Worldwide sales of biologic drugs exceeded 100 billion USD in 2011. About 32% is from therapeutic monoclonal antibody (mAb). With many blockbuster biopharmaceutical patents expiring over the next decade, there is a great opportunity for biosimilar to enter the worldwide especially emerging market. Both European Medicines Agency (EMA) and Food and Drug Administration (FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics. Rather than providing a highly abbreviated path, as in the case for small molecule chemical drug, approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis. Since mAb is the dominant category of biologic drugs, mAb will be the focus of this review. First, the United States (US) and European Union (EU) approved mAb and those in phase 3 trials will be reviewed, then strategies on how to win biosimilar competition will be reviewed.

BACKGROUND:At present, there is still lack of related reports about the aging process of in vitro cultured epidermal cells, since epidermal cells are seed cells necessary for the construction of tissue engineered skin, this articleis is aimed to investigate the biological property of normal human epidermal cells during aging process so as to provide a foundation for the selection of seed cells for tissue engineered skin OBJECTIVE: To observe the in vitro proliferation and aging property of human epidermal cells in order to provide a foundation for the proper selection of seed cells for tissue engineered skin.DESIGN: A self-comparative experiment.SETTING: Orthopedic Surgery Research Instioute of Weifang Medical College and the General Surgery Department of Weifang Medical College Affiliated Hospital.MATERIALS: This experiment was carried out at the Orthopedic Surgery Research Institute of Weifang Medical College, between September 2000and September 2002. Healthy foreskin tissue was obtained from 20 normal boys of 6-8 years old who received peritomy at the General Surgery Department of Weifang Medical College Affiliated Hospital.METHODS: Epidermal cells were obtained from normal young people for subculture. Cells were collected from different culture passages and taken as subjects, and their aging characteristics were assessed through morphological observation, population doubling time (PDT), immune cytochemistry and beta-galactosidase staining. MAIN OUTCOME MEASURES: ① The changes of the epidermal cell growth characteristics. ② The morphological changes of the epidermal cells. ③ The epidermal cell phenotypic changes. RESULTS: ① The clanges of the epidermal cell growth characteristics: Cells were in vitro cultured by monolayer for 9 passages, and PDT of P2 was the shortest. The cells showed strong proliferation in the first 5 passages.From P6, PDT was obviously prolonged, but the cells from P8 did not proliferate any longer. ② The morphological changes of epidermal cells: The primary cultured cells began to proliferate 3 days later, which accelerated 4 days later. The cells became approximately fused in about 1 week. The growth of epidermal cells was identified with a microscope and the immuno histological techniques. ③ The epidermal cell phenotypic changes: Along with the consecutive subculture, histological expression of beta-galactosidase was found to show an increasing tendency from weak expression (occupying 9% of the young cells) to strong expression (occupying 65% of aging cells), and the positive expression rate of beta-galactosidase was found to be remarkably correlated with cell passage age (r=0.87, P ＜ 0.01). CONCLUSION: ① Compared with young cells, aging cells displayed more obvious aging morphology and enzyme cyto-chemical characteristics.During the cell aging process, the PDT of cells showed an increasing tendency. ②Compared with young cells, the expression of beta-galactosidase in aging cells was remarkably increased, and this increase paralleled with the appearance of cell aging phenotype and the loss of cell proliferation capability, and reflects the aging degree of cells. ③ The in vitro cultured normal human epidermal cell aging model was established in this experiment. The results of this experiment indicated that epidermal cells from the 1st -5th passage (donators aged 16-18 years old) can be taken as the optimal seed cells for tissue engineered skin construction.

