[The cases of tracing the source of patients infected with Omicron variant of SARS-CoV-2 based on wastewater-based epidemiology in Shenzhen].

Wastewater-based epidemiology (WBE) is an emerging discipline, which has been applied to drug abuse tracking and infectious disease pathogen surveillance. During the COVID-19 epidemic, WBE has been applied to monitor the epidemic trend and SARS-CoV-2 variants etc. In order to detect hidden COVID-19 cases and prevent transmission in the community, wastewater surveillance system for monitoring SARS-CoV-2 RNA was developed in Shenzhen. The sewage sampling sites were set up in key places such as the port areas, urban villages and residential communities of Futian, Nanshan, Luohu and Yantian districts. From July 26 to November 30, 2022, a total of 369 sewage sampling sites were set up, covering 1.93 million people. Continuous sampling was carried out for 3 hours in the peak period of water use every day. Sewage virus enrichment and SARS-CoV-2 nucleic acid detection were carried out by polyethylene glycol precipitation method and RT-qPCR, and a positive water sample disposal process was molded. This article aims to introduce the case of source tracing of COVID-19 infected patients based on urban sewage in Shenzhen. The sewage monitoring of Honghu water treatment plant in Luohu District played an early warning role, and the source of infection was traced. In the disposal of positive water samples in Futian South Road, Futian District, the important experience of monitoring point layout was obtained. In the sewage monitoring of Nanshan village, Nanshan District, the existence of occult infection was revealed. Sharing the experience of tracing the source of COVID-19 patients to avoid the spread of COVID-19 in the community based on wastewater surveillance of SARS-CoV-2 RNA in Shenzhen, and summarizing the advantages and application prospects of sewage surveillance can provide new ideas for monitoring emerging or re-emerging pathogens that are known to exhibit gastrointestinal excretion in the future.

[Tumor Mechanomedicine].

Malignant tumors represent a significant health challenge, critically impacting human well-being. Historically, the focus has been on leveraging the biochemical cues of tumors for both diagnosis and treatment. While valuable, this strategy does not capture the full complexity of tumor diagnosis and management. Recently, the integration of biomechanics and mechanobiology with oncology has highlighted the importance of mechanical cues, which have emerged as new hallmarks of tumors, opening potential novel routes for cancer diagnosis and therapeutic interventions. Despite the advances, a thorough literature review suggests a pronounced gap in our understanding of the mechanical properties of tumors. The clinical community has not yet completely recognized the diagnostic and therapeutic relevance of the mechanical cues of tumors. To bridge this knowledge gap, we propose and introduce the paradigm of "Tumor Mechanomedicine". We provide a comprehensive overview of the multi-scale mechanical characteristics of tumors, exploring their influence on tumor biology, from the aspects of tumor biomechanics, tumor mechanobiology, tumor mechanodiagnostics, and tumor mechanotherapeutics. By elucidating the diagnostic and therapeutic potential of these mechanical cues, we aim to furnish the oncology community with fresh insights, paving the way for innovative solutions to persistent clinical conundrums.

[A case of poisoning caused by head and face bite of Deinagkistrodon acutus].

Snake bites kill and maim many people every year. Head and face venomous snake bite is rare, easy to misdiagnose and miss diagnosis, and the fatality rate is high. In this paper, 1 case of head and face venomous snake bite poisoning was reported and 10 similar cases were reviewed. The clinical characteristics of head and face venomous snake bite poisoning were summarized to provide guidance for clinical diagnosis and treatment. Head and face venomous snake bites may lead to airway injury, edema, and airway obstruction is the main cause of early death. Timely intubation or tracheotomy to maintain oxygen supply and early use of antivenin can improve prognosis.

[Radical vulvectomy and inguinal lymphadenectomy using a single incision for the treatment of vulvar malignancy].

