Objective: To analyze the serological phenotypes and molecular genetic characteristics of a proband with ABO forward and reverse typing discrepancy and their family members, and to investigate the molecular genetic mechanism underlying the Ael/AelB phenotype caused by the c. 575T>C variant on an ABO A1.02 background. Methods: Serological testing and adsorption-elution assays were performed for blood group identification of the proband and family members. PCR-SBT, cloning sequencing, and Nanopore third-generation sequencing were used to analyze ABO gene variants and haplotypes. Phyre2 and PyMOL were used to predict the effect of the p. Ile192Thr variant on the structure of glycosyltransferase A (GTA), and plasma GTA activity was evaluated using an in vitro transferase assay. Results: Among thirteen family members, four members showed a serological phenotype consistent with the Ael subtype and one with the AelB subtype. Cloning sequencing and third-generation sequencing confirmed that five family members carried the ABO A1.02 (c. 575T>C) variant, which co-segregated with the weak A phenotype in the family. Structural modeling suggested that p. Ile192Thr may alter the local spatial conformation and hydrogen-bond interactions of GTA. Plasma GTA activity testing showed markedly reduced GTA activity in the tested individuals. Conclusion: The ABO A1.02 (c. 575T>C) variant is associated with the Ael/AelB phenotype. The p. Ile192Thr substitution may weaken A antigen expression by altering GTA conformation and reducing enzymatic activity. This study provides additional evidence for the role of this variant in the Ael/AelB phenotype from the perspectives of familial inheritance, full-length haplotype analysis, and functional assessment, which is important for accurate ABO subtype identification and individualized transfusion strategies.

Objective To establish an effective and stable rat model of skin-infection for evaluating the therapeutic effects of topical anti-infection drugs.Methods SD rats were subcutaneously injected with cyclophosphamide to induce immunosuppression,and then surgical incisions were made on both sides of the spine.The rat model of skin-infection was established by applying methicillin-resistant Staphylococcus aureas suspension to the incisions for two consecutive days,and evaluated by analyzing infection status,pathological changes and healing time.Results The development of pyogenic infection was detected in all of the rats.Compared with the non-cyclophosphamide treated group,the cyclophosphamide treated group showed a more severe infection both from the visual inspection and the microscopic observation,moreover,its healing time was delayed more than 10 days.Conclusion The skin-infection model was successfully established in immunosuppressed rats induced by cyclophosphamide,which could be applicable to the efficacy evaluation of anti-infection drugs for external use on skin infection.

OBJECTIVETo investigate the specific cell-mediated immune efficacy of the an HPV16 prophylactic vaccine.

METHODSC57BL/6 mice were randomly divided into 3 groups: experimental group I (treated with pcDNA L1), control group II (treated with pcDNA3.1) and control group III (treated with PBS buffer). The mice were immunized three times during a three-week interval. Ten to fourteen days after the third inoculation, a footpad swelling test was used to detect delayed-type hypersensitivity (DTH) responses. Antigen-specific splenocyte proliferation assay and quantitation of IFN-gamma cells in splenocytes were performed by FACS assay.

RESULTSIn the experimental group, splenocytes actively proliferated after stimulation with HPV16 VLP, and had developed a markedly larger amount of CD8(+) IFN-gamma(+) cells, which is an index for special CTL. Also, the footpad was significantly thickened upon inoculation with HPV16 VLP.

CONCLUSIONNaked DNA vaccine of HPV16 L1 can induce specific cell-mediated immune responses in mice, which should be considered for evaluation of HPV16 DNA vaccine feasibility.

Animals ; Hypersensitivity, Delayed ; etiology ; Immunization ; Interferon-gamma ; biosynthesis ; Lymphocyte Activation ; Mice ; Mice, Inbred C57BL ; Papillomaviridae ; immunology ; Spleen ; cytology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

Objective It has been well accepted that human papillomavirus type 16 (HPV16) is associated with human cervical cancer and HPV16 L1 protein could induce both humoral and cellular immune responses. The objective of this study is to map epitopes on HPV16 L1 protein and to provide information to the design of HPV16 prophylactic peptide vaccine.Methods The epitopes on L1 protein were screened by polyclonal and 2 monoclonal antibodies (B8 and F4G3) against HPV16 L1 protein from a 6-mer phage display epitope library with the method of immuno-affinity screening (Bio-panning). After 3 rounds of Bio-panning, the positive phages were detected by L1 antibodies again with ELISA. The positive phages reacted strongly with L1 antibodies were then identified by DNA sequencing.Results Three mimotopes have been screened by polyclonal and two monoclonal antibodies. The mimotope (LSLFSC) which reacted with monoclonal antibody B8 showed 50% homology with the sequence 270～275a.a (DSLFFY) of prototype HPV16 L1. Another mimotope (LTSSYS) which reacted with polyclonal antibodies had 66% homology with the L1 sequence 516～521a.a (TTSSTS), also a mimotope (DRWDRF) was found to have the homologic RF with the known L1 sequense 441～446a.a. The mimotopes LSLFSC and DRWDRF were adjacent to the epitopes at 267～269a.a and 422～441a.a reported by other researchers previously.Conclusions Our results suggested that there might be a batch of epitopes on HPV16 L1 protein, and the predominant epitopes of HPV16 L1 protein were located in the above two domains. These results would be heplful for the design of HPV16 prophylactic peptide vaccines and HPV polyvalent vaccines.

The TNA mRNA expression in 33 samples of cervical carcinoma and 28 samples of condyloma have been observed by using ~3H and Digoxigenin labeled TNF—? cDNA probes and in situ hybridization technique, we found that the results of in situ hybrdfzation with these two kinds of labeled probes were alike, the sensitivity with ~3H labeled probe was slightly higher than that with Digoxigenin labeled probe. Nevertheless, the nonisotope probe would be used more and more in future because of its safety, rapidity and convenience in work.