BACKGROUND: Fibroblasts are considered as seed cells necessary for the construction of tissue engineering skin. The ultrastructure of cells of various generations was observed under the electron microscope in the hope of providing foundation for proper selection of seed cells for tissue engineering skin.OBJECTIVE: To observe the ultrastructural changes of normal human fibroblasts during in vitro culture.DESIGN: Self-control observation.SETTING: Institute of Plastic Surgery, Weifang Medical College; Department of General Surgery affiliated to Weifang Medical College.MATERIALS: This experiment was carried out in the Institute of Plastic Surgery, Weifang Medical College, between September 2000 and September 2002. Healthy prepuce specimens were collected during posthetomy from normal boys aged 6-8 years after the informed consent was obtained from their guardians.METHODS: The normal human diploid fibroblasts were used to carry out consecutive subculture; cells were collected from different generations for morphological and ultrastructural observation under the inverted phase contrast microscope and transmission electron microscope.ture under the transmission electron microscope.microscope: Cells could pass on for 65-70 generations and survive for 280-300 days. Cells within 45 generations could grow rapidly, but gradually grew slowly after the 45th generation, and even displayed no proliferaunder the transmission electron microscope: There were no obvious changes in cell ultrastructure within 40 generations, but cells presented inward tolds of nucleus membrane from the onset of generations 41-65, with the ratio of cell nuclear/plasma reduced as well as cell surface process and microvilli also reduced.CONCLUSION: The ultrastructural change of in vitro cultured fibroblasts varied between different generations, which became obvious after the 41st generation, suggesting that fibroblasts within 40 generations are considered preferable seed cells for the construction of tissue engineering skin.

BACKGROUND: As the seed cells for construction of tissue engineered skin, fibroblasts directly decide the quality of tissue-engineered skin. During in vitro culture, collagen gene expression and response to epidermal growth factor (EGF) stimulation of the fibroblasts in different passages can be indicative of their proliferative capability for use as the seed cells for skin tissue engineering.OBJECTIVE: To observe the expression of type Ⅰ and Ⅲ collagen mRNA in fibroblasts cultured in vitro and fibroblast response to EGF stimulation, and thereby providing reference for the selection of optimal seed cells for tissue engineering.DESIGN: Self-controlled experiment.SETTING: Institute of Plastic Surgery, Weifang Medical College.MATERIALS: This experiment was carried out at the Institute of Plastic Surgery, Weifang Medical College between September 2000 and June 2002. The specimens of normal prepuce tissues excised by circumcision were obtained from 20 healthy boys at the age between 6 and 8 years on a voluntarily basis in the Department of General Surgery, Affiliated Hospital of Weifang Medical College.surgically excised prepuce by trypsin and type Ⅰ collagenase digestion. After cultured till 80% confluence, the cells were digested with mixed digescontrast microscope was used for dynamic observation of the cell morphology and growth status, and transmission electron microscopy and anti-vigen gene expression: Reverse transcriptase PCR (RT-PCR) was performed for amplification of type Ⅰ and Ⅲ collagen cDNA derived from the total sis of fibroblast response to EGF stimulation: The fibroblasts of P10 and P60passage were divided into treatment group with stimulation by the conditioned medium containing EGF and control group with treatment with only the conditioned medium. 3H-TdR incorporation assay was performed for analyzing the growth of the fibroblasts in response to EGF stimulation.lasts of different passages to EGF stimulation.decreased with cell passaging and 3H-TdR incorporation was lower in P60cells without significant difference between the treatment group and control group (132.5±23.6 vs 124.9±16.8, P ＞ 0.05) than in P10 cells with,however, significant difference between the two groups (512.8±56.4 vs 306.4±22.5, P ＜ 0.01).EGF stimulation is weaker than P10 cells, moreover additional EGF in the condition medium has no obvious regulation on the proliferation of P60cell growth, but extremely remarkable on P10 cells, implying along with the increase of cell passage, tritium-thymidine incorporation reduced and regulative capability of EGF on aging fibroblastic growth was also attenuated.