Objective: To describe a novel procedure of radical vulvectomy and inguinal lymphadenectomy using a single incision (RVIL-SI) for the treatment of vulvar malignancy. Methods: In March, 2019, two cases affected with vulvar cancer (the first one is stage ⅢA squamous cell carcinoma and the second one is stage ⅠB with malignant melanoma) underwent this novel procedure, which was characterized by the combination of radical vulvectomy and bilateral inguinal lymphadenectomy without making additional incisions in groin areas. The boundaries of femoral triangle could be exposed perfectly using the initial incision of radical vulvectomy and the combined superficial and deep groin lymph node dissection were done subcutaneously from medial to lateral. Preoperative data and short term follow-up outcomes were collected. Results: The RVIL-SI was successfully conducted in two patients without any incisions of groin. The great saphenous veins were all spared. The operative time, average blood loss and median total regional lymph nodes of two cases were close. No major intraoperative complications occurred. Micrometastasis in one right superficial inguinal node was found in the first case with ipsilateral huge cancer lesion. No drain tube was left in inguinal areas intraoperatively. On postoperative day 3, the second case suffered mild lymphocele of right groin, which was resolved via repeated percutaneous needle puncture followed by elastic compression. Postoperative hospital stay of two cases were 10 and 11 days, respectively. With no skin complication at the time of writing this report. Conclusion: Our preliminary experience with the RVIL-SI has confirmed the reproducibility and minimal invasive therapeutic potential in the treatment for patients with vulvar cancer. But this novel procedure is in its infancy stage. Although short-term results are encouraging, a larger series with longer follow-up are required to fully evaluate the therapeutic efficacy.

Preliminary study of diffusion-weighted MRI in the preoperative diagnosis of cystitis glandularis.

AIM: To assess the efficacy of diffusion-weighted (DWI) magnetic resonance imaging (MRI) in distinguishing cystitis glandularis (CG) from bladder urothelial carcinoma. MATERIALS AND METHODS: Ultrasound, computed tomography (CT), conventional MRI, and DWI of 30 patients with histopathologically confirmed CG were analysed retrospectively and the imaging findings were correlated to the findings at histology. RESULTS: Ultrasound was non diagnostic in 11/18 and misdiagnosed malignancy in 7/18; CT was non diagnostic in 6/10 and misdiagnosed malignancy in 4/10; MRI was non diagnostic in 0 and misdiagnosed malignancy in 4/5 respectively. One patient with diffuse bladder wall thickening was correctly diagnosed as CG at MRI. All six patients who underwent additional DWI were accurately diagnosed as having CG with no or minimal reduction of diffusion. CONCLUSIONS: Diffusion is not reduced or shows minimal reduction in CG. DWI may aid the differential diagnosis of CG.

A replicating plasmid-based vector for GFP expression in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes porcine enzootic pneumonia (PEP) that significantly affects the pig industry worldwide. Despite the availability of the whole genome sequence, studies on the pathogenesis of this organism have been limited due to the lack of a genetic manipulation system. Therefore, the aim of the current study was to generate a general GFP reporter vector based on a replicating plasmid. Here, we describe the feasibility of GFP reporter expression in M. hyopneumoniae (strain 168L) controlled by the p97 gene promoter of this mycoplasma. An expression plasmid (pMD18-TOgfp) containing the p97 gene promoter, and origin of replication (oriC) of M. hyopneumoniae, tetracycline resistant marker (tetM), and GFP was constructed and used to transform competent M. hyopneumoniae cells. We observed green fluorescence in M. hyopneumoniae transformants under fluorescence microscopy, which indicates that there was expression of the GFP reporter that was driven by the p97 gene promoter. Additionally, an electroporation method for M. hyopneumoniae with an efficiency of approximately 1 x 10(-6) transformants/µg plasmid DNA was optimized and is described herein. In conclusion, our data demonstrate the susceptibility of M. hyopneumoniae to genetic manipulation whereby foreign genes are expressed. This work may encourage the development of genetic tools to manipulate the genome of M. hyopneumoniae for functional genomic analyses.

SNaPshot reveals high mutation and carrier frequencies of 15 common hearing loss mutants in a Chinese newborn cohort.

