Omicron variant (B.1.1.529) challenge the integrity of blood brain barrier: Evidence from protein structural analysis.

Studies on nonhuman primates, wild-type and transgenic mice have shown the presence of SARS-CoV-2 RNA components in the brains. Despite the Blood-Brain Barrier (BBB) provides protection there are less evidences on how the SARS-CoV-2 crosses the BBB. Given that there is an increase of Omicron reinfection rates, transmissibility rate and involvement to cause neurological dysfunctions, we hypothesized to investigate how the Omicron variant (B.1.1.529) binds structurally to key BBB-maintaining proteins and thus can possibly challenge the integrity and transportation to the brain. By using molecular dynamics simulation approaches we examined the interaction of Omicron variant (B.1.1.529) with different structural and transporter proteins located at the BBB. Our results show that in Zona Ocludin 1-RBD complex, we observe a distinct pattern. Omicron demonstrates a docking score of -88.9 ± 6.8 kcal/mol and six interactions, while the wild type (WT) presents a higher score of -94.0 ± 2.3 kcal/mol, forming eight interactions. Comparing affinities, the WT-RBD displays a stronger preference for Claudin-5, boasting a docking score of -110.2 ± 3.0 and nine interactions, versus Omicron-RBD's slightly reduced engagement, with a docking score of -105.6 ± 0.2 and seven interactions. Interestingly, the Omicron variant exhibits heightened stability in interactions with Glucose Transporter and ABC transporters, registering docking scores of -110.6 ± 1.9 and -112.0 ± 3.6 kcal/mol, respectively. This surpasses the WT's respective scores of -95.2 ± 2.2 and -104.0 ± 6.2 kcal/mol, reflecting a unique interaction profile. Rigorous molecular dynamics simulations validate our findings. Our study emphasizes the Omicron variant's increased affinity towards transporter proteins, illuminating potential implications for BBB integrity and brain transportation. While these insights offer a valuable framework, comprehensive experimental validation is indispensable for a comprehensive understanding.

anti-Markovnikov Iodofluorination of Alkenes.

The fluorination of alkenes with electrophilic N-F type reagents usually occurs through a Markovnikov-type addition, and the anti-Markovnikov-type addition may require the use of a transition metal catalyst or an expensive catalyst. Herein we describe a convenient anti-Markovnikov iodofluorination of alkenes with Selectfluor/n Bu4 NI. A wide substrate scope and good functional group tolerance were observed. The process allows for the construction of various C-F bonds, especially tertiary C-F bonds. The remarkable features make this protocol attractive, including convenient operations, simple reaction conditions, and the installation of an iodine atom which provides possibilities for further transformations.

Effects of enriched environment on anxiety- and depressive-like behavior and synaptic plasticity in neuropathic pain rats / 中华行为医学与脑科学杂志

