Objective To discuss a real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) wether if can be used to detect Brucella. Methods According to the BCSP31 gene sequences specific for Brucella, one pair of primers and one TaqMan probe were designed. A real-time PCR was developed with the BCSP31 fragments cloned into PMD18-T vector. The standard cure was established and the sensitivity, the species specificity and the stability of the assay were evaluated. The clinical blood specimens were detected by QT-PCR and compared with clinical diagnosis. Results The standard curve was established with the standard template and the relationship between the Ct and the DNA copy number was linear(r=0.999). The sensitivity of the real-time PCR was 5 copies/μl. The sensitivity of the common PCR was 5×102 copies/μl. The sensitivity was about 100 times higher than common PCR. Species specificity of this FQ-PCR assay evaluated using genomic DNA from 6 Bmcella strains and 5 non-Brucella strains and strong fluorescence was detected in all Brucella strains. The CV of intra-assay and inter-assay reproducibility were 0.71%,7.23%, reprectively. Twenty-four specimens from clinical brucellosis cases, 19 showed positive, the positive coincident rate was 79%(19/24). The negative results were obtained for all 31 negative control, and the negative coincident rate was 100%(31/31). Two were positive from all 30 specimens clinically suspected. Conclusions Highly specific, sensitive, repeatable and coincidental with clinic, this FQ-PCR is quite useful for rapid detection of tiny DNA of Brucella in various samples and laboratory diagnosis.

OBJECTIVETo identify the ETV6 gene rearrangement in patients with myelodysplastic syndromes (MDS) and explore its relationship with prognosis and disease stages.

METHODSETV6 rearrangement in 58 MDS cases were detected by conventional cytogenetics and Split-signal FISH. RT-PCR was used to detect 9p24-12p13 balance translocation with special designed primers ETV6F1/F2 and JAK2R1/R2. The relationship between ETV6 rearrangement and prognosis and disease staging in MDS patients was analyzed.

RESULTSETV6 rearrangement were found in 4 (6.9%) of 58 cases, among which ETV6/JAK2 fusion was identified by RT-PCR in 1 (1.7%) case. The mean follow-up duration was 12 months. All 4 patients (100%) with rearrangement transformed into acute leukemia, with a median survival time (MS) of 7 months; while 10 patients (17%) in the non-translocation group transformed to acute leukemia, with a MS of 28 months. In addition, all 4 patients (100%) with rearrangement were in advanced stage of MDS( RAEB), while 17 cases (31.5%) in non-rearrangement group were in that stage.

CONCLUSIONSETV6 rearrangement has higher expression rate (6.9%), and is closely associated with disease stage and prognosis in MDS.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; pathology ; Neoplasm Staging ; Prognosis ; Proto-Oncogene Proteins c-ets ; genetics ; Repressor Proteins ; genetics

A recombinant vaccinia virus (rWR-SARS-S)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. The expression of full length recombinant SARS spike protein (rSS) in HeLa cells possessing specific reaction ability to chicken anti-sera was confirmed by SDS-PAGE and Western-blot (190 kD). HeLa cells infected with rWR-SARS-S also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (IFA). The results above indicated that the availability of such a faithful model system offers particular advantages for the study of SARS in that it reduces the need for direct manipulation of an exotic pathogen. In the absence of infectious SARS, we may safely carry out detailed biochemical and genetic manipulations to investigate features of viral replication and gene function, as well as explore new avenue for vaccine development.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Blotting, Western ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; HeLa Cells ; Humans ; Immune Sera ; immunology ; Immunization ; methods ; Membrane Glycoproteins ; genetics ; immunology ; Mice ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; immunology

Antibody-drug conjugate (ADC) is a new class of therapeutics composed of a monoclonal antibody and small cytotoxin moieties conjugated through a chemical linker. ADC molecules bind to the target antigens expressed on the tumor cell surfaces guided by the monoclonal antibody component. The binding ADC molecules can be internalized and subsequently the toxin moieties can be released within the tumor cells via chemical and/or enzymatic reactions to kill the target cells. The conjugation combines the merits of both components, i.e., the high target specificity of the monoclonal antibody and the highly potent cell killing activity of the cytotoxin moieties. However, such complexities make the pharmacokinetic and metabolic studies of ADCs highly challenging. The major challenges should include characterization of absorption, distribution, metabolism and excretion, investigation of underlying mechanisms, assessment of pharmacokinetic- pharmacodynamic relationship, and analytical method development of ADC drugs. This review will discuss common pharmacokinetic issues and considerations, as well as tools and strategies that can be utilized to characterize the pharmacokinetic and metabolic properties of ADCs.
Antibodies, Monoclonal ; pharmacokinetics ; Cytotoxins ; pharmacokinetics ; Humans ; Immunoconjugates ; pharmacokinetics ; Neoplasms ; drug therapy