Genetic causes account for more than half of congenital hearing loss cases. The most frequent mutations found in non-syndromic hearing loss patients occur in GJB2 and SLC26A4. Mitochondrial genome mutations are also prevalent. However, the frequency of common hearing loss mutations in the Chinese population has not yet been well estimated. Here, we implemented the SNaPshot genotyping method to investigate the carrier frequency of 15 commonly reported hearing loss mutations in GJB2, SLC26A4 and the mitochondrial genome based on a cohort of 5800 neonates in China. Up to 15.9% (923/5800) of the newborns carry at least one mutant allele. The top three were GJB2-c.109G>A, GJB2-c.235delC, and SLC26A4-c.919A>G, with notably high carrier frequencies of 1/10, 1/53 and 1/62 respectively, and mt-7444G>A with 1/141 was the most frequent allele in the mitochondrial genome. In this cohort, 0.48% (28/5800) of neonates were genetically diagnosed with hearing loss, from which seven cases failed an OAE test. This is the first epidemiological study of non-syndromic hearing loss in Chinese newborns indicating a notably high carrier frequency (1 per 6.3 newborns) among these 15 mutant alleles. Our carrier frequency data also aid in effective risk assessment and genetic counseling for hearing loss patients in the Chinese population.

Function Clustering Self-Organization Maps (FCSOMs) for mining differentially expressed genes in Drosophila and its correlation with the growth medium.

The central task of this study was to mine the gene-to-medium relationship. Adequate knowledge of this relationship could potentially improve the accuracy of differentially expressed gene mining. One of the approaches to differentially expressed gene mining uses conventional clustering algorithms to identify the gene-to-medium relationship. Compared to conventional clustering algorithms, self-organization maps (SOMs) identify the nonlinear aspects of the gene-to-medium relationships by mapping the input space into another higher dimensional feature space. However, SOMs are not suitable for huge datasets consisting of millions of samples. Therefore, a new computational model, the Function Clustering Self-Organization Maps (FCSOMs), was developed. FCSOMs take advantage of the theory of granular computing as well as advanced statistical learning methodologies, and are built specifically for each information granule (a function cluster of genes), which are intelligently partitioned by the clustering algorithm provided by the DAVID_6.7 software platform. However, only the gene functions, and not their expression values, are considered in the fuzzy clustering algorithm of DAVID. Compared to the clustering algorithm of DAVID, these experimental results show a marked improvement in the accuracy of classification with the application of FCSOMs. FCSOMs can handle huge datasets and their complex classification problems, as each FCSOM (modeled for each function cluster) can be easily parallelized.

A rapid and sensitive loop-mediated isothermal amplification procedure (LAMP) for Mycoplasma hyopneumoniae detection based on the p36 gene.

The aim of this study was to establish a method for sensitive and rapid diagnosis of Mycoplasma hyopneumoniae in clinical specimens. To this effect, we employed three sets of primers specifically designed for amplification of nucleic acids under isothermal conditions. After optimization of reaction conditions, M. hyopneumoniae could be successfully detected at 63°C in 45 min through use of the loop-mediated isothermal amplification (LAMP) assay. A positive reaction was identified visually as white precipitate and confirmed by gel electrophoresis. The detection limit for this assay was 10 copies/µL, as observed by electrophoretic analysis. The accuracy of the LAMP reaction was confirmed by restriction endonuclease digestion as well as by direct sequencing of the amplified product. This method can specifically detect M. hyopneumoniae; other species with high homology and other bacterial and virus strains gave negative results. To test the utility of this procedure, the LAMP assay was applied to 40 clinical samples collected from swine lung tissues experimentally challenged with M. hyopneumoniae isolates, and compared to the results from a real-time polymerase chain reaction (PCR) assay. A concordance of 100% was observed between the two assays. In conclusion, the results from our study demonstrated that the LAMP assay provided a rapid reaction and was inexpensive to perform, with no need of complex instruments or systems such as Geneamp PCR. The LAMP assay may therefore be applied in routine diagnosis in the clinical laboratory and for in-field detection of M. hyopneumoniae infection.