Objective:To explore the effect of enriched environment on pain sensitivity, anxiety- and depressive-like behavior in selective nerve injury(SNI) rats model and its potential mechanism.Methods:A total of 36 male clean grade SD rats aged 6-8 weeks were randomly divided into three groups( n=12 in each group): sham operation+ standard environment group (sham group), SNI+ standard environment group (standard environment group), SNI+ enriched environment group (enriched environment group). The rat model of neuropathic pain was established by SNI.The rats in the enriched enviroment group were placed in an enriched enviroment 7 days before operation until 21 days after operation.The paw withdraw threshold(PWT) and paw withdraw latency (PWL) were performed to assess hyperalgesia.The open field test, elevated plus maze test, novelty suppressed feeding test and forced swimming test were used to assess anxiety and depression like behavior.The expressions of cAMP response element binding protein (CREB), p-CREB, brain-derived neurotrophic factor (BDNF), postsynaptic density-95 (PSD-95) and neuroligin 2 (NLGN2) were detected by Western blot.The expression of CREB and BDNF in contralateral ACC were measured by immunofluorescence.GraphPad prism 8.0 and SPSS 23.0 were used for data analysis.One way ANOVA was used for inter group comparison, repeated measurement ANOVA was used to analyze PWT and PWL results, and Tukey test was used for pairwise comparison. Results:(1) In PWT and PWL experiments, the interaction effect between group and time, group main effect and time main effect of PWT were significant ( F=13.4, 39.6, 369.6, all P<0.05), and the interaction effect between group and time, group main effect and time main effect of PWL were significant ( F=3.8, 10.3, 58.8, all P<0.05). Compared with sham group, PWT((8.0±3.5) g, (2.4±1.4) g, (2.3±1.1) g, (2.2±1.6) g, (1.6±0.5) g) and PWL((8.6±1.3) s, (7.3±1.5) s, (7.9±1.0) s, (6.6±1.1) s, (7.7±1.4) s) in standard environment group decreased at each time point (all P<0.05). (2) Compared with sham group, the number of entrying into the central area (1.3±1.7), the time of entrying into the central area((1.6±1.3) s), the proportion of entering open arms ((8.0±7.8) %) and the proportion of time in the open arms ((1.3±1.2) %) all significantly decreased in standard environment group ( t=4.585, 5.423, 4.682, 5.202, all P<0.05). The eating latency ((365.2±94.4) s) and immobility time ((127.6±24.3) s) dramatically increased ( t=6.008, 14.290, both P<0.05). The number and time of entrying into central area of enriched environment group were both higher than those of standard environment group(both P<0.05), while the eating latency and immobility time of enriched environment group were both lower than those of standard environment group(both P<0.05). (3) Compared with sham group(CREB: (1.6±0.2), (0.8±0.5); BDNF: (0.8±0.5), (1.0±0.4)), the expression of CREB ((1.8±0.1), (1.5±0.2)), BDNF ((0.9±0.6), (1.4±0.3)) in spinal cord and ACC of standard environment group increased (spinal: t=3.283, 4.989; ACC: t=5.502, 4.257, all P<0.05). The expression of PSD-95 ((1.6±0.2), (1.0±0.2) and NLGN2 ((1.5±0.5), (1.1±0.2)) also increased in ACC of standard enviroment group ( t=4.257, 2.214, both P<0.05). Compared with standard environment group, the expression of CREB (1.3±0.3), BDNF (0.7±0.4), PSD-95(1.0±0.3) and NLGN2(1.1±0.4) in spinal cord of enriched environment group decreased ( t=5.007, 2.166, 2.358, 2.322, all P<0.05). The expression of PSD-95(1.2±0.3) and NLGN2(1.1±0.2) also decreased in ACC of enriched environment group ( t=2.674, 2.944, both P<0.05). However, the expression of p-CREB (1.7±0.6) and BDNF (2.4±0.2) increased in ACC ( t=4.180, 7.610, P<0.05). Conclusion:Enriched environment can improve neuropathic pain and anxiety- and depressive-like behavior in SNI rats, which may be related to the change of synaptic plasticity in spinal cord and ACC.

Role of ten-eleven translocation methylcytosine dioxygenase 3 in trigeminal ganglion in maxillofacial inflammatory pain in mice / 中华麻醉学杂志

Objective:To evaluate the role of ten-eleven translocation methylcytosine dioxygenase 3 (TET3) in trigeminal ganglion in maxillofacial inflammatory pain in mice.Methods:Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 19-23 g, were divided into 5 groups ( n=8 each) using a random number table method: control group (group C), inflammatory pain group (group IP), control+ TET3-siRNA group (group C+ siTET3), inflammatory pain+ TET3-siRNA group (group IP+ siTET3) and inflammatory pain+ negative control Scrambled-siRNA group (group IP+ siNC). Normal saline or complete Freund′s adjuvant (CFA) 10 μl was injected into the temporomandibular joint of mice, respectively, and the mechanical paw withdrawal threshold (MWT) was measured at 1, 4, 8 and 12 days after injection (T 1-4). Before injection of normal saline or CFA, 0.75 μl siTET3 or siNC was injected into the trigeminal ganglion and the animals were then sacrificed and trigeminal ganglion was removed at T 2 for determination of the expression of TET3 by Western blot in C+ siTET3, IP+ siTET3 and IP+ siNC groups. Results:Compared with group C, MWT was significantly decreased at T 1-3 , the expression of TET3 in trigeminal ganglion was up-regulate in group IP ( P<0.05 or 0.01). Compared with IP and IP+ siNC groups, MWT was significantly increased at T 2, 3, and the expression of TET3 in trigeminal ganglion was down-regulate in group IP+ siTET3 ( P<0.05 or 0.01). Conclusion:TET3 in trigeminal ganglion is involved in the development of maxillofacial inflammatory pain in mice.

