A SARS-CoV-2-specific CAR-T-cell model identifies felodipine, fasudil, imatinib, and caspofungin as potential treatments for lethal COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cytokine storm is closely associated with coronavirus disease 2019 (COVID-19) severity and lethality. However, drugs that are effective against inflammation to treat lethal COVID-19 are still urgently needed. Here, we constructed a SARS-CoV-2 spike protein-specific CAR, and human T cells infected with this CAR (SARS-CoV-2-S CAR-T) and stimulated with spike protein mimicked the T-cell responses seen in COVID-19 patients, causing cytokine storm and displaying a distinct memory, exhausted, and regulatory T-cell phenotype. THP1 remarkably augmented cytokine release in SARS-CoV-2-S CAR-T cells when they were in coculture. Based on this "two-cell" (CAR-T and THP1 cells) model, we screened an FDA-approved drug library and found that felodipine, fasudil, imatinib, and caspofungin were effective in suppressing the release of cytokines, which was likely due to their ability to suppress the NF-κB pathway in vitro. Felodipine, fasudil, imatinib, and caspofungin were further demonstrated, although to different extents, to attenuate lethal inflammation, ameliorate severe pneumonia, and prevent mortality in a SARS-CoV-2-infected Syrian hamster model, which were also linked to their suppressive role in inflammation. In summary, we established a SARS-CoV-2-specific CAR-T-cell model that can be utilized as a tool for anti-inflammatory drug screening in a fast and high-throughput manner. The drugs identified herein have great potential for early treatment to prevent COVID-19 patients from cytokine storm-induced lethality in the clinic because they are safe, inexpensive, and easily accessible for immediate use in most countries.

Analysis of Bacterial Biofilm Formation in Patients with Malignancy Undergoing Double J Stent Indwelling and Its Influencing Factors.

OBJECTIVE: To analyze the bacterial biofilm (BF) formation in patients with malignancy undergoing double J stent indwelling and its influencing factors. METHODS: A total of 167 patients with malignant tumors who received double J stent indwelling in the hospital from January 2018 to January 2021 were included in the study. The urine and double J stent samples were collected for bacterial identification and observed for BF formation on the surface of the urinary catheter under a scanning electron microscope (SEM). Univariate and multivariate logistic regression analyses were used to analyze the influencing factors of BF. RESULTS: The BF formation rate was 34.73% (58/167). The BF formation rate of positive specimens cultured in urine and double J stent was significantly higher than that of negative ones (P<0.05). Staphylococcus was the main BF bacteria in double J stent and urine culture specimens, followed by Enterococcus, Pseudomonas, Enterobacter, and Acinetobacter. Compared with the non-BF group, the number of viable bacteria in the double J stent and urine and the catheterization time in the BF group rose markedly (P<0.05). Advanced age, chemotherapy, anemia, indwelling time ≥90d, and urinary tract infection were risk factors for BF formation in patients with malignancy undergoing double J stent indwelling (P<0.05). CONCLUSION: There is a high rate of BF formation in patients with malignancy undergoing double J stent indwelling, with Staphylococcus as the dominant species. Treatment requires enhanced urinary catheter management and nutritional status to inhibit BF formation and lower the rate of urinary catheter-related infections.

Observations of Bacterial Biofilm on Ureteral Stent and Studies on the Distribution of Pathogenic Bacteria and Drug Resistance.

OBJECTIVE: This study aims to observe the morphological characteristics of bacterial biofilm on the surface of ureteral stents and analyze distribution characteristics of pathogens on the bacterial biofilm and drug resistance. METHODS: Ureteral stents were sampled from 129 patients. A scanning electron microscope was used to observe the morphological characteristics of bacterial biofilms, and the Congo red medium was applied to screen bacterial biofilm strains on the renal pelvis section, ureter section, and bladder section respectively. Urine culture was performed, and the drug sensitive test analysis was carried out for the pathogenic bacteria detected. RESULTS: Bacterial biofilms can be observed on the surface of ureteral stents, and these bacteria are embraced by large amounts of fiber membranes. A total of 107 patients were found to be positive for biofilms with a positive rate of 82.9%. The positive rates of the bladder section, ureter section, renal pelvis section, and urine culture were 85.0, 42.9, 67.3, and 24.3% respectively. Pathogenic bacteria mainly concentrated on Escherichia coli, and the drug resistance rate of the bacterial biofilm strains on the ureteral stent was relatively higher. CONCLUSION: The bacterial biofilm on the ureteral stent is an important factor that induces catheter-associated urinary tract infections.