Association between SNPs in genes involved in folate metabolism and preterm birth risk.

We investigated the association between 12 single nucleotide polymorphisms (SNPs) in 11 genes involved in folate metabolic and preterm birth. A subset of SNPs selected from 11 genes/loci involved in the folic acid metabolism pathway were subjected to SNaPshot analysis in a case-control study. Twelve SNPs (CBS-C699T, DHFR-c594+59del19, GST01-C428T, MTHFD-G1958A, MTHFR-C677T, MTHFR-A1298C, MTR-A2756G, MTRR-A66G, NFE2L2-ins1+C11108T, RFC1-G80A, TCN2-C776G, and TYMS-1494del6) in 503 DNA samples were simultaneously tested, and included 315 preterm births and 188 controls. None of the 12 SNP genotype distributions related to the folic acid metabolism pathway showed a significant difference between preterm and term babies. The frequency of the compound mutation genotype of MTHFD-G1958A, MTR-A2756G and RFC1-G80A in preterm babies was 7.3%, which was significantly higher than the 2.7% in term babies. Seven babies carried the compound mutation genotype of MTHFD-G1958A, MTR-A2756G, and CBS-C699T, but this was not observed in term babies. The frequency of the combined wild-type genotype of MTHFD-G1958A, MTR-A2756G, MTRR-A66G, MTHFR-A1298C, NFE2L2-ins1+C11108T, and RFC1- G80A in preterm babies was 3.17%, which was significantly lower than the 7.4% in term babies. The 12 SNPs screened in this study were not independent risk factors of preterm birth. Compound mutation genotypes, including MTHFD-G1958A, MTR-A2756G, and RFC1- G80A and MTHFD-G1958A, MTR-A2756G, and CBS-C699T, may increase the risk of preterm birth. The combined wild-type genotype MTHFD-G1958A, MTR-A2756G, MTRR-A66G, MTHFR-A1298C, NFE2L2-ins1+C11108T, and RFC1-G80A may decrease the risk of preterm birth.

Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections.

Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris.

Myostatin propeptide can inhibit the biological activity of myostatin protein and promote muscle growth. To express myostatin propeptide in vitro with a higher biological activity, we performed codon optimization on the sheep myostatin propeptide gene sequence, and mutated aspartic acid-76 to alanine based on the codon usage bias of Pichia pastoris and the enhanced biological activity of myostatin propeptide mutant. Modified myostatin propeptide gene was cloned into the pPIC9K plasmid to form the recombinant plasmid pPIC9K-Msp. Recombinant plasmid pPIC9K-Msp was transformed into Pichia pastoris GS115 by electrotransformation. Transformed cells were screened, and methanol was used to induce expression. SDS-PAGE and western blotting were used to verify the successful expression of myostatin propeptide with biological activity in Pichia pastoris, providing the basis for characterization of this protein.

Value of normalized apparent diffusion coefficient for estimating histological grade of vesical urothelial carcinoma.

AIM: To compare the efficacy of apparent diffusion coefficient (ADC) and normalized ADC (nADC) for estimating the histological grade of vesical urothelial carcinoma and to identify an optimal reference for nADC calculation. MATERIALS AND METHODS: Thirty patients with histologically confirmed vesical urothelial carcinomas underwent preoperative diffusion-weighted magnetic resonance imaging (DW-MRI) of the pelvis. nADC of the tumour was calculated as ADC (tumour)/ADC (reference) using urine in the bladder lumen, and the obturator internus and gluteus maximus muscles as reference. Receiver operating characteristic (ROC) curves were constructed and compared to identify an optimal reference for nADC calculation. RESULTS: Both ADC and nADC of low-grade tumours (1.112 ± 0.159 × 10(-3) mm(2)/s, 0.403 ± 0.047 × 10(-3) mm(2)/s) were significantly (p < 0.001) higher than those of high-grade tumours (0.772 ± 0.091 × 10(-3) mm(2)/s, 0.276 ± 0.033 × 10(-3) mm(2)/s). The area under the nADC ROC curve using urine as reference was significantly (p = 0.000) larger (0.995) than those using obturator internus (0.960) and gluteus maximus (0.945). CONCLUSIONS: nADC is superior to ADC for estimating the histological grade of bladder carcinoma using urine in the bladder lumen as an optimal reference for nADC calculation.

