OBJECTIVE To detect the changes in young children,with diarrhea including rotavirus A antigens in stool specimens,serum myocardial enzymes,high-sensitivity C-reactive protein(hs-CRP) and alteration of intestinal flora.METHODS Rotavirus A antigens in stool specimens were detected by immunologic colloidal gold,serum myocardial enzymes were measured by full automatic analyzer,hs-CRP level of serum specimens was measured by gold labeled test,and alteration of intestinal flora was also analyzed.The same method was also applied in control group.RESULTS Among 234 diarrhear cases of young children,124 cases with positive rotavirus A antigens appeared the high level of serum myocardial enzyme and hs-CRP compared with the control group.Candida albicans from stool specimens was detected in 45 cases with positive rotavirus A.CONCLUSIONS The results indicate that rotavirus A can cause gastrointestinal tract infection in young children,lead in alteration of intestinal flora,damage myocardium and lead to high level of hs-CRP.All these laboratory results are significant for clinical diagnosis and treatment.

Objective To analyze the differences of size and charge heterogeneities between origi-nal humanized anti-TNF-αantibody and four similar biotherapeutic products ( SBP ) .Methods The size exclusion chromatography ( SEC-HPLC ) and weak cation exchange chromatography ( WCX-HPLC ) were used to analyze the size and charge heterogeneities , respectively.Carboxypeptidase B (CpB) treatment was employed to analyze the source of charge heterogeneity of the antibody products .Results Four SBPs showed the same pattern with the originator in SEC-HPLC, and no significant difference with the percentage of mono-mer was observed .The percentages of the aggregates of SBP-3 and SBP-4 were a little higher than those of the originator .The charge distribution of SBPs was significantly different from the originator ′s, especially in the basic region .The results from the samples treated with CpB indicated that the difference of charge distri -bution in the basic region might be caused by the C-terminal lysine variants .Conclusion Four SBPs showed similar size heterogeneity with the originator , but significant differences with charge heterogeneity were observed among them .The study suggested that more attention should be paid to the charge heterogene -ity analysis of the biosimilar products .

Objective To compare the capability of capillary electrophoresis-sodium dodecyl sul-fate ( CE-SDS) and size exclusion-high performance liquid chromatography ( SE-HPLC) for analysis of size heterogeneity of monoclonal antibody products .Methods The size heterogeneity of one humanized anti-VEGF monoclonal antibody was analyzed by using non-reduced and reduced CE-SDS, and conventional , de-natured and denatured reduced SE-HPLC.Results The percentage of aggregates detected by non-reduced CE-SDS (0.82%±0.01%) was equal to that by using denatured SE-HPLC (1.05%±0.02%), but it was significantly lower than that by using conventional SE-HPLC analysis (5.08%±0.10%).With regard to fragments analyzed with non-reduced antibodies, its percentage was (7.12±0.04)% measured by non-re-duced CE-SDS analysis that was significantly higher than that by conventional SE -HPLC analysis (0.02%± 0.01%) and denatured SE-HPLC analysis (0.62%±0.01%).Using reduced antibodies , the percentage of fragments was (3.19±0.50)%tested by reduced CE-SDS analysis that was significantly higher than that by using denatured reduced SE-HPLC analysis (0.07%±0.01%).Conclusion Conventional SE-HPLC was more objective than CE-SDS for content analysis of aggregates , as both the covalent and non-covalent forms of aggregates could be detected .Non-reduced CE-SDS could demonstrate the content of clips , while reduced CE-SDS showed the degraded fragments .Therefore, CE-SDS had an advantage over conventional SE-HPLC for content analysis of fragments .The use of the two analytical methods in combination provided solid techni-cal supports for the quality control of size heterogeneity of monoclonal antibodies .

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.

Here we discuss whether N terminal sequencing is appropriate as one of the conventional control methods for monoclonal antibody products. We determined the N terminal sequences of two monoclonal antibody products targeting two antigens separately with both Edman degradation and mass peptide spectrometry. We also identified the characteristic peptide fragments with mass spectrometry. Furthermore, we analyzed their heterogeneity with ion exchange chromatography, capillary zone electrophoresis and Imaged Capillary Isoelectric Focusing. Edman degradation method showed that the N terminal 15 amino acids of heavy and light chains of the two monoclonal antibodies were identical. Peptide mass spectrometry demonstrated that T1 peptide fragments of heavy and light chains of the two antibodies were also the same. But in contrast, peptide mapping and the three analytical methods for heterogeneity analysis could effectively identify and differentiate the two antibodies. The N terminal sequences of two monoclonal antibodies are identical because the number of framework sequences of humanized or human monoclonal antibodies is relatively limited, so whether N terminal sequencing analysis could be regulated as one of the practical control methods should be carefully discussed. Our work also proves that the above analytical methods could combinatorially applied to the identification of monoclonal antibody products, and are more objective compared to N terminal sequencing.
Amino Acid Sequence ; Antibodies, Monoclonal ; isolation & purification ; Chromatography, Ion Exchange ; Humans ; Isoelectric Focusing ; Mass Spectrometry ; Peptide Mapping ; Peptides ; Sequence Analysis, Protein ; methods

This paper reports the determination of the drug antibody ratio in an antibody-drug conjugate with two methods, i.e. LC-MS and UV/VIS, and to provide a reliable method to scientifically evaluate and effectively control the drug antibody ratio. Deglycosylated sample was analyzed with C4 column followed by MS, and the number of conjugated drugs in the antibody was determined by the molecular weight increase due to the addition of different number of drugs to the antibody, and then drug antibody ratio was calculated by weighted average of different number of drugs conjugated to the antibody. Optical density at 252 and 280 nm was measured with UV/VIS, and due to the difference of extinction coefficients between the antibody and the drug, the drug antibody ratio was calculated from linear equation with two unknowns. The drug antibody ratio was 3.21 and 3.25 respectively measured by the two methods, and the results were similar with the two methods. Our study indicated that both methods, LC-MS and UV/VIS, could be applied to the analysis of drug antibody ratio of the antibody drug conjugate.

