Regaining chondrocyte phenotype in thermosensitive gel culture

Chondrocyte tissue engineering continues to be a challenging problem. When chondrocytes are duplicated in vitro, it is imperative to obtain an adequate number of cells of optimal phenotype. A temperature-sensitive polymer gel, a copolymer of poly(N-isopropylacrylamide) and acrylic acid (PNiPAAm-co-Aac), has the ability of gelling at 37 degrees C (the lower critical solution temperature, LCST) or above and liquefying below that temperature (Vernon and Gutowska, Macromol. Symp. 1996;109:155-167). The hypothesis of this study was that chondrocytes could (1) duplicate in the copolymer gel; (2) regain their chondrocyte phenotype; and (3) be easily recovered from the gel by simply lowering the temperature below 37 degrees C. Chondrocytes from adult rabbit scapular cartilage were harvested and cultured in a monolayer culture until confluency (approximately 2 weeks). Next, the cells were harvested and seeded into the copolymer gel and cultured for 2-4 weeks. The phenotype of the cultured cells was then characterized. Two groups of control cultures, monolayer and agarose gel, were used to compare their ability to maintain chondrocyte phenotype. The results showed that chondrocytes isolated from rabbit scapula can re-express chondrocyte phenotype in agarose culture and polymer gel culture but not in monolayer culture. Also, cultured chondrocytes can be easily recovered from polymer gel culture by simply lowering the temperature. This new in vitro method of chondrocyte culture is recommended for chondrocyte propagation and regaining chondrocyte phenotype before cell seeding or transplantation.     

The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts

The metalloprotease ADAM 10 is an important APP alpha-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPs alpha generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative alpha-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific 'teams' of different proteases exerting alpha-secretase activity.     

利用神经网络集成预测MHC-Ⅰ类分子结合肽

目的：利用神经网络集成（NNE）预测MHC-Ⅰ类分子结合肽。 方法： 基于HLA-A*0201编码的MHC-Ⅰ类分子结合肽数据库（含有628个9聚物）及其结合能力分类，利用NNE分别对具有无、低、中和高4类亲合性的结合肽进行分类预测；同时还进一步利用T细胞真实表位集（含50个表位）评估了NNE的预测性能。 结果： 集成数为12的NNE对上述分类的平均预测命中率可达0.8,而且NNE对潜在T细胞表位的预测能力也较高，约84%的真实表位归于高和中等亲合性的潜在抗原肽一类。 结论： 可以利用神经网络集成预测MHC-Ⅰ类分子结合肽，并进而预测相应的T细胞表位。经适当修改，NNE预测工具可扩展为能涵盖任意长度的Ⅰ类分子结合肽甚至可扩展到Ⅱ类分子结合肽的预测。     

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of highly malignant colon cancer from an aggressive to a controlled disease by oral administration of a metalloproteinase inhibitor

In this study, we describe the activity of CT1746, an orally-active synthetic MMP inhibitor that has a greater specificity for gelatinase A, gelatinase B and stromelysin than for interstitial collagenase and matrilysin, in a nude mouse model that better mimics the clinical development of human colon cancer. The model is constructed by surgical orthotopic implantation (SOI) of histologically-intact tissue of the metastatic human colon tumor cell line Co-3. Animals were gavaged with CT1746 twice a day at 100 mg/kg for 5 days after the SOI of Co-3 for 43 days. In this model CT1746 significantly prolonged the median survival time of the tumor-bearing animals from 51 to 78 days. Significant efficacy of CT1746 was observed on primary tumor growth (32% reduction in mean tumor area at day 36), total spread and metastasis (6/20 treated animals had no detectable spread and metastasis at autopsy compared to 100% incidence of secondaries in control groups). Efficacy of CT1746 could also be seen on reducing tumor spread and metastasis to individual organ sites such as the abdominal wall, cecum and lymph nodes compared to vehicle and untreated controls. We conclude that chronic administration of a peptidomimetic MMP inhibitor via the oral route is feasible and results in inhibition of solid tumor growth, spread and metastasis with increase in survival in this model of human cancer, thus converting aggressive cancer to a more controlled indolent disease.     