Objective To investigate the flexibility of both the covered stents specially designed for use in intracranial vasculature and the delivering system in passing through the bone tube and the physiological curves of the cranial internal carotid artery(CICA)to reach the targeted area,the performance (adherence)of the covered stents in occluding vascular wall diseases and the impact on the vascular branches of the covered segment.Methods The covered stents specially designed for use in intracranial vaseulature were used to treat 13 patients with CICA diseases using endovascular techniques.There were 4 huge pseudoaneurysms,4 giant aneurysms,3 small wide-necked aneurysms,1 giant pseudoaneurysm with concurrent internal carotid cavernous fistula(CCF),and 1 CCF.Prior to the detachment of the covered stents,balloon occlusion test(BOT)of the internal carotid artery on the diseased side and whole-brain digital subtraction angiography(DSA)were performed in all the patients.Three to 16 months following procedure,DSA and clinical follow-ups were performed.Results Thirteen patients all tolerated the BOT well with the DSA demonstrating well-opened anterior and posterior communicating arteries.The covered stents and the delivering systems all successfully passed CICA to reach the targeted diseased area,with the diseased segments of the internal carotid artery including C3—C4 in 4 cases,C4—C5 in 4 and C6—C7 in 5.Immediately following the detachment of the covered stents,DSA demonstrated that 7 aneurysms were completely occluded,4 aneurysms had slight endoleak,and 1 CCF had markedly-decreased blood flow through the fistula.In the patient with concurrent pseudoaneurysm and CCF,the pseudoaneurysm disappeared and the blood flow through the fistula was markedly-reduced immediately following the stenting procedure.Apart from one patient with aneurysmal subarachnoid hemorrhage who died due to extensive vascular spasm on the 9th day following the stenting procedure,all the other 12 patients had unobstructed stented vessels on the follow-up DSA images,with 2 demonstrating slight stenosis.In the 6 patients with post-procedure endoleak,DSA showed that the endoleak in 4 patients had disappeared,one endoleak disappeared following the second stenting,and one CCF remained low-flow fistula.There was no sequela related to the occlusion of branches in the covered arterial segment.Conclusion The covered stents specially designed for use in the intracranial vasculature and the delivering system are both flexible enough to pass the tortuous CICA to reach the intracranial diseased artery,and are effective in managing CICA diseases.Further follow-up is still needed to determine the long-term effect of the covered stents,and the adherence of the covered stents needs further investigation.

The full-length porcine interferon gamma(PoIFN-gamma) cDNA, including the secretion signal peptide coding region was recloned into honor plasmid pFastBac 1 of Bac-To-Bac Baculovirus Expression System. These recombinant plasmids, pFastBac -PoIFN-gamma, were transformed into DH(10Bac) host bacteria to get recombinant shuttle plasmids, rBacmid-PoIFN-gamma. Recombinant baculovirus, rBac-PoIFN-gamma, was generated for expressing PoIFN-gamma, by transfecting rBacmid-PoIFN-gamma with Cellfectin Reagent into sf9 insect cells. The expression of PoIFN-gamma in insect cells was confirmed by Western Blot, indirect immunofluorescence assay and indirect ELISA. The antiviral activity assay shows that PoIFN-gamma expressed by the rBac-PoIFN-gamma can efficiently inhibit the replication of the recombinant Vesicular Stomatitis Virus expressing green fluorescence protein in PK-15 cells. The antiviral activity of PoIFN-gamma can be specifically blocked by anti-PoIFN-gamma mouse serum. The antiviral titer of culture supernatant of insect cells infected by rBac-PoIFN-gamma is 2 x 10(4) IU/mL. The results demonstrat that the rBac-PoIFN-gamma can express rPoIFN-gamma efficiently and rPoIFN-gamma has high antiviral activity.
Animals ; Antiviral Agents ; metabolism ; pharmacology ; Baculoviridae ; genetics ; Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; Green Fluorescent Proteins ; genetics ; metabolism ; Immune Sera ; immunology ; Interferon-gamma ; genetics ; immunology ; metabolism ; pharmacology ; Mice ; Mice, Inbred BALB C ; Microscopy, Fluorescence ; Recombinant Proteins ; Spodoptera ; Swine ; Vesiculovirus ; genetics ; Virus Replication ; drug effects

