Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at [~]3 [A] resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

Magnitude and timing of the antiviral response determine SARS-CoV-2 replication early in infection

The interferon response is a potent antiviral defense mechanism, but its effectiveness depends on its timing relative to viral replication. Here, we report viral replication and host response kinetics in patients at the start of SARS-CoV-2 infection and explore the impact of these kinetics experimentally. In both longitudinal patient nasopharyngeal samples and airway epithelial organoids, we found that SARS-CoV-2 initially replicated exponentially with a doubling time of [~]6hr, and induced interferon stimulated genes (ISGs) with delayed timing relative to viral replication. Prior exposure to rhinovirus increased ISG levels and blocked SARS-CoV-2 replication. Conversely, inhibiting ISG induction abrogated interference by rhinovirus and enhanced SARS-CoV-2 replication rate. These results demonstrate the importance of initial interferon-mediated defenses in determining the extent to which SARS-CoV-2 can replicate at the start of infection and indicate that biological variables that alter the airway interferon response, including heterologous induction of innate immunity by other viruses, could profoundly impact SARS-CoV-2 susceptibility and transmission.

Single-Cell Omics Reveals Dyssynchrony of the Innate and Adaptive Immune System in Progressive COVID-19

A dysregulated immune response against the SARS-CoV-2 virus plays a critical role in severe COVID-19. However, the molecular and cellular mechanisms by which the virus causes lethal immunopathology are poorly understood. Here, we utilize multiomics single-cell analysis to probe dynamic immune responses in patients with stable or progressive manifestations of COVID-19, and assess the effects of tocilizumab, an anti-IL-6 receptor monoclonal antibody. Coordinated profiling of gene expression and cell lineage protein markers reveals a prominent type-1 interferon response across all immune cells, especially in progressive patients. An anti-inflammatory innate immune response and a pre-exhaustion phenotype in activated T cells are hallmarks of progressive disease. Skewed T cell receptor repertoires in CD8+ T cells and uniquely enriched V(D)J sequences are also identified in COVID-19 patients. B cell repertoire and somatic hypermutation analysis are consistent with a primary immune response, with possible contribution from memory B cells. Our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19, which may contribute to delayed virus clearance and has implications for therapeutic intervention.

Neuroinvasive potential of SARS-CoV-2 revealed in a human brain organoid model

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate in vivo that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.

Causes and management for male urethral stricture / 中南大学学报(医学版)

Objective:To explore the etiology of male urethral stricture,analyze the therapeutic strategies of urethral stricture,and summarize the complicated cases.Methods:The data of 183 patients with urethral stricture were retrospectively analyzed,including etiology,obstruction site,stricture length,therapeutic strategy,and related complications.Results:The mean age was 49.7 years,the average course was 64.7 months,and the constituent ratio of51 to 65 years old patients was 38.8％ (71/183).The traumatic injury of patients accounted for 52.4％ (96/183),in which the pelvic fracture accounted for 35.5％ (65/183) and the straddle injury accounted for 16.9％ (31/183).There were 54 cases of iatrogenic injury (29.5％).The posterior urethral stricture accounted for 45.9％ (84/183),followed by the anterior urethral stricture (44.8％,82/183) and the stenosis (6.6％,12/183).A total of 99 patients (54.1％) received the end to end anastomosis,and 40 (21.9％) were treated with intracavitary surgery,such as endoscopic holmium laser,cold knife incision,endoscopic electroknife scar removal,balloon dilation,and urethral dilation.In the patients over 65-years old,the urethral stricture rate was 14.8％ and the complication rate (70.4％) for transurethral resection of the prostate (TURP) was significantly higher than that of all samples (P＜ 0.01).Conclusion:Both the etiology of male urethral stricture and the treatment strategy have changed and the incidence of traumatic and iatrogenic urethral stricture has increased in recent 3 years.The main treatment of urethral stricture has been transformed from endoscopic surgery into urethroplasty.

Effect of calcium channel blockers on primary cultured human urethra scar fibroblasts / 中南大学学报(医学版)

To investigate effects of verapamil on primary cultured human urethral scar fibroblasts (USFs) and to provide basis for protecting the formation of urethra scar.
 Methods: The cell proliferation was evaluated with the cell counting kit (CCK)-8 method after USFs were incubated various verapamil concentrations (50, 100, 150, 200, or 250 μmol/L) or solvent for 12, 24, or 48 h. The protein level of matrix metalloproteinase (MMP) was evaluated with ELISA after cells were incubated with verapamil (100 μmol/L) or solvent (control cells) for 24 h.
 Results: The proliferation of USFs was obviously suppressed after verapamil treatment, which was in a dose-dependent and time-dependent manner. Meanwhile, the protein levels of MMP-2 and MMP-9 in the verapamil treatment group increased obviously compared with those of the control groups (P<0.05).
 Conclusion: Calcium channel blockers may prevent the excessive formation of urethra scar by inhibiting the proliferation of urethral scar fibroblasts and enhancing the activity of MMP.