Effect of pulsed radiofrequency on spinal ATP-P2X4-NLRP3 signaling pathway in rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the effect of pulsed radiofrequency (PRF) on spinal adenosine triphosphate (ATP)-P2X4-NLRP3 signaling pathway in rats with neuropathic pain.Methods Forty healthy clean-grade adult male Sprague-Dawley rats,aged 2-3 months,weighing 220-260 g,were divided into 4 groups (n =10 each) using a random number table method:sham operation group (group S),neuropathic pain group (group NP),sham PRF group (group SPRF) and PRF group.Neuropathic pain was induced by chronic constriction injury to the left sciatic nerve of anesthetized rats.Rats received PRF treatment on 7th day after establishing the model in group PRF.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before establishing the model (T0) and at 3,7,10,14,21 and 28 days after establishing the model (T1-6).The rats were then sacrificed and the spinal cord was removed for determination of P2X4 and NLRP3 expression (by Western blot) and interleukin-1beta (IL-1β),IL-2,IL-6 and tumor necrosis factor-alpha (TNF-α) contents (by enzymelinked immunosorbent assay).Results Compared with group S,the MWT and TWL were significantly decreased at T1-6,the expression of P2X4 and NLRP3 was up-regulated,and the contents of IL-1β,IL-2,IL-6 and TNF-α were increased in NP,SPRF and PRF groups (P＜0.05).Compared with group NP and group SPRF,the MWT and MWT were significantly increased at T3-6,the expression of P2X4 and NLRP3 was down-regulated,and the contents of IL-1 β,IL-2,IL-6 and TNF-α were decreased in group PRF (P＜0.05).Conclusion The mechanism by which PRF alleviates neuropathic pain is related to inhibiting ATP-P2X4-NLRP3 signaling pathway in rats.

MicroRNA-3963 Promotes Adipogenic Differentiation of Mouse-Derived Mesenchymal Stem Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of MicroRNA-3963(miR-3963) on the adipogenic differentiation of mouse bone-derived mesenchymal stem cells(MSC).</p><p><b>METHODS</b>MSCs were isolated from C57BL/6 mice bone fragment and transfected with miR-3963 mimic, miR-3963 inhibitor and negative control. The expression of miR-3963 and transfection efficiency were detected by q-PCR. These transfected cells were induced to adipocytes and stained with oil red O after 14 days culture. q-PCR and Western blot were used to detect the expression of adipogenic differentiation marker genes C/EBPα and PPARγ at transcriptional level and protein level.</p><p><b>RESULTS</b>The results of q-PCR revealed that miR-3963 expression level was up-regulated after transfection with miR-3963 mimic (P<0.0001), and down-regulated after transfection with miR-3963 inhibitor (P<0.0001). After oil red staining, overexpression of miR-3963 in MSCs could promote the formation of lipid droplet. The q-PCR and Western blot analyses showed the significant increase of expression of adipogenic marker genes C/EBPα and PPARγ in MSC transfected with miR-3963 mimic. Additionally, compared with the control group, miR-3963 inhibitor could decrease adipogenic differentiation of MSC.</p><p><b>CONCLUSION</b>miR-3963 can regulate and promote adipogenic differentiation of mouse bone-derived MSC.</p>

Role of protein kinase C in maintenance of chronic inflammatory pain in rats: the relationship with expression of Nav1.8 in dorsal root ganglion / 中华麻醉学杂志

Objective To evaluate the role of protein kinase C in the maintenance of chronic inflammatory pain in rats and the relationship with the expression of Nav1.8 in the dorsal root ganglion (DRG).Methods Thirty pathogen-free healthy female Sprague-Dawley rats,weighing 180-220 g,were divided into 3 groups using a random number table:control group (group C),chronic inflammatory pain group (group CIP) and PKC inhibitor group (group P).Normal saline 20 μl was injected into the plantar surface of the right hindpaw every day for 14 consecutive days in group C.Prostaglandin E2 100 ng was injected into the plantar surface of the right hindpaw every day for 13 consecutive days to establish the model of chronic inflammatory pain,and dimethyl sulfoxide 20μl was injected into the plantar surface of the right hindpaw on 14th day in group CIP.Prostaglandin E2 100 ng was injected into the plantar surface of the right hindpaw every day for 13 consecutive days,and PKC inhibitor GF1O9203X 100 nmol/20 μl was injected into the plantar surface of the right hindpaw on 14th day in group P.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before injection (T0) and 1,3,7 and 14 days after the last injection (T1-4).The DRGs of the lumbar segment (L4.5) were removed for determination of Nav1.8 expression using immunofluorescence and Western blot.Results Compared with group C,the MWT was significantly decreased at T1-4 in CIP and P groups,and the expression of Nav1.8 in DRGs was significantly up-regulated in group CIP (P＜0.05).Compared with group CIP,the MWT was significantly increased at T4,and the expression of Nav1.8 in DRGs was down-regulated in group P (P＜0.05).Conclusion Up-regulated expression of Nav1.8 after PKC activation in DRGs is involved in the maintenance of chronic inflammatory pain in rats.