OBJECTIVETo investigate the impact of aquaporin 9 (AQP9) on the proliferation,apoptosis,invasiveness and migration of hepatocellular carcinoma cells using the HepG2 cell line.

METHODSA lentiviral vector targeting the coding region of human AQP9 was constructed. The recombinant lentiviral vector was harvested from the 293T cell line and transfected into the HepG2 cell line; resistant cell clones were selected with puromycin. Three groups of cells were established, including the CC group (control without lentiviral vector), the PWPI group (control with empty carrier virus), and the AQP9 overexpression group (experimental with the AQP9 recombinant virus). Transfection efficiency was validated by laser confocal microscopy.Expression of AQP9 was detected in the transfected HepG2 cells by westem blotting (protein) and real-time qPCR (mRNA). AQP9 effects on proliferation, migration, invasion and apoptosis of the HepG2 cell line were assessed by plate colony formation assay, woumd healing assay, transwell assay and flow cytometry.

RESULTSThe green fluorescent protein of the recombinant lentiviral vector was appropriately distributed in the cell membrane. The AQP9 overexpression group showed significantly higher AQP9 mRNA and protein levels than the PWPI group and the CC group (both P < 0.01). Cells with AQP9 overexpression showed a lower colony formation rate (16.93±3.19% vs. CC group: 23.53±2.10% and PWPI group: 23.00±2.02%; F=6.46, P=0.032) and a lower overall apoptosis rate (44.96±3.53% vs. CC group:19.7±2.49% and PWPI group: 24.37±2.38%; F=66.88, P < 0.01). The AQP9 overexpression group also showed significantly higher number of cells in the G1 stage and significantly lower number of cells in the S stage (G1: 66.58±0.99% and S:15.25±1.81%), significantly smaller cell migration distance (P=0.01 < 0.05), and significantly suppressed invasiveness (17±8 vs. CC group:109+/-9 and PWPI group: 95±11; P=0.01 < 0.05).

CONCLUSIONIn HepG2 cells, AQP9 significantly reduces the migrative and invasive capabilities, induces cell apoptosis, and inhibits cell proliferation via cell cycle arrest at the G1/S phases.

Apoptosis ; Aquaporins ; Cell Movement ; Cell Proliferation ; Genetic Vectors ; Hep G2 Cells ; Humans ; Lentivirus ; Neoplasm Invasiveness ; RNA, Messenger ; Transfection

Objective:To investigate the clinical significance of intraoperative prepositioning of dual mediastinal drains in elderly patients developing anastomotic leakage(AL)after a total endoscopic Ivor-Lewis procedure.Methods:This retrospective case-control study analyzed the clinical data of 500 elderly patients who underwent total endoscopic Ivor-Lewis surgery for esophageal or cardia cancer from January 2020 to December 2022.In the control group, one mediastinal drainage tube was placed intraoperatively, while in the study group, two mediastinal drainage tubes were placed.Both groups had a chest tube placed conventionally.The study compared the incidence of anastomotic leak(AL)at 1 month postoperatively, inflammatory indexes in patients with AL, grading of AL, rate of nasal fistula placement, incision infection, anastomotic stenosis, and incidence of hoarseness.Additionally, it compared ICU occupancy, ventilator use, and ICU length of stay between the two groups.Results:The analysis included clinical data from 455 elderly patients.Among the patients who developed AL, the study group had significantly lower peak body temperature[(39.58±1.03)℃ vs.(38.05±0.56)℃, t=4.298, P<0.05], white blood cell count[(18.63±3.35)×10 9/L vs.(14.28±2.78)×10 9/L, t=3.450, P<0.05], and C-reactive protein(CRP)levels[(154.66±41.64)mg/L vs.(122.19±31.29)mg/L, t=2.131, P<0.05]. The study group also had a significantly lower grading of AL and rate of nasal fistula placement(82.4% vs.30.0%, P<0.05). In terms of ICU indicators, the study group had a significantly lower ICU admission rate(64.7% vs.10.0%, P<0.05), shorter period of ventilator use[(6.47±8.15)days vs.(0.90±2.23)days, t=2.62, P<0.05], and shorter ICU stay[(11.70±8.89)days vs.(4.70±6.27)days, t=2.184, P<0.05]. Conclusions:Double mediastinal drainage tubes, have been found to significantly alleviate inflammation, decrease the rate of nasal fistula placement and ICU admission, and shorten the length of ICU stay in elderly patients.Therefore, they are considered safe and deserving of clinical promotion.

Obesity-related hypertension is a common hypertension as well as a common chronic disease with wide distribution and great harm to human health. In recent years, this disease has become one of the hot issues of public health due to the significant increase in prevalence. The pathogenesis and pathway of obesity-related hypertension are not yet clear, and the research on its pathogenesis has received extensive attention. Studies have shown that they are regulated in most biological processes, including differentiation, proliferation, migration, and apoptosis. The miR-200 family is a group of miRNAs, which have been suggested to play a crucial role in obesity-related hypertension and glucolipid metabolism dysfunction in recent years. This paper reviews relevant research results, suggesting that the expression level of miR-200 family in obese patients with hypertension is higher than that in healthy people, which regulates the occurrence and development of hypertension through mediating oxidative stress response and GATA expression level. This review reveals the relationship between miR-200 family and obesity-related hypertension, which offers new clues to explore potential therapeutic targets for obesity-related hypertension.