Objective To investigate the application value of enhanced recovery after surgery(ERAS) in laparoscopic pancreaticoduodenectomy (LPD).Methods The retrospective cohort study was adopted.The clinical data of 64 patients who underwent LPD from January 2014 to January 2016 in the Shangjin Hospital of West China Hospital of Sichuan University were collected.Of the 64 patients,41 patients managed with ERAS program between March 2015 and January 2016 were allocated into the ERAS group,23 patients managed with traditional perioperative treatment between January 2014 and Febuary 2015 were allocated into the traditional group.The following indexes were observed:(l) intraoperative status:operation time,volume of intraoperative blood loss,conversion to open surgery,pylorus preservation.(2) Postoperative status:the time to out-of-bed activity,time to postoperative flatus,time of drainage tube removal,postoperative complications (pancreatic leakage,bile leakage,hemorrhage,delayed gastric emptying,abdominal infection,cardiovascular complications),duration of postoperative hospital stay,death within the postoperative 30 days.(3) Follow-up status:incidence of complications after discharge and survival of patients.The follow-up including incidence of complications after discharge and survival of patients was conducted by outpatient examination and telephone interview up to March 2016.Measurement data with normal distribution were presented as (x) ± s and analyzed by t test.Count data were analyzed using the chi-square test or Fisher exact probability.Results (1) Intraoperative status:the operation time,volume of intraoperative blood loss,number of patients with conversion to open surgery and pylorus preservation were (377 ± 38) minutes,(164 ± 48) mL,1,40 in the ERAS group and (392 ± 53) minutes,(152 ±31)mL,2,21 in the traditional group,showing no statistically significant difference between the 2 groups (t =5.02,8.43,x2=1.29,1.29,P ＞ 0.05).(2) Postoperative status:the time to out-of-bed activity,time to postoperative flatus,time of drainage tube removal and duration of postoperative hospital stay were (1.7 ± 0.6)days,(2.5 ± 0.6) days,(5.3 ± 1.7) days,(9.1 ± 1.3) days in the ERAS group and (2.1 ± 0.9) days,(3.8 ±1.2) days,(8.2 ± 2.6) days,(11.9 ± 1.8) days in the traditional group,showing statistically significant differences between the 2 groups(t =-5.28,-7.01,-16.20,-10.67,P ＜ 0.05).The numbers of patients with pancreatic leakage in stage A,B and C,bile leakage,hemorrhage,delayed gastric emptying,abdominal inflection,cardiovascular complications and death in the postoperative 30 days were 8,0,0,0,1,3,0,1,1 in the ERAS group and 5,1,0,1,1,3,2,1,0 in the traditional group,respectively,showing no significant difference between the 2 groups (x2=0.37,1.81,0.18,0.57,3.68,0.18,P ＞0.05).(3) Follow-up status:the 64 patients were followed up for a median time of 11 months (range,1-25 months).During the follow-up,number of patients complicated with diabetes,local tumor recurrence,liver metastasis and death were 5,4,1,0in the ERAS group and 2,5,2,3 (2 died of tumor recurrence and 1 died of myocardial infarction) in the traditonal group.Conclusion Application of ERAS in the perioperative management of LPD is safe and effective,meanwhile,it can accelerate the recovery of patients who underwent LPD and shorten the duration of hospital stay.

Objective To investigate the effect of the polymorphisms of multidrug resistance 1 (MDR1) C3435T and G2677T on Tacrolimus (Tac) individualized treatment and prognosis of grafts in the renal transplantation recipients (RTRs).Methods One hundred and twenty-seven RTRs who treated with Tac regimen and had a stable graft function were enrolled,and were divided into adjuvant treatment group and non-adjuvant treatment group according to whether given adjuvant drugs to raise Tac trough concentrations. MDR1 C3435T and G2677T SNPs were detected by using sequence specific primers PCR.Tac trough concentrations of whole blood were measured by using enzymelabeled immunosorbent assay.Tac concentration-to-dose ratio (C/D) standardized by body weight was compared according to the various genotypes and haplotypes of MDR1 C3435T and G2677TA SNPs.Results Adjuvant treatment group including 36 recipients had a higher frequency of C genotype of C3435T than un-adjuvant treatment group (68.05％ vs 48.35％,P ＜ 0.01 ). The frequency of G2677TA polymorphisms was of no significant difference between the two group recipients (P＞ 0.05).As to non-adjuvant treatment recipients,the mean Tac DD required and C/D were not significantly different among various polymorphisms of MDR1 G2677T/A and C3435T or various haplotypes (P＞0.05).During A follow-up period of 4 years,13 recipients suffered graft dysfunction in which 84.6％ (11/13) carried 3435C genotype (P＞0.05).Conclusion The frequency of MDR1 C3435T polymorphisms in RTRs is high in the recipients given adjuvant treatment to raise Tac concentrations.Recipients with 3435C genotype were prone to graft dysfunction.