Ketamine induces apoptosis of human uroepithelial SV-HUC-1 cells / 中南大学学报(医学版)

Objective:To determine the effect of ketamine on the apoptosis of human uroepithelial cells (SV-HUC-1) and the pathogenesis of ketamine-associated cystitis. Methods:SV-HUC-1 cells were cultured under various concentrations of ketamine and different time. Flow cytometry was used to analyze the rate of cell apoptosis. hTe protein levels of Bax, Bcl-2, pro-caspase-3, and cleaved caspase-3 were detected by Western blot. Results:Compared with the control group, the apoptotic rate of ketamine cultured SV-HUC-1 cells increased. hTe expression of Bax increased, Bcl-2 expression decreased, and Bax/Bcl-2 in the ketamine cultured SV-HUC-1 cells was signiifcantly higher. hTe protein level of pro-caspase-3 was signiifcantly lower, and that of cleaved caspase-3 was signiifcantly higher than that in the control group (P<0.05), positively correlated with the dose of ketamine and time of culture (P<0.05). Conclusion:Ketamine can induce the apoptosis of SV-HUC-1 cells in a dose and time dependent manner.

The application of standard remnant liver volume to assessment of cirrhotic liver function reserve / 中华肝胆外科杂志

Objective To investigate the effect of standard remnant liver volume (SRLV) on liver insufficiency after hepatectomy in cirrhotic patients with hepatocellular carcinoma (HCC).Methods Sixty-seven HCC patients with liver cirrhosis were involved in this study.The following parameters were obtained in all cases:total liver volume (TLV),resected liver volume by surgery,body surface area (BSA),remnant liver volume (RLV)and SRLV.Compared analysis of relationship between liver insufficient and the parameters as well as the age of patients,duration of operation and blood lose etc.was carried out,in order to establish the security threshold of SRLV.Results According to the postoperative liver function,the patients were divided into 2 groups:Group A,52cases with mild liver dysfunction; Group B,15cases among them 12 with moderate and 3 with severe liver insufficiency.Statistical analysis showed that the difference of TLV,duration of operation,intra-operative blood lose and age between Group A and B were insignificant(P＞0.05).However,that of RLV and SRLV were significant(P＜0.05).The average SRLV in Group A was 562±89 ml/m2 and 410±87 ml/m2 in Group B (P＜＜0.01).The security threshold of SRLV was 438 ml/m2 calculated by receiver operating characteristic (ROC)in our patients.Then randomly selected sixty HCC patients,the incidences of moderate and severe liver insufficiency postoperative in the SRLV≤438 ml/m2 and SRLV＞438 ml/m2 patients were 92.3％and8.5％ (P＜0.01).Conclusions It is suggested from our present study that SRLV is a good predictor for post-operative liver function reserve in patients with cirrhotic HCC.Its security threshold is 438 ml/m2,and the risk of occurring hepatic failure will be high postoperatively when patient,s SRLV is less than this value.

Diagnosis and treatment of ketamine-associated cystitis：A report of 3 cases and literature review / 中南大学学报(医学版)

Objective:To determine the diagnosis, treatment, and pathogenesis of ketamine-associated cystitis. Methods:Clinical data from 3 patients with ketamine-associated cystitis were analyzed retrospectively and discussed in light of relevant literature. Results:In the 3 cases, 2 presented severe lower urinary tract symptoms, including frequency, urgency, dysuria, urge incontinence, and painful haematuria. Urinalysis and urine culture were negative. Imaging examination demonstrated thickening of the bladder wall and a small capacity. Inflammatory changes in the bladder mucosa were observed by cystoscopy and biopsies. After cessation of ketamine use, with the addition of steroids or hydrodistension, the symptoms in the 3 patients improved. The symptoms recurred in 2 patients, as 1 was exposed to ketamine again and 1 had severe bladder contraction after for 3-4 month follow-up. Conclusion:Ketamine-associated cystitis is a new urinary system inlfammatory damage. Its etiology and treatment methods are not clear. early abstinence from ketamine use and early treatment are crucial for patients with ketamine-associated cystitis to avoid irreversible damage.