shRNA targeting PLCε inhibits bladder cancer cell growth in vitro and in vivo.

OBJECTIVES: To investigate the role of phospholipase Cε (PLCε) by silencing PLCε with short hairpin RNA (shRNA) in human bladder cancer cells BIU-87 in vitro and in vivo. METHODS: A PLCε shRNA expression vector was transfected into BIU-87 cells, and the expression of PLCε protein was detected by Western blotting. Cell proliferation was determined using the MTT assay, and the cell cycle was detected using flow cytometry. A tumor xenograft experiment was established to evaluate the tumor growth under the condition of PLCε knockdown, and the expression of PLCε, proliferating cell nuclear antigen, and cyclin D1 were detected by Western blotting or immunohistrochemistry. RESULTS: PLCε shRNA reduced the protein level of PLCε, leading to marked proliferation inhibition and significant cell cycle arrest. Furthermore, PLCε shRNA reduced the tumor xenograft growth implanted with BIU-87 cells. The protein expression of PLCε, proliferating cell nuclear antigen, and cyclin D1 were downregulated in the bladder tumor xenograft. CONCLUSIONS: The knockdown of PLCε by shRNA could inhibit bladder tumor growth and might be an alternative approach for human bladder cancer therapy.

Effects of silencing transforming growth factor-ß1 by RNA interference plasmid on rat renal allograft fibrosis using Smads pathway.

OBJECTIVES: To evaluate the effects of transforming growth factor (TGF)-ß1 RNA interference plasmid on rat renal allograft fibrosis and to explore its mechanisms. METHODS: A Sprague-Dawley to Wistar rat transplant kidney-sclerosis accelerated model was constructed and transfected with short hairpin RNA-TGF-ß1 based on the hydromechanics. Kidney and blood samples were collected at the first, second, and third months after transplantation. Reverse transcriptase-polymerase chain reaction and Western blotting were used to detect the expression of TGF-ß1, phosphorylated Smad3/7, E-cadherin, and type I collagen. The fibrosis extent was assessed using Masson staining. The immunohistochemical staining of E-cadherin and α-smooth muscle actin were used to label the tubular epithelial cells and fibroblast, respectively. RESULTS: The blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P <.05 and P <.01, respectively). The expression of TGF-ß1 was significantly inhibited by the plasmid and its target gene type I collagen (P <.05 or P <.01), in which the signal proteins of phosphorylated Smad3 was downregulated and phosphorylated Smad7 was upregulated. Also, the fibrosis of the renal allograft was improved and milder fibrosis was present in the plasmid group. In addition, short hairpin RNA-TGF-ß1 plasmid maintained the expression of E-cadherin on tubular epithelial cells, resulting in inhibition of cell transdifferentiation from epithelial cells to fibroblast. CONCLUSIONS: Our results suggest that short hairpin RNA-TGF-ß1 plasmid could prevent the fibrosis of renal allografts. The mechanism might be associated with its effects of downregulating phosphorylated Smad3 and upregulating phosphorylated Smad7, leading to the suppression of epithelial-myofibroblast transdifferentiation and extracellular matrix synthesis.

[Glycosyl-phosphatidylinositol-anchored interleukin-2 expressed on tumor-derived exosomes induces anti-tumor immune response].