Objective To develop the hypothesis ‘saturated or non-saturated cytotoxicity model' and explain the various phenomena of antibody mediated immunoresponses in recipients,including rejection and accommodation.Methods The imitating complement dependent cytotoxicity.The threshold set to identify as saturated or non-saturated cytotoxicity depends on antigen-antibody complex(R)whether or not above lethal number(D)in effective time.Feasibility of the hypothesis was examined through explaining various phenomena mediated by anti-donor antibodies,especially some contradictory phenomena.Results Hyperacute rejection,accelerated rejection and acute rejection could be well explained by saturated cytotoxicity.Accommodation of ABO imcompatible transplantion,de novo antibody induced injury,change of protein profile,and C4d deposition in graft could be well elucidated by the hypothesis.Conclusion The hypothesis saturated or nonsaturated cytotoxicity model' help to interpret and interconnect various phenomena of antibodies mediated immune response,such as rejection and accommodation.

Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.

Objective To investigate the protective effect of hydrodynamics-based injection with plasmids of IL-10, TGF-β1 and TGF-β1 + IL-10 in murine skin transplantation model. Methods Plasmids were constructed by inserting coding sequences of IL-10 and TGF-β1. In F1 mice (Balb/c×C57BL/6, H-2b/d) to Balb/c (H-2d) murine skin transplant model, 20 μg plasmid (blank or IL-10 or TGF-β1 or IL-10 + TGF-β1) was injected to donors by hydrodynamics-based method in first day and every interval 2 days for 6 times. The survival of grafts was observed after 7 days of transplantation. After C56BL/6 spleen cells transfused Balb/c accepted 5 times hydrodynamics-based injection as above,CD4+ CD25+ T regulatory cells of spleen were measured by FACS. Results The survival time of graft in each group was (13.50±1.04)days (blank group), (13.83±1.16)days (IL-10 group), (15.33±1.50) days (TGF-β1 group), and (21.33±3.20) days (IL-10 + TGF-β1 group),respectively (P<0.05). The percentage of CD4+ CD25+ cells was (6. 58±1.86)% (blank group),(10.52±1.13)% (IL-10 group),(14.44±0.42)% (TGF-β1 group),and (14.25±1.24)% (IL-10+TGF-β1 group) respectively (P<0.05). Conclusion Hydrodynamics-based transfection of IL-10 combined with TGF-β1 can synergistically enhance the percentage of CD4+ CD25+ T cells and prolong the graft survival.

ObjectiveTo understand the characteristics of mutations in BCR-ABL1 kinase domain mutation,these chronic myeloid leukemia (CML) and Ph positive acute lymphoblastic leukemia (ALL)patients who got imatinib treatment had poor effect.MethodsTotally 177 CML patients and 33 Ph( + )ALL patients were selected at Beijing Dao-Pei Hospital from Sep.2007 to Dec.2010.All of them were Chinese patients.Totally 243 bone marrow or peripheral blood specimens were collected from the patients,who had early effect,then resistance emergenced,or for more than 3 months of poor efficacy.Extracted total RNA from the specimens' nuclear cells,reversed transcription to cDNA.Amplified the whole span of BCRABL1 fusion kinase gene by nest PCR (from 242 to 493 amino acid coding sequence),used the type AB3130XL gene sequencing instrument determinate the gene sequence of ABL1 kinase region and then used the Variant Reporter V1.0 software to analyze the results of gene mutations.ResultsThirty-two kinds of different mutations were detected of ABL1 gene mutations,accounting for 34.2％ (83/243 cases).Among them,the T315I was 12％ (10/83),mutation rate was the highest,followed by Y253H was 11％ (9/83),G250E was 7％ (6/83),E255K was 7％ (6/83),M351T was 6％ (5/83),E459K was 5％ (4/83) ;Q252H,D276G,F317L,E355G,F359V,H396R were all 4％ (3/83).Three cases of insertion mutations were found,including 2 cases of 357-358insk,1 case of V304RfsX17.Seven patients had found existence two or more point mutations.The multiple drug resistance mutations might exist in the same leukemia clone.The same individual was not only contain common resistance mutations,but also rare point mutations,insertion mutations.The mutations might be lead to loss of kinase activity.ConclusionsUnder the imatinib drugs pressure,the ABL1 gene mutation in leukemia cells appears randomly,and results in different resistant clones.Different resistant clones can coexist in the same patients in vivo; resistant clones not only contain point mutations,but also contain inserted deletion mutations.