Effect of atorvastatin in the treatment of nonalcoholic fatty liver disease / 中国基层医药

Objective To explore the effect of atorvastatin in the treatment of nonalcoholic fatty liver disease and its clinical application value.Methods 110 patients with nonalcoholic fatty liver disease were randomly divided into observation group and control group,55 cases in each group.The control group was given compound dichloroace-tate diisopropylamine orally,the observation group was given atorvastatin therapy.The effects of the two groups were recorded.Results In the observation group after treatment,the alanine aminotransferase was (39.78 ±3.45) U/L, aspartate aminotransferase was (29.17 ±3.17) U/L,gamma glutamine transpeptidase was (54.28 ±4.11) U/L.In the control group after treatment,the alanine aminotransferase was (52.78 ±6.81) U/L,aspartate aminotransferase was (39.96 ±6.21)U/L,gamma glutamine transpeptidase was (68.69 ±8.31)U/L,there were statistically signifi-cant differences between the two groups(t=12.6290,11.4770,11.5273,all P<0.05).In the observation group after treatment,the triglycerides was (1.66 ±0.32)mmol/L,total cholesterol was (3.27 ±0.37)mmol/L,low density lipo-protein was (1.94 ±0.45)mmol/L.In the control group after treatment,the triglycerides was (2.38 ±0.92)mmol/L,total cholesterol was (5.74 ±1.49)mmol/L,low density lipoprotein was (3.46 ±1.17)mmol/L,there were statis-tically significant differences between the two groups(t=5.4818,11.9316,8.9925,all P<0.05).In the observation group after treatment,the ultrasonic score was (1.33 ±0.12),liver/spleen CT ratio was (0.33 ±0.08).In the con-trol group after treatment,the ultrasonic score was (1.78 ±0.35),liver/spleen CT ratio was (0.47 ±0.21),there were statistically significant differences between the two groups(t =9.0197,4.6202,all P <0.05).Conclusion Atorvastatin in the treatment of nonalcoholic fatty liver disease can effectively improve the liver function,reduce blood lipid concentration,minor adverse reactions,and it is worth popularizing in clinical use.

Effects of Dexmedetomidine on Intraoperative Wake-up Quality of Patients Underwent Neurosurgical Oper-ation / 中国药房

OBJECTIVE:To observe the influence and safety of dexmedetomidine (DEX) on intraoperative wake-up quality of patients underwent neurosurgical surgery. METHODS:126 patients with general anesthesia in neurosurgery were enrolled and randomized equally into observation group and control group,with 63 cases in each group. Control group was given target con-trolled infusion of propofol with plasma target concentration of 3-5 μg/ml and remifentanil with target effect site concentration of 2-6 ng/ml for anesthesia induction and maintenance,and then plasma target concentration of remifentanil decreased to 0.5 ng/ml 30 min before wake-up. Observation group received target controlled infusion of propofol with plasma target concentration of 3-5 μg/ml and remifentanil with target effect site concentration of 2-6 ng/ml for anesthesia induction and maintenance,and then given DEX 0.3 μg/kg intravenously 30 min before wake-up and maintained at 0.1 μg/(kg·h). MAP,HR,SBP,SaO2,serum levels of IgA,IgM,IgG,IL-6,IL-8 and TNF-α were observed in 2 groups 2 h before operation(T1)and after extubation(T2)as well as the occurrence of ADR during wake-up. RESULTS:There was no statistical significance in HR,MAP,SBP,SaO2,IgA,IgM, IgG,IL-6,IL-8 and TNF-α levels at T1 and SaO2 levels at T2 between 2 groups(P>0.05). HR,MAP,SBP,IL-6 and TNF-α lev-els of observation group decreased significantly at T2 and lower than those of control group;IgA,IgM and IgG increased signifi-cantly and higher than those of control group,with statistical significance (P0.05). CONCLUSIONS:DEX influence intraoperative wake-up quality of patients underwent neurosurgical surgery slightly,and can reduce inflammatory reaction with less ADR.