Comparative analysis of mucosal immunity to Mycoplasma hyopneumoniae in Jiangquhai porcine lean strain and DLY piglets.

The Jiangquhai porcine lean strain (JQHPL) is a new pork meat-type strain that has been developed in recent years from the parent lines Duroc, Fengjing, and Jiangquhai pigs (DurocxFengjing pigxJiangquhai pig). Enzootic pneumonia (EP) in pigs induced by Mycoplasma hyopneumoniae (M. hyopneumoniae) is a chronic respiratory disease of pigs, generating high economic losses in the swine industry. Here, we investigated the degree of resistance to M. hyopneumoniae for the Jiangquhai porcine lean strain and the Duroc x Landrace x Yorkshire (DLY) pigs, which are Western commercial pigs that have been introduced in China. A total of 209 DLY piglets and 221 JQHPL piglets from 19 Landrace x Yorkshire and 22 JQHPL M. hyopneumoniae positive gestating sows with different expected dates of confinement were selected and raised in the same M. hyopneumoniae positive farrowing barn. When the oldest suckling piglets were 37 days old, nasal swabs were collected from all the piglets (ranging from 4 to 37 days old) to detect the M. hyopneumoniae pathogen using n-PCR and M. hyopneumoniae specific SIgA using ELISA. Positive M. hyopneumoniae infection rates in both the strains increased with age; however, positive rates for JQHPL were lower compared to DLY at 14 to 35 days old. The level of the specific SIgA rose rapidly in JQHPL respiratory tracts, particularly in piglets 21 to 35 days in age compared to DLY piglets of the same age; however, the level of the specific SIgA in DLY also marginally increased. In conclusion, JQHPL pigs exhibits higher resistance to M. hyopneumoniae compared to DLY. It is possible that this characteristic is caused by the faster and stronger mucosal immunity phenotype of the JQHPL strain.

Spironolactone prevents dietary-induced metabolic syndrome by inhibiting PI3-K/Akt and p38MAPK signaling pathways.

OBJECTIVES: Aim of the study is to evaluate the impact of spironolactone (SPL) on indexes of metabolic syndrome (MS) and further investigate the mechanisms underlying its protective effects. METHODS: A rat model of MS was established by administering a fat- and salt-enriched diet (FS diet). The occurrence of MS was examined by measurement of blood pressure (BP), aldosterone (ALD) content, blood lipid (BL), glucose and insulin levels. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Pancreatic gland tissue injury was assessed by ß-cell apoptosis. Mineralocorticoid receptor (MR) activity, phosphatidylinositol 3- kinase/Akt (PI3-K/Akt), and phosphorylation of p38MAPK (Pp38MAPK) in pancreatic gland tissue were evaluated by western blot analysis. RESULTS: SPL prevented hypertension, and dyslipidemia during MS induced by the intake of FS diet, but had no effect on K+ and Na+ disturbances. Furthermore, SPL significantly attenuated ALD and MR expression levels after FS diet. Finally, SPL inhibited phosphorylation protein kinase B (p- PKB) activation in the pancreatic gland tissue, a downstream target of PI3-K, and phosphorylation of p38MAPK pathway, critical for cellular apoptosis. CONCLUSIONS: This study demonstrates that SPL exerts a protective effect on hypertension and dyslipidemia. This protective effect may depend, at least in part, on MAPK and PI3-K pathways.

[Effects of glucocorticoids on the proliferation of CD19 CAR-T cells targeting B-cell tumor cell lines].