Molecular mechanism of FGF8b regulation of epithelial-mesenchymal transition in prostate cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To explore the molecular mechanism of fibroblast growth factor 8b (FGF8b) in promoting epithelial-mesenchymal transition in prostate cancer DU145 cells.@*METHODS@#Cells were selected in three groups as follows: a block control group (DU145 cells), a negative control group [DU145 cells transfected with empty plasmid (pcDNA3.1/DU145)], and an experimental group [DU145 cells transfected with FGF8b (FGF8b/DU145)]. The activity of extracellular regulated protein kinases1/2( ERK1/2) pathway was detected by western-blot in the three groups. The FGF8b-DU145 cells and DU145 cells were cultured with PD98059 (an ERK kinase inhibitor) to observe microscopically the morphology changes within the cells. The experimental samples were also divided into four groups: FGF8b/DU145 cells cultured with 2% FBS (Group A); FGF8b/DU145 cells cultured with 2% FBS+PD98059 (50 μmol/L) (Group B); DU145 cells cultured with 2% FBS (Group C); DU145 cells cultured with FBS+PD98059 (50 μmol/L) (Group D). The expression of epithelial- mesenchymal transition (EMT) markers (E-cadherin, vimentin) were detected by western-blot analysis and the cell's mobility were detected by the Transwell chamber.@*RESULTS@#The activity of ERK1/2 in the experimental group was significantly higher than that in the other two control groups; when ERK kinase inhibitor PD98059 was added to FGF8b/ DU145 cells, the expression of epithelial marker E-cadherin protein was significantly increased in group B compared with that in the group A (P<0.05). The expression of mesenchymal marker vimentin protein was significantly reduced in group B compared with that in group A (P<0.05). The cell migration assay suggested that cell migration was markedly decreased in group B (P<0.05) compared with that in group A.@*CONCLUSION@#EMT in prostate cancer induced by FGF8b can be mediated by ERK kinase pathway, in which mitogen-activated/extraceluer signal regulated kinase 1 (MEK1) may be a key factor. MEK1 could be an effective target in regulating the invasion and migration of prostate cancer.

Diagnosis and treatment of carcinoma of renal pelvis accompanied with kidney stones / 中华泌尿外科杂志

Objective To discuss the diagnosis and treatment of carcinoma of renal pelvis ac-companied with kidney stones. Methods Twenty-one cases of carcinoma of renal pelvis with kidney stones were reviewed. The urinary stones history was from 10 d to 24 years with an average of 27 months. Four cases had recurrent fever, weight loss. Seventeen cases had gross hematuria. CT scan-ning was performed in 17 cases, which indicated 9 cases of carcinoma of renal pelvis, 4 cases of possi-ble renal mass and enlarged renal hilum lymph nodes. MRU was performed in 10 cases, which showed 9 cases of stones concomitant with carcinoma of renal pelvis. IVU indicated 13 cases of light filling of contrast and 8 cases of complete negative filling. Nine cases who had been diagnosed before surgery were performed radical nephrectomy and local lymph nodes dissection. Five cases were carried out ne-phrostomy first because of pyonephrosis, then secondary subcapsule nephrectomy was performed, 2 of them who were diagnosed with carcinoma of renal pelvis after surgery were performed with ureterecto-my and excision of bladder cuff. Three cases were performed with subcapsule nephrectomy because of dense perinephric adhesion. Three cases with complicated calculi and nonfunctional kidney were per-formed with nephroureterectomy, 2 of them who were diagnosed with carcinoma of renal pelvis were carried out with ureterectomy and excision of bladder cuff. One case who was performed PCNL under ultrasound guidance was found a mass in the renal pelvis. The pathological examination indicated ade-nocarcinoma. Radical nephrectomy and lymphadenectomy of renal hilum were performed afterwards. Resnlts Twenty-one cases were performed with pathological examination, in which there were 4 ca-ses of transitional cell carcinoma, 16 cases of squamous cell carcinoma and 1 case of adenocarcinoma. All the 21 cases were discharged after surgery. Nine of them were followed up with 4 to 28 months. Six cases were dead. The post-surgery survival time was 3 to 21 months. Two of them died of heart infarction, 4 died of metastasis. Conclusions The carcinoma of renal pelvis should be considered be-fore surgery in the patients with long history of renal calculi, hydronephrosis and infection. CT scan-ning and MRU are helpful for the diagnoses of renal calculi concomitant with carcinoma of renal pel-vis. Earlier diagnosis, earlier management of renal calculi can extend survival time of the patients with renal calculi concomitant with carcinoma of renal pelvis.

