BACKGROUND:Nuclear transcription factor(NTF) ?B is a transcription factor,which exists universally in eukaryocyte.It has been identified that its overacting is correlated to the ongoing and development of many diseases.Among the pathomechanism of rheumatoid arthritis,the abnormal activation of NTF ?B has a lot to do with the inflammatory hyperplasia of synovium and the erosion of tissue around articulation.OBJECTIVE:To review the recent progress of NTF ?B,pathogenesis and treatment of rheumatoid arthritis.RETRIEVAL STRATEGY:A computer-based online search of PubMed database was undertaken to identify the articles published in English from January 2000 to July 2007,with the Key words of "nuclear transcription factor ?B,rheumatoid arthritis,pathogenesis,treatment".Meanwhile,Wanfang database was searched for the related Chinese articles published between January 2000 and July 2007,with the same key words in Chinese.A total of 72 articles were finally selected for the first trial.Inclusive criteria:the articles focus on the NTF ?B,the pathogenesis and treatment of rheumatoid arthritis.Exclusive criteria:repeated experiments.LITERATURE EVALUATION:Among the 30 inclusive literatures,7 were related to reviews while the others were clinical or basic researches.DATA SYNTHESIS:①NTF ?B is a significant transcription factor,which participates in regulating many genes related to immune function and inflammation reaction.The promoters of many genes have the binding sites of NTF ?B.②It is indicated that the abnormal activation of NTF ?B plays an important role in the pathogenesis of rheumatoid arthritis.In recent years,many researches inhibiting its activation through different components of signaling pathways have been doing,which become very hot in anti-inflammatory and anti-rheumatism treatment.③In recent years,people have been making great progress in the therapy of rheumatoid arthritis aiming at cytokine and its receptor,however,interfering NTF ?B to treat rheumatoid arthritis is still in study stage.CONCLUSION:NTF ?B target therapy provides a new therapeutic strategy for the treatment of rheumatoid arthritis,which is a very exciting prospect indeed.

BACKGROUND:The etiological factor for rheumatoid arthritis remains unclear, but the effects of nuclear factor-κB on the onset of rheumatoid arthritis have been gradual y paid great attention by rheumatologists.
OBJECTIVE:By using the RNA interference (RNAi) technique to block the signal pathway of nuclear factor-κB
mRNA of human rheumatoid arthritis synovial cells, this study explored its application prospect in the treatment of rheumatoid arthritis.
METHODS:The synovial cells were isolated, digested, and cultured for further use. In accordance with the
design principle of smal interfering RNA (siRNA), target sequences of siRNA of nuclear factor-κB were identified, and siRNA expression vector of nuclear factor-κB was synthesized and constructed. The four pGenesil-1/nuclear factor-κB siRNA expression vectors were transfected into the first passage of synovial cells that wel grew. Blank and negative control groups were set. cells at 12, 24, 48, 72 hours, 5 and 7 days after transfection were col ected, and RNA was extracted. Intracellular nuclear factor-κB mRNA expression levels were measured, and siRNA plasmid vector that could effectively inhibit nuclear factor-κB mRNA expression was screened out.
RESULTS AND CONCLUSION:Nuclear factor-κB highly expressed in synovial cells after human rheumatoid arthritis. 3#pGenesil-1/nuclear factor-κB apparently suppressed nuclear factor-κB mRNA expression in synovial cells with human rheumatoid arthritis. RNAi technique blocked nuclear factor-κB mRNA expression. Therefore, the block of nuclear factor-κB signal pathway might be a good target for rheumatoid arthritis gene therapy.

Modified ORF5 (MORF5) and ORF6 gene of PRRSV were cloned into two multiple cloning sites of MVA transfer vector pLR-gpt to construct the recombinant plasmid pLR-MORF5/ORF6. Homologous recombination between pLR-MORF5/ORF6 and the wtMVA on BHK-21 cell line was mediated with liposome by infecting the cell with 0.01 MOI wtMVA two hours before transfecting the recombinant plasmid into the cell. When the cytopathic effect (CPE) was obvious, virus was collected from the cell plate and the recombinant virus was selected with drug selecting medium (2% MXHAT). After 12 cycles of selection, rMVA with a selection marker Eco gpt was obtained and named as rMVAgpt-MGP5/M. By infecting BHK-Cre expressing Cre recombinant enzyme, the Eco gpt marker in rMVAgpt-MGP5/M was deleted and this rMVA was named as rMVA-MGP5/M. The insertion of MORF5 and ORF6 into the MVA genome was confirmed with PCR analysis and the expression of MGP5 and M protein was identified with Western blot and IFA. Through biological study on the recombinant MVA, no obvious difference was observed between rMVA-MGP5/M and the wtMVA regarding to the CPE and growth curve. The recombinant MVA constructed in this study could coexpress the modified GP5 and M protein and the expressed product had good immunocompetence. Furthermore, the insertion of the MORF5 and ORF6 into MVA genome had no obvious effect on the replication and biological characteristics of this virus.
Animals ; Cell Line ; Genetic Vectors ; genetics ; Porcine Reproductive and Respiratory Syndrome ; prevention & control ; Porcine respiratory and reproductive syndrome virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Swine ; Transfection ; Vaccines, DNA ; genetics ; immunology ; Vaccinia virus ; classification ; genetics ; metabolism ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Matrix Proteins ; biosynthesis ; genetics