Objective: To evaluate the effects of glucocorticoids (dexamethasone and methylprednisolone) on the proliferation of CD19 Chimeric antigen receptor (CAR) modified T cells in vitro. Methods: Peripheral blood mononuclear cells from healthy volunteers were collected as T cells. CD19 CAR-T cells were prepared by CD3 magnetic beads sorting and CD19 CAR lentivirus transfection. The transfection rates and the proportion of CD19 CAR-T cells in the culture system were analyzed using a flow cytometer. The mean fluorescence intensity (MFI) of CD19 CAR-T cells was measured after staining with Carboxyfluorescein diacetate succinimidyl ester cell proliferation tracer fluorescent probe, Lactate dehydrogenase (LDH) cytotoxicity assay was used to detect the effects of different concentrations of glucocorticoid on the killing activity of B-cell tumor cell lines. Results: In this study, the CD19 CAR transfection rate of CD19 CAR-T cells was (51.34±5.28) %. The killing activities of different doses of methylprednisolone on Nalm6, Pfeiffer, and U2932 tumor cells were higher than that of dexamethasone at 24 h. The killing activities of 4 mg/mL methylprednisolone on Nalm6, Pfeiffer, and U2932 were higher than that of 0.75 mg/ml group, while the killing activity of 12 mg/ml methylprednisolone was lower than that of 2.25 mg/ml dexamethasone at 48 h. However, the killing activities of different doses of methylprednisolone on EHEB tumor cells were lower than those of different doses of dexamethasone at 24 and 48 h. The average MFI and proportion of CD19 CAR-T cells under different concentrations of glucocorticoid the proliferation inhibition of CD19 CAR-T cells by dexamethasone was higher than that of methylprednisolone. The proliferation inhibition of CD19 CAR-T cells of the two glucocorticoids in high concentration groups were more obvious than that in low concentration groups. When CD19 CAR-T cells were co-cultured with different tumor cells, the proportion and average MFI of CD19 CAR-T cells showed that the proliferation inhibition of dexamethasone was higher than that of methylprednisolone. The proliferation inhibition of CD19 CAR-T cells of the two glucocorticoids in high concentration groups was more obvious than that in low concentration groups. Conclusion: Dexamethasone inhibits the cell proliferation of CD19 CAR-T cells more than methylprednisolone during the targeting of different tumor cell lines. The inhibition effect of dexamethasone on the proliferation and amplification of CD19 CAR-T cells was greater than that of methylprednisolone during the targeting of CD19 CAR-T cells to different tumor cell lines. Moreover, the inhibition effect of the high dose group was more obvious.

[In vitro studies on the transfer of CAR into leukemia cells due to their residue in the autologous CAR-T cell preparation system for acute B-cell acute lymphoblastic leukemia].

Objective: To investigate the characteristics and cytotoxicity in vitro of the residual leukemia cells in the culture system that caused the accidental transfer of CD19 chimeric antigen receptor (CAR) into leukemia cells during the preparation of autologous CD19 CAR-T cells of relapsed/refractory B-cell acute lymphoblastic leukemia. Methods: ①Peripheral blood mononuclear cells (PBMC) of 30 patients with relapsed/refractory B-cell acute lymphoblastic anemia (R/R B-ALL) who accepted CD19 CAR-T cell therapy and six healthy volunteers were collected. ②The residual leukemia cells were analyzed by flow cytometry in the system after the PBMCs of R/R B-ALL patients were sorted by CD3 magnetic beads. ③ CD3(+) T cells from patients and healthy volunteers were transfected with CD19 CAR and CD22 CAR lentivirus to prepare CD19 CAR-T and CD22 CAR-T cells. ④The Nalm-6 cell line was resuscitated and the Nalm-6 cells with CD19 CAR lentivirus were transfected to prepare CD19 CAR-Nalm-6 cells. The patient's primary ALL cells were transfected with CD19 CAR lentivirus at the same time. ⑤The transfection rates were analyzed by flow cytometer, the cell proliferation was analyzed by the CCK-8 method, and the cell-killing activities were detected by the lactate dehydrogenase method. Results: ① Among the 30 R/R B-ALL patients who received CD19 CAR-T cell therapy, two patients had 2.04% and 3.32% residual leukemia cells in CD3(+) T cells. After 4 days in culture, the residual leukemia cells disappeared and could not be detected by a flow cytometer with prolonged cultivation in vitro. ② The proliferation of CD19 CAR-Nalm-6 cells was higher than that of the Nalm-6 cells. ③ The killing activity of the CD19 CAR-T cells on Nalm-6 cells was higher than that of the CD19 CAR-Nalm6 cells at a target ratio of 1∶1 on 24, 48, 72 h, respectively. The cytotoxicity of CD22 CAR-T cells on CD19 CAR-Nalm-6 cells was significantly higher than that of CD19 CAR-T cells. ④ The cytotoxicity of CD22 CAR-T alone on CD19 CAR-Nalm-6 cells was higher than that of CD19 CAR-T combined with CD22 CAR-T at the same target ratio. Conclusion: The residual leukemia cells in the culture system in the preparation of CD19 CAR-T cells may lead to the introduction of CD19 CAR into leukemia cells and results in the failure of the CD19 CAR-T cell therapy. Detecting the residual leukemia cells in the culture system via flow cytometry before transfection with CD19 CAR lentivirus is needed. Thus, CD22 CAR-T cell therapy could be used as one of the salvage treatments.