Isolation and culture methods of mouse adipose-derived stem cells / 重庆医学

Objective To establish a simple and effective method for isolation and culture of mouse adipose-derived stem cells (mASCs)in vitro,in order to provide the sufficient sources of seed cells for the research of mesenchymal stem cells.Methods The mouse inguinal fat tissues were isolated in vitro and performed a digestion with 0.1% collagenase type NB4,then adipose-derived stem cells(ASCs)were seeded and adhered to the culture dishes in low glucose DMEM containing 10% fetal calf serum.The cellu-lar morphology,in vitro proliferation capacity,multidifferentiation potential and immunophenotype were assessed.Results The mASCs showed good cell morphology,extremely strong proliferation capacity and potential of adipogenesis,osteogenesis and chon-drogenesis via in vitro three-dimensional induction.The cellular surface antigen phenotype was consistent with that reported by lit-erature,and the expression of CD34 and CD105 was positive,Sca-1 was highly expressed,CD45 and SSEA-1 were not expressed. Conclusion Using the experimental methods in this research can culture the high purity of mASCs with the excellent stem cell properties and extremely strong proliferative ability.

Effect of propofol on expression of c-fos mRNA in hippocampus in rats with visceral pain / 中华麻醉学杂志

Objective To investigate the effect of propofol on the expression of c-fos mRNA in hippocampus in rats with visceral pain (VP).Methods Thirty male Sprague-Dawley rats,weighing 180-240 g,were randomly divided into 3 groups (n =10 each):VP group,propofol 7.5 mg/kg group (group P1) and propofol 75.0 mg/kg group (group P2).0.9％ normal saline was injected intravenously via the caudal vein in group VP.Propofol 7.5 and 75.0 mg/kg were injected intravenously via the caudal vein in groups P1 and P2,respectively.VP was produced by colorectal distension in anesthetized rats.The threshold of VP was assessed by the intra-balloon pressure which was limited to 100 mm Hg to avoid damage to intestine before and after administration.The rats were sacrificed after measurement of the pain threshold,their brains were removed and hippocampi were isolated for determination of the expression of c-fos mRNA by RT-PCR.Results Compared with group VP,the threshold of VP was significantly increased and the expression of c-fos mRNA in hippocampus was down-regulated in groups P1 and P2 (P ＜ 0.05).The threshold of VP was significantly higher and the expression of c-fos mRNA in hippocampus was lower in group P2 than in group P1 (P ＜ 0.05).Conclusion Propofol can reduce VP through down-regulating the expression of c-fos mRNA in hippocampus in rats.

[Study on the change of urinary iodine of different salt concentrations among the target population in an intervention trial].

OBJECTIVE: To analyze the change of urinary iodine in a cohort of intervention trial and to observe the role of different doses on salt iodization and related impact factors on nutritional condition of iodine. METHODS: Multistage cluster sampling was used to sample three townships in two counties for community intervention with different doses (15 ± 5, 25 ± 5, 35 ± 5) mg/kg. RESULTS: Compared to the (35 ± 5) mg/kg group, the urine iodine levels of three experimental townships were gradually declining in county B when time went on, and the (15 ± 5) mg/kg group showed an obvious results, at 6, 12, 18 and 24 months, with the urine iodine level as 180.00, 186.10, 150.04, 191.28 µg/L respectively, which were in accordance with the WHO standard and reached to appropriate range (187.96 µg/L) at the 18 month. The townships at county Y under intervention had declined slightly, but the urine iodine levels did not reach the WHO standard. The thyroid volume declined from 3.65 ml to 3.40 ml in two counties and the difference between them was statistically significant. CONCLUSION: To some extent, reducing the iodine concentration in salt, had a role of lowering the urine iodine level and reducing the strumous rate.

[Determination of leukemia stem cells in childhood acute myeloid leukemia and its clinical significance].