OBJECTIVE: To prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2. METHODS: To construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2. RESULTS: The pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05). CONCLUSIONS: GPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

Glycosyl-phosphatidylinositol-anchored interleukin-2 expressed on tumor-derived exosomes induces antitumor immune response in vitro.

AIMS AND BACKGROUND: Tumor-derived exosomes (TEXs) have been considered as a new kind of cancer vaccine, but the antitumor effects are not satisfactory. In order to improve the efficacy of TEXs, we investigated whether exosomes derived from glycosyl-phosphatidylinositol-anchored interleukin 2 (GPI-IL-2) gene-modified bladder cancer cells can increase the antitumor effects. METHODS AND STUDY DESIGN: We transfected melanoma antigen-1 (MAGE-1)-expressing T24 tumor cells with a plasmid encoding GPI-IL-2 and prepared the TEXs. Exosomes expressing GPI-IL-2 were characterized by electron microscope and Western blot analysis. RESULTS: IL-2 was present on the cell surface in the GPI-anchored form as demonstrated by fluorescent microscope and ELISA analyses. Exosomes expressing GPI-IL-2 naturally contained bioactive GPI-IL-2 and tumor-associated antigen MAGE-1. Moreover, exosomes expressing GPI-IL-2-pulsed dendritic cells could induce the proliferation of T cells and the antigen-specific cytotoxic T-lymphocyte immune response more efficiently. CONCLUSIONS: GPI-IL-2 gene-modified tumor cells can make the TEXs contain GPI-IL-2, resulting in increased antitumor effects. Our study provided a feasible approach for exosome-based tumor immunotherapy.

[Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro].

OBJECTIVE: To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro. METHODS: A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay. RESULTS: Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro. CONCLUSION: A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

Interleukin-12-anchored exosomes increase cytotoxicity of T lymphocytes by reversing the JAK/STAT pathway impaired by tumor-derived exosomes.

Tumor-derived exosomes express tumor antigens, leading to their promising utility as tumor vaccines, but they also can suppress T-cell signaling molecules and reduce cytotoxic effects. We investigated whether interleukin-12 (IL-12)-anchored exosomes (EXO/IL-12) reverse tumor exosome-mediated inhibition of T-cell activation and cytotoxicity was associated with inhibition of JAK3 and p-STAT5. A co-expression plasmid of pBudCE4.1/IL-12A/ IL-12B-GPI was constructed. EXO/IL-12 was identified by transmission electron microscopy and Western blotting, which induced proliferation and cytotoxicity of T-cells and were analyzed by CFSE-based flow cytometry. Expression of JAK2, JAK3 and p-STAT5 was detected by Western blotting. Our results showed that EXO/IL-12 was much more efficient in induction of the proliferation, release of IFN-gamma and cytotoxic effect of T lymphocytes than conventional exosomes in vitro. Exosomes inhibited the expression of JAK3 and phosphorylation of STAT5 in high doses in T-cells, but not JAK2, while EXO/IL-12 had much less attenuated reduction of the expression of p-STAT5. The enhanced cytotoxic effects of T lymphocytes might partly depend on EXO/IL-12 reversing the suppressed expression of p-Stat5 by Jak2/Stat5 pathway. These findings might provide an alternative approach for developing exosomes into tumor vaccines.

Exosomes derived from IL-12-anchored renal cancer cells increase induction of specific antitumor response in vitro: a novel vaccine for renal cell carcinoma.

Exosome-based immunotherapy for cancer holds promise, but needs improvements, especially for tumor-derived exosomes. We investigated, whether exosomes derived from IL-12-anchored human renal cancer cells could enhance their immunogenicity and increase induction of specific antitumor response. A mammalian co-expression plasmid of glycolipid-anchored-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence of human placental alkaline phosphatase-1 (hPLAP-1) and IL-12B chain gene (P40 subunit) in pBudCE4.1. Then exosomes were prepared from renal cancer cells modified to express GPI-IL-12. The results showed that exosomes derived from IL-12-anchored renal cancer cells expressed renal cell carcinoma-associated antigen G250 and GPI-IL-12. The incorporation of GPI-IL-12 onto exosomes (exosomes-GPI-IL-12, EXO/IL-12) significantly promotes proliferation of T cells, and subsequently increased the release of IFN-gamma. Notably, stimulation with EXOs/IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTLs), resulting in more significant cytotoxic effects in vitro. These results suggested that exosomes derived from IL-12-anchored renal cancer cells bore GPI-IL-12 and G250, which have tumor rejection antigen with enhanced immunogenicity and antitumor effects, representing a novel strategy of exosomes-based vaccine for renal cell carcinoma.

