OBJECTIVETo study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection.

METHODSFab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of V(H) and V(L) and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-kappa-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G.

RESULTSSDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein.

CONCLUSIONIgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 microg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

Amino Acid Sequence ; Animals ; Base Sequence ; Cell Line ; Cloning, Molecular ; Cytomegalovirus ; immunology ; DNA, Viral ; genetics ; Genes, Viral ; Humans ; Immunoblotting ; Immunoglobulin G ; immunology ; Immunoprecipitation ; Insecta ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; immunology

Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human eytomegalovirus (HCMV) infection. Methods Fab monochinal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of Vhand Vland neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-κ-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibodyrecognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

BACKGROUNDCloning recombinant human Fab fragment against HCMV for the purpose of prophylaxis and control of HCMV infection.

METHODSThe authors constructed a HCMV phage display library with 2 x 10(6) clones, then used purified HCMV viral lysates to pan the library, then screened by ELISA.

RESULTSThree clones showed positive responses in ELISA, they also showed high specificity in IFA, two of them could neutralize HCMV in neutralizing assays.

CONCLUSIONThe specific binding of Fab antibodies to HCMV was demonstrated by ELISA, IFA and neutralizing activities. These results provide us the basis for further research of neutralizing recombinant human whole IgG molecule.

Antibodies, Viral ; genetics ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; immunology ; virology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Neutralization Tests ; Peptide Library

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; CHO Cells ; Cricetulus ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Thrombomodulin ; immunology ; Transfection

OBJECTIVETo investigate the constitution and health status of the Beijing residents, and the primary influencing factors on nutrition and health, as to finding out the epidemiological characters and the changing trend of chronic diseases and to establish scientific basis for the establishment of public health and disease prevention policies of Beijing.

METHODSA stratified multi-stage cluster randomly sampling method was used. In total, 18 districts were surveyed, and among them, 9 were conducted dietary survey. The investigation was based on household, and all family members were surveyed at their home. After having got the compliance from the family members, question asking, medical examination, laboratory test and dietary investigation were carried out in these people.

RESULTSThe incidence rate of hypertension was 25.0% for the resident > or = 15 year-old, and over 50% for middle-aged and elderly people (> or = 45 years old). The incidence of diabetes for those not younger than 15 years old was 7.7%. The over-weight rate and the obesity rate for adults were 35.1% and 20.6%, respectively.

CONCLUSIONThe following risk factors of chronic disease, such as unreasonable diet pattern, deficient intake of some micronutrients, over-weight and obesity, lack of physical examination, smoking and over-drinking, should be the important factors influencing the health of Beijing residents severely.

China ; epidemiology ; Diabetes Mellitus ; epidemiology ; Female ; Health Status ; Humans ; Hypertension ; epidemiology ; Incidence ; Life Style ; Male ; Nutritional Status ; Obesity ; epidemiology ; Surveys and Questionnaires

OBJECTIVETo observe amplitude changes of low frequency fluctuation in brain spontaneous nervous activities induced by needling at Hand Taiyin Lung Channel, and to preliminarily explore the possible brain function network of Hand Taiyin Lung Channel.

METHODSBy using functional magnetic resonance imaging (fMRI), 16 healthy volunteers underwent resting-state scanning (R1) and scanning with retained acupuncture at Hand Taiyin Lung Channel (acupuncture, AP). Data of fMRI collected were statistically calculated using amplitude of low frequency fluctuations (ALFF).

RESULTSUnder R1 significantly enhanced ALFF occurred in right precuneus, left inferior parietal lobule, bilateral superior temporal gyrus, bilateral middle frontal gyrus, left superior frontal gyrus, left inferior frontal gyrus, left medial frontal gyrus. Under AP significantly enhanced ALFF occurred in right precuneus, bilateral superior frontal gyrus, cerebellum, bilateral middle frontal gyrus, right medial frontal gyrus, and so on. Compared with R1, needing at Hand Taiyin Lung Channel could significantly enhance ALFF in right gyrus subcallosum and right inferior frontal gyrus. Significant decreased ALFF appeared in right postcentral gyrus, left precuneus, left superior temporal gyrus, left middle temporal gyrus, and so on.

CONCLUSIONNeeding at Hand Taiyin Lung Channel could significantly change fixed activities of cerebral cortex, especially in right subcallosal gyrus, right inferior frontal gyrus, and so on.

Acupuncture Therapy ; Brain ; physiology ; Brain Mapping ; Humans ; Magnetic Resonance Imaging

Objective To observe the clinical efficacy of infrared laser combined with alpha-zinc sulfate for the treatment of diabetic peripheral neuropathy ( DNP).Methods A total of 106 patients with DPN were randomly divided into observation group ( n =53 ) and control group (n=53).Besides base hypoglycemic drug therapy, patients in the control group received alpha-zinc sulfate injection 0.6 g once a day.The patients in observation group were given the above treatment plus anodyne therapeutic apparatus treatment.The treatment period lasted for 12 days.The data of clinical symptoms, temperature, and nerve conduc-tion velocity of two groups were observed before and after treatment.Results The effective rate and total effective rate were 52.83% and 90.57%in the observation group, significantly better than 41.51%and 86.66%in control group ( P<0.05 ).The data of peroneal nerve and sural nerve conduction velocity in observation group were much signifi-cantly improved than before treatment(P <0.05).No significant adverse effect was observed.Conclusion Infrared laser combined with alpha-zinc sulfate is effective on the treatment of diabetic peripheral neuropathy.