Adolescent ; Fucosyltransferases ; genetics ; Gene Rearrangement ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Male ; Protein-Tyrosine Kinases ; genetics

In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.
Animals ; Antigens, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nipah Virus ; chemistry ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

The two mammalian codon optimized genes, F and G genes of Nipah virus, were generated by assembly PCR, and inserted into mammalian expression vector pCAGGS under chicken beta-actin promoter to construct pCAGG-NiV-F and pCAGG-NiV-G. Syncytium formation was induced in BHK cells by plasmid pCAGG-NiV-F and pCAGG-NiV-G transfection, which indicate recombination proteins F and G were expressed in BHK cell and possessed good biologic activity. Six-week-old female BALB/c mice were intramuscularly primed with 100 microg pCAGG-NiV-F, pCAGG-NiV-G or pCAGG-NiV-F+ pCAGG-NiV-G respectively, and boosted with same dose after 4 weeks. The sera were collected at 3 weeks post second boost. The serum IgG against Nipah virus F and G proteins was detected by indirect ELISA using recombinant Baculovirus expressed Nipah F and G glycoproteins. The results showed that specific antibodies possessed good sensitivity and specificity. Furthermore, the G and F proteins' specific antibodies could neutralize the infectivity of VSVdeltaG* F/G (the NiV F and G envelope glycoproteins psudotyped recombinant vesicular stomatitis virus expressing green fluorescence protein). And, pCAGG-NiV-G also induced higher titer of neutralizing antibody response than pCAGG-NiV-F did. The result indicates that DAN immunization is an efficient vaccine strategy against Nipah virus.
Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Mice ; Mice, Inbred BALB C ; Nipah Virus ; immunology ; Vaccines, DNA ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Vaccines ; immunology

Objective:To evaluate the effect of different doses of cisatracurium on the monitoring of motor-evoked potentials (MEPs) in the patients undergoing intracranial aneurysm clipping.Methods:Eighty patients of both sexes, aged 18-64 yr, with body mass index <30 kg/m 2, of American Society of Anesthesiologists physical statusⅠor Ⅱ, with Hunt-Hess grade 0-Ⅱ, undergoing intracranial aneurysm clipping under general anesthesia, were divided into 4 groups ( n=20 each) using a random number table method: conventional group (group R) and different doses of cis-atracurium groups (Cis 1-3 groups). After anesthesia induction, muscle relaxation was monitored, and train-of-four (TOF) stimulation (frequency 2 Hz, wave width 0.2 ms, interval 15 s) was applied to the ulnar nerve of forearm, and the TOF ratio was recorded as the baseline value (T 1). MEPs were assessed with a nerve electrophysiology monitor after induction of anesthesia.In Cis 1-3 groups, cis-atracurium 0.625, 0.833 and 1.000 μg·kg -1·min -1 were intravenously infused, while the equal volume of normal saline was given instead in group R when TOF ratio returned to the baseline value and MEPs could be effectively elicited.At T 1, immediately after dural incision (T 2), immediately after aneurysm occlusion (T 3) and immediately after dural closure (T 4), the TOF ratio, effective elicitation of intraoperative MEPs, and occurrence of intraoperative cardiovascular events, recovery of spontaneous breathing and body movement were recorded. Results:Compared with group R, no significant change was found in TOF ratio at each time point in group Cis 1 ( P>0.05), and TOF ratio was significantly decreased at T 2-4 in group Cis 2 and at T 2-4 in group Cis 3 ( P<0.05). Compared with group Cis 1, TOF ratio was significantly decreased at T 3, 4 in group Cis 2 and at T 2-4 in group Cis 3 ( P<0.05). The effective elicitation rate of MEPs was 100% in the four groups.There was no significant difference in the incidence of intraoperative cardiovascular events, recovery of spontaneous breathing and body movement among the four groups ( P>0.05). Conclusion:Continuous intravenous infusion of cis-atracurium 0.833-1.000 μg·kg -1·min -1 can maintain a certain degree of muscle relaxation without affecting MEP monitoring in the patients undergoing intracranial aneurysm clipping.