UNLABELLEDA novel double expression shuttle vector named pLR-gpt was constructed for marker-free recombinant modified vaccinia virus Ankara generation. A delectable Eco gpt marker was adopted with Cre/LoxP DNA recombination system and a BHK-21 cell line that can express Cre enzyme. Eco gpt gene controlled by P7.5 promoter from Vaccinia virus was cloned between two LoxP sites in the same direction. Additionally, two multiple cloning site under control of other two Vaccinia virus promoters were constructed outside LoxP sites. With this new transfer vector, Eco gpt marker in rMVA can be deleted on BHK-Cre with interaction between Cre enzyme and LoxP sequence. In order to verify the efficacy of this system, ORF5 and ORF6 gene of Porcine reproductive and respiratory syndrome virus (PRRSV) NJ-a strain were cloned into two multiple cloning sites of pLR-gpt to construct recombinant plasmid pLR-ORFS/ORF6. Homologous recombination between pLR-ORF5/ORF6 and wtMVA on BHK-21 cell was mediated by liposome by infecting cells with 0.01 MOI wtMVA two hours before transfection. After twelve cycles of selection, recombinant MVA with selecting marker Eco gpt was obtained and named as rMVAgpt-GP5/M. By infecting BHK-Cre, the Eco gpt marker in rMVAgpt-GP5/M was deleted and this rMVA was named as rMVA-GP5/M. Expression of GP5 and M protein was identified with Western blotting and IFA. Results from PCR and biological study for rMVA indicated that Eco gpt marker was completely deleted.

CONCLUSIONSdouble expression transfer vector for marker-free recombinant Modified vaccinia virus Ankara generation was successfully constructed, and works well in MVA expression system.

Cell Line ; Cloning, Molecular ; DNA, Recombinant ; genetics ; Escherichia coli Proteins ; genetics ; Genetic Vectors ; genetics ; Pentosyltransferases ; genetics ; Porcine respiratory and reproductive syndrome virus ; genetics ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; Viral Matrix Proteins ; genetics

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.
Animals ; Antigens, Viral ; biosynthesis ; genetics ; Artificial Gene Fusion ; Baculoviridae ; genetics ; Capsid Proteins ; biosynthesis ; genetics ; Mice ; Parvoviridae Infections ; prevention & control ; Parvovirus, Porcine ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Somatostatin ; genetics ; Swine ; Vaccines, Virus-Like Particle ; biosynthesis ; immunology ; Viral Vaccines ; biosynthesis ; immunology ; Virion ; genetics ; immunology

Objective:To explore the efficacy and safety of single-incision transobturator bulbourethral sling suspension without skin tunnel puncture in male patients with urinary incontinence.Methods:The clinical data of 6 male patients with urinary incontinence who underwent single-incision transobturator bulbourethral sling suspension without skin tunnel puncture in Beijing Hospital from August 2023 to August 2024 were retrospectively analyzed.The age of the patients ranged from 66 to 76 years old, with an average of 71.7 years old. The disease duration ranged from 18 to 48 months, with an average of 30 months. Six patients used 1 to 3 pads per day, with an average of 2.3 pads. The International Continence Incontinence Questionnaire Short Form (ICI-Q-SF) scored 13 to 19, with an average of 15.8. The Incontinence Quality of Life Questionnaire (I-QOL) scored 5.3 to 30.6, with an average of 18.8. Three patients underwent transurethral resection of the prostate for benign prostatic hyperplasia and three patients underwent radical prostatectomy for prostate cancer. The degree of urinary incontinence was mild in 2 cases and moderate in 4 cases. The technical points are as follows: the puncture method has been changed from the traditional outside-in approach to an inside-out approach. After the puncture needle passes through from beneath the skin at the incision, the sling is guided in, avoiding the need for skin tunneling punctures. Upon completion of the puncture, the ends of the sling on both sides are tied with a certain tension at the midline of the incision, and the incision is then closed layer by layer. The efficacy and safety of surgery were evaluated by recording the number of daily pad use, subjective scoring scale [International Committee on Urinary Incontinence Questionnaire-Short Form (ICI-Q-SF), Incontinence Quality of Life (I-QOL)] and complications at 1 month after surgery. Social continence was defined as 0 to 1 pad use per day. Successful treatment was defined as social continence. Treatment improvement was defined as no social continence, but 50% or more improvement of symptoms compared with that before surgery. Other conditions were defined as treatment failure.Results:All operations were successfully completed. After 1 to 11 months of follow-up, all patients achieved social continence. The patients' postoperative daily use of urinary pads ranged from 0 to 1 piece, with a mean of 0.5 piece. ICI-Q-SF scores ranged from 1 to 7, with a mean of 3. I-QOL scores ranged from 72.1 to 85.2, with a mean of 77.0. All the indicators were significantly improved compared with those before operation. In terms of postoperative complications, one patient had dysuria and urinary retention 2 days after the removal of the catheter, which was improved after symptomatic treatment of anti-inflammatory, detumescence, and indwelling catheter. At the last follow-up, there were no surgical related complications.Conclusions:The single-incision transobturator bulbourethral sling suspension without skin tunnel puncture for the treatment of male urinary incontinence is safe and effective. Compared to the traditional surgical method, it does not increase the difficulty of the procedure and is technically feasible, offering clinicians a new approach and perspective.

Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 μg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.