[Exosomes derived form bladder transitional cell carcinoma cells induce CTL cytotoxicity in vitro].

OBJECTIVE: To isolate and purify exosomes derived from human bladder transitional cell carcinoma T24 cells, analyze the morphology and protein composition, and investigate the antitumor effect of specific cytotoxic T lymphocytes induced by exosomes. METHODS: Exosomes were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and characterized by electron microscopy and Western blot. Dendritic cells were amplified and purified from peripheral blood and pulsed with exosomes. Then they were co-cultured with T cells, and divided into 3 groups: exosome-pulsed DC group, unplused DC group and control group. Alamar-Blue assay was used to evaluate the specific cytolytic activity. RESULTS: The exosomes were in size about 30 approximately 90 nm saucer-shaped membranous vesicles. HSP70, ICAM-1 and CK20 were detected by Western blot. The CTL induced by DC pulsed with exosomes had significant cytolytic activity (P < 0.01). CONCLUSION: The exosomes derived from T24 cells are loaded with immunoprotein HSP70 and ICAM-1, and DC pulsed with exosomes can promote the anti-tumor effect of CTLs in vitro.

[Effects of silencing transforming growth factor-beta1 by RNAi upon transdifferentiation of renal allograft tubular epithelial cells in rats].

OBJECTIVE: To investigate the effects of shRNA-transforming growth factor (TGF)-beta1 plasmid upon epithelial-myofibroblast transdifferentiation of renal allograft in rats. METHODS: Divided the Wistar rats into 4 groups: Group J (sham-operated group), T (plasmid group), H (vacant plasmid group) and Y (simply transplantation group). The SD to Wistar rat transplant kidney-sclerosis accelerated model was constructed and transfected with the plasmid based on hydromechanics. Transplanted kidneys were collected at Months 1, 2 and 3 post-transplantation. The gene transcriptional levels of TGF-beta1 and E-cadherin were detected by RT-PCR and the protein variation of E-cadherin was examined by Western blotting. The pathological changes and infiltrated inflammatory cells were assessed by HE staining and the immunohistochemical staining of E-cadherin and alpha-SMA used to label epithelial cells and fibroblast in order to exhibit cell transdifferentiation. RESULTS: Compared with Group H and Y, the mRNA transcription of TGF-beta1 was obviously inhibited in the Group T: at Month 3, the TGF-beta1 mRNA of Group T is 0.73 +/- 0.08, significantly lower than Group H and Y (0.92 +/- 0.07 and 0.95 +/- 0.04, both P < 0.01); the expression of E-cadherin was maintained at a high level: at the Month 3, the E-cadherin mRNA of Group T is 0.39 +/- 0.11, significantly higher than Group H and Y (0.15 +/- 0.07, and 0.17 +/- 0.06, both P < 0.01); the E-cadherin protein of Group T is 0.38 +/- 0.08, significantly higher than group H and Y (0.15 +/- 0.07 and 0.15 +/- 0.07, both P < 0.01); epithelial cells were much more and fibroblast was much less than that of Group H and Y; there were also less infiltrated chronic inflammatory cells and extracellular matrix deposition in the Group T. CONCLUSION: The shRNA-TGF-beta1 plasmid can inhibit the epithelial-myofibroblast transdifferentiation of renal allograft in rats. The mechanisms may be associated with its effects of down-regulating TGF-beta1 and up-regulating E-cadherin.