Development of connector between anesthetic machine and oxygen provider used in field hospital / 医疗卫生装备

Objective To develop a connector between anesthetic machine and oxygen provider for continual oxygen supply to anesthetic machine during field operation.Method According to the principle of three-way block and one-way qas wave,the new connector with two-routes but one-way for the anesthetic machine was made.Result This setting enable the anesthetic machine to connect two oxygen providers at one time.Conclusion With low cost and being easy to install and dissemble,it can be connected to all kinds of anesthetic machine,thus ensuring the persistence work of the anesthetic machine when changing oxygen provider.

Positive inotropic effect of methyl polyglycoside on isolated guinea pig atria / 中国药理学通报

AIM　To study the positive inotropic effect of methyl polyglycoside (Mpg) on isolated guinea pig atria and its mechanism. METHODS　The effects of Mpg on contractility, beat rate of right atria, post-rest contraction and positive staircase phenomenon were observed in isolated guinea pig left and right atria. Na+-K+-ATPase activity of rat myocardial cell membrane was measured. RESULTS　Mpg (0.01～3 mmol*L-1) produced a concentration-dependent increase in myocardial contractility of atria and decrease in beat rate of right atria. Mpg markedly enhanced the post-rest contraction and the positive staircase phenomenon induced by increasing stimulation frequency in left atria, and inhibited Na+-K+-ATPase activity of rat myocardial cell membrane. CONCLUSION　The results suggest that Mpg can strengthen myocardial contractility of atria and decrease beat rate of right atria. Its positive inotropic effect may be related to inhibiting Na+-K+-ATPase activity of myocardial cell membrane, increasing the transmembrane influx of calcium and the amount of calcium released from intracellular stores.

Joint report of the Jilin 192Ir radiation accident : A clinical study on a case of moderate degree bone marrow form of acute radiation sickness with extremely severe local radiation injury

After the medical handling of a patient exposed to extremely uneven total body irradiation from an 192ir source (the activity was 2.765 TBq) on January 5, 1996 in Jilin City in order to afford new experience and information for similar case in the future. The authors emphasize the early amputation as the key to success, the importance of rhG-CSF effect and the significance of rational nutrition for the support of the patient, who had total body irradiation, massive local radiation injury and extensive surgical intervention to sustain. The document describe the accident, early clinical manifestation and posterior clinical manifestations (peripheral blood changes and bone marrow examination), evolution of local radiation injury; the assay of myocardial and musculature enzymes; assay of hemopoietic activity in the patient's sera and the treatment

High-Efficiency Retroviral Vector Containing Human Mutated Dihydrofolate Reductase cDNA and Its Expression in Murine Hematopoietic Progenitor Cells / 中国肿瘤生物治疗杂志

A double-copy Moloney leukemia virus-based retroviral vector containing both the Neo~(R) gene and a mutant human dihydrofolate re-ductase(S31 mutation) cDNA was packaged into the Amphotropic packaging cell hne GP-EAM12( AM12), and a Amphotropic producer cell hne (named AM12-S31)was obtained. In this study, we investigated its drug resistant characteristics, viral titer and for murine hematopoietic progenitor cells transduction as well. MTT assay verified that the AM12-S31 cells were resistant to G418 and methotrexate(MTX), the IC50 were more than 800 ?g/ml and 100 ?M respectively while the control cell line AM12 was sensitive to both drugs, the IC50 were 180 ?g/ml and 10 ?M, respectively. The viral titer for this cell line was approximately 7.8? 104~4.2? 105 G418-resistant colony forming units/ml. The replication-competent virus can not be detected in this producer cell line. We also use the AM12-S31 cells to transfect murine hematopoietic cells (By coculture) . The positive colonies were found in all the G418 concentrations using CFU-GM assay. No G418-resistant colony was found using AM12 transfection. The infected murine marrow cells were returned to lethally irradiated(900rad)recipients. The murine transplanted with AM12-S31 infected marrow cells showed protection from lethal MTX toxicity as compared with AM12 infected animals. Evidence for integration and the proviral DNA was obtained by PCR amplification of proviral DNA. These results indicated this producer cell hne could produce high titer, high-efficiency and non-replcational competent virus. The murine marrow cells could be transfected successfully using this system, and express the foreign gene. The lethal irradiated murine marrow function could be reinstitution by infusing the hematopoietic progenitor cells tranducted with human mutant dihydrofolate reductase. In my opinion, this system would play an important role in research the long-term protection of murine marrow hematopoietic function and drug resistant gene therapy.