The aim of this study was to detect the presence of human AML leukemia stem cells (LSC) in childhood patients with acute leukemia (AL) and analyze the correlation between LSC concentrations and minimal residual disease (MRD) levels in AML cases after remission. The multi-parameter flow cytometry (FCM) and a panel of monoclonal antibody combination were used to detect the AML LSC or AML LSC immunophenotype-identical cell (AML LSC-IPIC) concentrations in childhood AML or ALL leukemia both at new diagnosis and at remission and correlated AML LSC to the MRD levels at different time points after remission. The results indicated that the AML LSC or AML LSC-IPIC concentrations [in average 166 (range 14 - 1459)/100 000 mononuclear cells (MNCs)] in AML at initial diagnosis were significantly higher than those in ALL [7 (range 0 - 560)/100 000 MNCs, p < 0.017] and control [0 (range 0 - 6)/100 000 MNCs, p < 0.017], respectively. The AML LSC concentrations in AML at non-CR were in average 36 (range 5 - 224)/100 000 MNCs. No statistical difference (p > 0.05) was found between the AML LSC or AML LSC-IPIC concentrations in AML (in average 6 (range 0 - 41)/100, 000 MNCs) and ALL [10 (range 0 - 105)/100, 000 MNCs] after CR. The significantly negative correlation was noticed between AML LSC concentrations and MRD levels. It is concluded that the AML LSCs exist in newly diagnosed AML, which are significantly reduced when complete remission has achieved, but the low levels of these populations still remain. The phenotypically similar (CD34(+)CD38⁻CD123(+)) AML LSC populations have also been found in the bone marrow from ALL patients, but their concentrations are not significantly different when CR has achieved. The significantly negative correlation between AML LSC concentrations and MRD levels is observed in AML patients after remission.

Purification and characterization of recombinant human lactoferrin expressed in a cattle mammary bioreactor / 生物工程学报

Novel ion exchange adsorbents were synthesized by immobilizing sulfopropyl derivative onto homemade highly cross-linked agarose beads. The effects of different ligand densities (from 0.05 to 0.24 mol/L) on static and dynamic adsorption of the adsorbents were investigated using lysozyme as a model protein. Based on these results, rHLF was purified from the transgenic milk by our SP media. 1 mL high density (0.24 mol/L) adsorbent could handle 50 mL rHLF-containing milk. The mass recovery of rHLF was 86.5% and the purity was 98.5%. CD spectra demonstrated that the native structure of rHLF was not affected in the purification process. The biological functions of the purified rHLF, including iron binding, releasing and antimicrobial activities were then investigated. The results showed that rHLF had comparable iron binding and releasing activity to that of native HLF. 5 g/L concentration of rHLF significantly inhibited the growth of Escherichia coli. These studies lay a solid foundation for the wide application of our self-prepared ion exchange adsorbents in protein purification.

[Targeted killing of the Nalm-6 cells with 2E8-Genistein immunotoxin and its mechanism].

OBJECTIVE: Leukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma. METHODS: 2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing. RESULTS: After 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture. CONCLUSION: 2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

[Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14].

OBJECTIVE: Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity. METHODS: Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9). RESULTS: The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A. CONCLUSIONS: The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.

[Preparation of the immunotoxin 2E8-norcantharidin and its targeting killing effect in vitro].