The effect of RANTES chemokine genetic variants on early HIV-1 plasma RNA among African American injection drug users

HIV-1 plasma RNA is a prognostic indicator of HIV-1, and increased levels of HIV-1 plasma RNA are associated with rapid progression to AIDS. Because chemokines and chemokine receptors are involved in the binding and entry of HIV-1, possible effects of host genetics on viral RNA levels should be visible in early infection. HIV-1 plasma RNA was measured within 2 years of seroconversion in 198 seroincident injection drug users followed in the AIDS Link to Intravenous Experience cohort. Genetic variants were identified in the chemokine receptors (CCR2, CCR5, and CCR5 promoter) and the chemokine RANTES using TaqMan and restriction fragment length polymorphism assays. Linear regression of RANTES haplotypes on early HIV-1 plasma RNA identified individuals homozygous for the RANTES R1 haplotype as having a lower viral load by almost one-half log10 unit compared with those bearing non-RANTES R1 haplotypes (-0.43, 95% confidence interval: -0.74, -0.12). Genetic variants in RANTES may downregulate RANTES gene expression and increase early HIV-1 plasma RNA. Because RANTES is a critical chemokine and competitively inhibits HIV-1 by binding to its receptor CCR5, treatment to enhance RANTES expression may assist in delaying the progression of AIDS by decreasing the initial viral load.     

The relationship of vascularity and water content to tensile strength in a patellar tendon replacement of the anterior cruciate in dogs

The methods and materials for ACL reconstruction are important issues for the practicing orthopaedic surgeon. In this study a model was developed to study the biological and biomechanical characteristics of a patellar tendon autograft used for ACL reconstruction. Specifically it was hypothesized that since vascularity of these grafts reflects their "healthiness," strength and vascularity should be inversely related in the early period after implantation. Using an over the top technique, a patellar tendon graft was placed in three groups of dogs and studied at 37, 57, and 120 days. Vascularity of the grafts was measured using technetium-tagged red blood cells, and percent water by weight was determined by dessication. Tensile testing to failure was performed using an MTS machine. The grafts became more vascular, more hydrated, less stiff, and less strong (by 4 weeks) than controls. By 16 weeks the vascular response was subsiding but the grafts remained only 40% as strong as controls. Percent water increased significantly over controls for all time periods. Decrease in strength correlated poorly with vascularity but correlated well with increase in percent water. These findings suggest that the change in strength of an intraarticular ACL replacement relates more to a basic rearrangement of its collagen-ground substance relationships, and that vascularity may reflect the inflammatory response bringing about these changes. The model developed in this study serves as a basis for further studies, and the findings reveal important information about the behavior of ACL grafting materials.     

老年人髋部骨折的骨密度及影响因素的分析

目的调查分析成都地区老年人髋部骨折骨密度变化和影响因素,为预防髋部骨折提供科学依据.方法骨折组答卷式调查有关指标,髋部作X线正斜侧位摄片,骨折组与非骨折组进行配对,均用DEXA测量L2～4,股骨上段骨密度(BMD),骨矿含量(BMC),骨面积(Area)等,并进行统计分析.结果股骨上段及L2～4的BMD、BMC骨折组明显低于非骨折组(P＜0.05),两组各进行男女性比较,女性均明显低于男性(P＜0.05),骨折组L2～4BMD男、女分别为0.872±0.178g/cm2,0.607±0.155g/cm2;非骨折组男女分男为0.921±0.147g/cm2,0.741±0.152g/cm2.同性别的骨面积Area两组比较无差异,骨折组骨质疏松症发生率76%明显高于非骨折组63%(P＜0.05);滑倒和被人撞倒股骨颈骨折发生率较高,自行拌倒者粗隆间骨折发生率较高.结论预防治疗骨质疏松症和避免摔倒,以防骨折的发生.