[Analysis of local reactions and efficacy of CD19 chimeric antigen receptor-modified T cells therapy in recurrent/refractory B-cell lymphoma with >7.5 cm lesions].

Objective: To observe the local reactions and efficacy of CD19 CAR-T therapy in recurrence/refractory B-cell non-Hodgkin's lymphoma (R/R NHL) patients with >7.5 cm lesions. Methods: 32 R/R NHL patients with >7.5 cm lesions were enrolled and injected with CD19 CAR-T cells. Flow cytometry was used to detect and observe the amplification of CD19 CAR-T cells in vivo. Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines in peripheral blood of patients. The side effects of CD19 CAR-T cell therapy included systemic side effects and local reactions of tumor. The local side effects were observed by Ultrasound, Computed tomography and Magnetic resonance imaging. Treatment options included glucocorticoid, interleukin-6 antibody and drainage of exudate. Overall response rate (ORR) and overall survival rate (OS) were observed. Results: ①Among the 32 patients, CR (40.63%) , PR (31.25%) and ORR (71.88%) were 13, 10 and 23, respectively. ②In all 23 patients received ORR, 13 patients had grade 1-2 CRS, while 10 patients had grade 3-4 CRS. All the 9 patients in the SD+PD group had grade 1-2 CRS (P=0.030) . ③A total of 15 patients with tumor local reactions, included 9 patients with CR, 5 patients with PR and 1 patient with SD. The local reactions of the tumor included that the diameter of the superficial lesions increased with redness, swelling and heat pain. The deep lesions presented abdominal pain, abdominal distension, suffocation and local pain, and burning of the tumor. The deep lesions were enlarged or accompanied by local edema. The local exudative lesions were found in the abdominal cavity and pleural cavity. ④ Peak proportion of CD19 CAR-T cells in ORR group was higher than that of in SD+PD group[16.8% (5.3%-48.2%) vs 2.9% (1.5%-5.7%) , z=-4.297, P<0.001]. The peak proportion of CD19 CAR-T cells in ORR group with local reactions was higher than that of in patients without local reactions [22.2% (10.5%-48.2%) vs 12.6% (5.3%-21.6%) , z=-3.213, P=0.001]. The peak proportion of CD19 CAR-T cells in multiple lesion group was higher than that of in single lesion group [35.8% (1.5%-48.2%) vs 16.8% (10.5%-18.5%) , z=-2.023, P=0.040]. ⑤Occurrence of local reactions and tumor shrinkage time were both delayed compared with systemic side effects. ⑥In the ORR group, the OS of patients with tumor local reactions was longer than that of patients without tumor local reactions, but there was no difference in the two groups (75% vs 34.6%, P=0.169) . Conclusions: CD19 CAR-T cell therapy in R/R NHL patients with >7.5 cm lesions might cause tumor local reactions later than systemic side effects. Clinicaltrial:: ChiCTR1800018059.