OBJECTIVE: Monoclonal antibody (mAb) conjugated with certain toxin to generate immunotoxin bears an important and promising effect as a new therapy for patients with hematopoietic malignancies. However, most toxic moieties conjugated with antibody proteins reported in the literature were toxic proteins which presented immunogenicity to patients capable of inducing anti-toxin antibody. Norcantharidin (NCTD) is a small molecule toxin. It does not have the immunogenicity to human body so that it bears a promising potential for development of new targeting drug. In this study, a new clone of self-made anti-CD19 mAb named ZCH-4-2E8 conjugated with NCTD was used to investigate its targeting efficacy against CD19+ lymphoid malignant Nalm-6 cells in vitro to provide the experimental data for the further development of this new targeting agent. METHODS: A monoclonal antibody named 2E8 was prepared from mouse ascites and purified by gel chromatography. The purity of the antibody protein was checked by SDS-PAGE assay. Immunotoxin 2E8-NCTD was successfully generated through conjugating CD19 mAb protein and Norcantharidin by the activated ester method. The binding activity of the immunoconjugate (2E8-NCTD) against CD19 antigens on cell surface and the expression levels of CD19 antigens on Nalm-6 and K562 cells were examined by flow cytometry. Comparisons of the inhibitory effects among PBS, purified 2E8 antibody, norcantharidin and immunotoxin 2E8-NCTD groups on cell growth of either Nalm-6 cells or K562 cells were made. RESULTS: The purity of the purified 2E8 antibody was higher than 99.00% demonstrated by SDS-PAGE assay. 2E8 antibody in the supernatant reacted with 99.34% of Nalm-6 cells, while only 0.98% of K562 cells reacted with this antibody. The newly generated immunotoxin (2E8-NCTD) had a positive rate of 99.90% on Nalm-6 cells with little reduction of binding activity. From the in vitro study, both 2E8-NCTD and norcantharidin were shown to have significant inhibitory effects on the growth of CD19+ Nalm-6 cells (P < 0.001), while the purified 2E8 antibody did not show any significant influences on the growth of Nalm-6 cells. Since no significant inhibitory effects were identified among immunotoxin 2E8-NCTD, 2E8 antibody and control groups on CD19(-) K562 cells, a significant targeting effect of the 2E8-NCTD against Nalm-6 cells was confirmed. CONCLUSIONS: The immunotoxin 2E8-NCTD was successfully synthesized by activated ester method with an excellent targeting killing effect on CD19+ Nalm-6 leukemia cells in vitro, which provides some experimental data for the further development of this new targeting agent.

[Establishment of animal model with B lineage acute leukemia in nude mice for evaluation of new therapeutic agents].

OBJECTIVE: To establish an acute leukemia animal model for testing new therapeutic agents in vivo. METHODS: Nude mice were intraperitoneally injected with 2 mg cyclophosphamide, 24 h later 5 x 10(6) acute B-cell leukemia Nalm-6 cells was inoculated via the tail vein, then monitored daily. When animals were paralyzed or dying, the organs including the liver, spleen, lung, heart, kidney, brain, bone marrow, pancreas, testes were removed and fixed with formalin, examined by routine histopathology. RESULTS: After Nalm-6 cells were inoculated the mean survival of mice were( 19.4+/-0.55)d (n=6). The paralysis of mice was followed by weight loss, bent spines, hogback, cachexia and death. Histopathological examination showed that the tumor cells infiltrated liver, spleen, kidney, lung, meninges, interior cerebrum, the liver and kidney were the most affected organs. CONCLUSION: B lineage acute leukemia animal model has been successfully established in the nude mice, which is suitable for testing new therapeutic agents.

[Construction and expression of prokaryotic vector of single chain antibody derived from a new clone of anti-CD14 antibody ZCH-7-2F9].

OBJECTIVE: To construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies. METHODS: Primers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively. RESULT: The cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice. CONCLUSION: The ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Construction and expression of single-chain antibody derived from a new clone of monoclonal antibody against human CD14 in CHO cells.

Our aim was to construct the eukaryotic expressional system of single-chain antibody (ScFv(2F9)) derived from a new clone of monoclonal antibody named ZCH-7-2F9 against human CD14 antigen, and to obtain the ScFv(2F9) protein with a high biological activity for further studies on ScFv(2F9) immunotoxin and other kinds of genetically engineered antibodies. Primers were synthesized according to the gene sequence of ScFv(2F9), 4 tandem glysines and 1 serine (Gly4Ser)3 linker and multiple cloning site(MCS) of pSectag2A vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. ScFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pSectag2A vectors. Positive recombinants (pSectag2A/ScFv(2F9)) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells for expression. The relative molecular weight (MW) and the binding activity of the interesting protein were determined by SDS-PAGE, Western-blot and flow cytometry (FCM), respectively. The scFv(2F9) V(H) is a member of the IgH gene family V(1) subgroup and the scFv(2F9) V(L) gene belongs to the Ig(kappa) gene family V(10) subgroup. The recombined ScFv(2F9) gene was identified to be functional by sequencing and expressing. The interesting protein could be detected in the culture supernatant of transformed CHO cells with a MW of 31 kDa. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.3%and 63.38%, respectively after blockage of ScFv(2F9). The ScFv(2F9) gene was cloned and the interest protein against human CD14 antigen has been successfully expressed in eukaryotic cells with a high biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.