Objective To evaluate the molecular mechanism of You-guiyin inhibit the activity of rat osteoclasts through inhibiting Annexin A 2 ( AnxA2) and Atp61.Methods One hundred and five rats of collagen-induced arthritis ( CIA) were divided in to 7 groups:high, moderate and low doses Youguiyin groups , methotrexate group , lentiviruses ( +) group, lentiviruses ( -) group and model control group.And 15 rats without any treatment were included as blank group.Rats in the Yougui-yin groups were given Youguiyin intragastric.Rats in the methotrexate group were given methotrexate 1 μg· g -1 intragastric.Rats in the lenti-viruses (+) group were given 2.5 ×10 6 TU AnxA2 and Atp6 i lentiviru-ses double knee joints injection.Rats in the lentiviruses (-) group were given lentiviruses double knee joints injection.At the time of 8 weeks af-ter administration the rats were put to death.The serum level of tartrate resistant acid phosphatase -5b(TRACP-5b), the interleukin -1 beta ( IL-1β) , and knee joint bone density were evaluated.The change of the knee joint bone cortex in rats was evaluated by molybdenum target X-ray before and after administration.Results After 8 weeks, the IL-1βand TRACP -5b levels in CIA model group were much higher than that of blank group (P<0.05).IL-1βand TRACP-5b levels in Youguiyin, methotrexate, and lentiviruses ( +) groups were much lower than that of CIA model group ( P<0.05 ).The bone density in the Youguiyin and methotrexate groups were lower than the blank group ( P<0.05 ) but significant improve in the high dosages Youguiyin group than that of moderate and low dosage groups ( P<0.05 ) .After administration with Youguiyin , bone cortex is complete , bone mass distribution is uni-form according to X-ray in the high dosage group compared with the moderate and low groups.Conclusion In the process of osteoclasia in rats with rheumatoid arthritis , Youguiyin can inhibit the osteoclasia effects by regulate and con-trol the AnxA2 and IL-1βexpression.

The study was aimed at the exploration of relationship between T cells expressing killer cell inhibitor receptors (KIR, CD158 and CD94) and graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. The expression rates of CD158a, CD158b and CD94 on T cells and NK cell were detected by flow cytometry and donor/recipient HLA-Cw was analyzed using PCR after peripheral blood stem cell transplantation (PBSCT) and umbilical cord blood transplantation (UCBT). After both PBSCT and UCBT, the rates of CD3(+)CD158a(+) and CD3(+)CD158b(+) T cells increased, especially the rate of CD8(+)CD158b(+) T cells. In both acute and chronic GVHD groups, the rate of CD3(+)CD158b(+) T cells increased, especially in acute GVHD. The CD94 mainly expressed on CD3(+)CD8(+) T cells. The percentage of the expression of CD94 on CD4(+) and CD8(+) cells after UCBT and PBSCT increased significantly. The expression of KIR in GVHD (early stage of transplantation) increased but the expression of KIR in chronic GVHD (advanced stage of transplantation) decreased. Five patients who HLA-Cw matched had no severe GVHD. In four patients who underwent allo-PBSCT and UCBT from related HLA-matched donors, only 2 patients had no aGVHD. Four patients underwent transplantation from unrelated HLA-matched donors had GVHD. These observations suggested that there is some relationship between GVHD and KIR expression on T cells. CD158b might be an inhibitory molecule of T cell activated at early stage after transplantation. Understanding the mechanism of GVHD with the expression of KIR on T cells, especially those binding the HLA-Cw might shed light on the establishment of the specific immunotolerance for the prevention of GVHD. To pay attention to HLA-Cw typing is very important to reduce GVHD and increase GVL effect in related or unrelated HLA-matched transplantation.
Antigens, CD ; analysis ; Antigens, Differentiation, T-Lymphocyte ; analysis ; Genotype ; Graft vs Host Disease ; etiology ; HLA-C Antigens ; genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Lectins, C-Type ; Receptors, Immunologic ; analysis ; Receptors, Interleukin-2 ; analysis ; Receptors, KIR ; Receptors, KIR2DL1 ; Receptors, KIR2DL3 ; T-Lymphocytes ; immunology

OBJECTIVETo explore the effects of surgical procedure combined with the intravascular minimal invasive technique for the treatment of deep vein thrombosis (DVT) of lower extremity.

METHODSAt the curse of disease from six hours to ninety days, one hundred and two patients with DVT including one hundred and three lower extremities had received surgical procedure and intravascular minimal invasive treatment.

RESULTSThere were not procedure-related morbidities in 102 cases, and symptoms disappeared, all procedures were successful based on angiography. The detecting head for the intravascular ultrasound ablation was entered to inferior vena cava (IVC) in 74 cases (78%), Forgarty catheter was entered to IVC in 21 cases (21%), the stenosis in the confluence of the common iliac vein and IVC was dilated by sacculus rotundus catheter in 89 cases (88%), including 9 patients underwent percutaneous transluminal stenting. One hundred and two patients followed up for twenty months, follow-up by angiography showed no restenosis in 91 cases, restenosis in ilio-femoral vein in 1 cases, and thrombus recontouring in 4 cases, as well as 6 cases died caused by primary disease.

CONCLUSIONSurgical procedure combined with the intravascular minimal invasive technique is a safe and effective therapeutic method for DVT.

Adult ; Aged ; Aged, 80 and over ; Angioplasty, Balloon ; Catheter Ablation ; methods ; Combined Modality Therapy ; Female ; Follow-Up Studies ; Humans ; Lower Extremity ; Male ; Middle Aged ; Minimally Invasive Surgical Procedures ; Stents ; Ultrasonic Therapy ; Venous Thrombosis ; surgery ; therapy