essential. Problems of cooperation between the two naturally arise, and a robotic assistant that automatically controls the laparoscope can offer a highly desirable alternative to this situation. METHODS: A self-guided robotic camera control system (SGRCCS) based upon a color tracking method has been developed and its use evaluated in 20 cases of laparoscopic cholecystectomy and compared with using human camera control. RESULTS: In 83% of the patients the procedures were successfully completed with the SGRCCS. Set-up time for the robot averaged 21 minutes; and the surgical time with and without the robot averaged 54 and 60 minutes, respectively. Using the robot instead of a human camera assistant significantly reduced both the frequency of the camera correction, 2.2 per hour compared with 15.3 per hour, and frequency of the lens cleaning, 1.0 per hour compared with 6.8 per hour. Subjective assessment by the surgeon revealed that the robot performed better than the human assistant in 71 % of the cases. CONCLUSIONS: In laparoscopic surgery, the SGRCCS offered optimal camera guidance and helped to maintain the surgeon's concentration during the operation.

Multisensory visual servoing by a neural network.

Conventional computer vision methods for determining a robot's end-effector motion based on sensory data needs sensor calibration (e.g., camera calibration) and sensor-to-hand calibration (e.g., hand-eye calibration). This involves many computations and even some difficulties, especially when different kinds of sensors are involved. In this correspondence, we present a neural network approach to the motion determination problem without any calibration. Two kinds of sensory data, namely, camera images and laser range data, are used as the input to a multilayer feedforward network to associate the direct transformation from the sensory data to the required motions. This provides a practical sensor fusion method. Using a recursive motion strategy and in terms of a network correction, we relax the requirement for the exactness of the learned transformation. Another important feature of our work is that the goal position can be changed without having to do network retraining. Experimental results show the effectiveness of our method.

Learning shape from shading by a multilayer network.

The multilayer feedforward network has often been used for learning a nonlinear mapping based on a set of examples of the input-output data. In this paper, we present a novel use of the network, in which the example data are not explicitly given. We consider the problem of shape from shading in computer vision, where the input (image coordinates) and the output (surface depth) satisfy only a known differential equation. We use the feedforward network as a parametric representation of the object surface and reformulate the shape from shading problem as the minimization of an error function over the network weights. The stochastic gradient and conjugate gradient methods are used for the minimization. Boundary conditions for either surface depth or surface normal (or both) can be imposed by adjusting the same network at different levels. It is further shown that the light source direction can be estimated, based on an initial guess, by integrating the source estimation with the surface estimation. Extensions of the method to a wider class of problems are discussed. The efficiency of the method is verified by examples of both synthetic and real images.

[Dilatation of stricture of anastomosis after reconstruction of esophagus].

This article reports the experience of the esophagus dilating technique in the treatment of 658 cases of postoperative anastomotic stricture for esophageal cancer and cardiac cancer during the period from May 1983 to May 1993, being delated add up to 3240 times with total effective rate 96.5%, among them 61.6% of the cases less than five, 84.5% of the cases less than ten. Of them, 27 cases (4.1%) had postoperative complications, of which the main ones were esophagus perforation 8 cases (1.2%), hemorrhage of anastomotic stoma 4 cases (0.6%), mediastinitis 1 case (0.2%) and loca trauma 14 cases (2.1%). The operative indication, surgical technique and complication were discussed.

[Prognostic evaluation of lymph node metastasis in thoracic esophageal cancer--an analysis of 212 cases].

The authors studied retrospectively lymph node metastatic status impacts on survival of the 212 patients with thoracic esophageal squamous cell carcinoma and lymph node metastasis after curative resection. 663 (19.4%) of the total 3419 lymph nodes examined (an average of 16.1 per patient) were proved to be positive. The overall 5-year survival rate was 19.3%. The results showed that no difference in survival was observed in relation to the site of the involved lymph node. Difference in survival based on the number of metastatic lymph nodes (1 or > or = 2) and the frequency of positive nodes (< or = 10% or > 10%). The results indicate that staging of esophageal cancer should be made according to the absolute number and the relative frequency of lymph nodes involved. The effectiveness and limitation of extended lymph node dissection in relation to prognosis was discussed.

[A randomized clinical study of preoperative accelerated radiotherapy versus routine radiotherapy for late esophageal cancers--an analysis of 177 cases].

From March 1989 to Oct. 1993, 177 patients with esophageal cancer were treated with preoperative radiotherapy. They were randomly divided into two groups: preoperative routine radiotherapy (RR) group and accelerated radiotherapy (AR) group. In the former group, radiation was given 2 Gy/day, 5 sessions/week with a total of 40Gy in 4 wks, while in the AR group, radiation was given 2 Gy/session, two sessions/day at an interval of 6-8 hours, 10 sessions/week for 2 weeks with a total dose of 40 Gy in 2 weeks. The patients were operated 3-4 weeks after radiation. The resectability rate was 95% in AR group, and 90% in RR group. The 1-, 2-, 3- and 4-year survival rates in AR group were 89%, 85%, 69% and 60%, while 80%, 65%, 48% and 41% in RR group respectively. The degree of tumor regression examined histopathologically was: Grade I in 9%, Grade II in 41% and Grade III in 50% of AR-treated patients, while in 34%, 39%, 27% of RR-treated patients. Periesophageal lymph node metastasis was 13% in AR group, while 24% in RR group. The results of AR are better than that of RR indicate that preoperative accelerated radiation gave better therapeutic results than routine radiotherapy.

[Surgical treatment of recurrent esophageal carcinoma after curative radiotherapy].

34 patients with recurrent esophageal carcinoma received surgery after curative radiotherapy. The overall resectability was 82.3%, and anastomotic leakage pulmonary complication, recurrent laryngeal nerve paralysis, empyema occurred in 32%. The operative mortality was 17.8%, four of the five postoperative deaths were due to pulmonary complication. The 1-, 3-, 5-year survival rates were 71.4%, 36.3%, 25% respectively. Surgical technique, indications and operative factors were discussed.