Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering

Cartilage tissue engineering plays an important role in the generation of grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality, and the molecular basis is still not well understood. Therefore, the aim of our study was to investigate the expression of matrix modulators in human chondrocytes during expansion. Human chondrocytes were isolated from septal cartilage (n=32) and held in primary cell culture. Cells were harvested after 1, 6 and 21 days. The differentiation of cells using light microscopy, the expression patterns of various proteins (MMPs, BMPs, and TIMPs) using immunohistochemistry, and the expression of distinct genes using microarray technique, were investigated. The chondrocytes showed strong in vitro proliferation. After 6 and 21 days, BMP-5 and -8 were up-regulated, BMP-2 was down-regulated and BMP-6 was inactivated. Other BMPs were not expressed. The expression of MMP-2, -3 and -13 was up-regulated from day 1 to 21, and MMP-12 and -20 were down-regulated. Other MMPs were not expressed. TIMP-1 was up-regulated and TIMP-3 was down-regulated during expansion. Differential expression of matrix modulators might influence the matrix composition of engineered cartilage. Improving the basic knowledge in this area may ultimately help clinicians to identify and proactively intervene in an attempt to prevent bioartificial cartilage from losing stability.     

Enhanced factor H binding to sialylated Gonococci is restricted to the sialylated lacto-N-neotetraose lipooligosaccharide species: implications for serum resistance and evidence for a bifunctional lipooligosaccharide sialyltransferase in Gonococci

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galalpha1 --> 4 [corrected] Galbeta1 --> 4Glcbeta1 --> 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galbeta1 --> 4GlcNAcbeta1 --> 3Galbeta1 --> 4Glcbeta1 --> 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an alpha2 --> 6 linkage and, as expected, to the terminal Gal of LNT LOS via an alpha2--> 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.     

DNA microarray technique for detection and identification of seven flaviviruses pathogenic for man

A flavivirus microarray was developed for detection and identification of yellow fever (YF), West Nile, Japanese encephalitis (JE), and the dengue 1-4 viruses, which are causing severe human disease all over the world. The microarray was based on 500-nucleotide probe fragments from five different parts of the seven viral genomes. A low-stringent amplification method targeting the corresponding regions of the viral genomic RNA was developed and combined with hybridization to the microarray for detection and identification. For distinction of the generated virus-specific fluorescence-patterns a fitting analysis procedure was adapted. The method was verified as functional for all seven flaviviruses and the strategy for the amplification, combined with the long probes, provided a high tolerance for smaller genetic variability, most suitable for these rapidly changing RNA viruses. A potentially high detection and identification capacity was proven on diverged strains of West Nile and dengue viruses. The lower limit for detection was equivalent, or better, when compared to routinely used RT-PCR methods. The performance of the method was verified on human patient samples containing dengue viruses, or normal human serum spiked with YF or JE viruses. The results demonstrated the ability of the flavivirus microarray to screen simultaneously a sample for several viruses in parallel, in combination with a good lower limit of detection.     

Development and characterisation of neutralising monoclonal antibody to the SARS-coronavirus

There is a global need to elucidate protective antigens expressed by the SARS-coronavirus (SARS-CoV). Monoclonal antibody reagents that recognise specific antigens on SARS-CoV are needed urgently. In this report, the development and immunochemical characterisation of a panel of murine monoclonal antibodies (mAbs) against the SARS-CoV is presented, based upon their specificity, binding requirements, and biological activity. Initial screening by ELISA, using highly purified virus as the coating antigen, resulted in the selection of 103 mAbs to the SARS virus. Subsequent screening steps reduced this panel to seventeen IgG mAbs. A single mAb, F26G15, is specific for the nucleoprotein as seen in Western immunoblot while five other mAbs react with the Spike protein. Two of these Spike-specific mAbs demonstrate the ability to neutralise SARS-CoV in vitro while another four Western immunoblot-negative mAbs also neutralise the virus. The utility of these mAbs for diagnostic development is demonstrated. Antibody from convalescent SARS patients, but not normal human serum, is also shown to specifically compete off binding of mAbs to whole SARS-CoV. These studies highlight the importance of using standardised assays and reagents. These mAbs will be useful for the development of diagnostic tests, studies of SARS-CoV pathogenesis and vaccine development.     

Influence of resistance to antidepressant pharmacotherapy on short-term response to electroconvulsive therapy

BACKGROUND: Few studies assessing the influence of resistance to antidepressant pharmacotherapy on the response to subsequent electroconvulsive therapy (ECT) are found in the literature. Results are somewhat conflicting and may not be applicable to the population of depressed patients in The Netherlands. The aim of this study is to assess the influence of medication resistance on the short-term response to ECT in a population of severely depressed inpatients in The Netherlands, where ECT is an exceptional treatment, often used as a final treatment option. METHODS: We reviewed the records of 41 consecutive inpatients with major depression according to DSM-III-R criteria and rated each patients' antidepressant pharmacotherapy prior to ECT. We examined the extent to which medication resistance was related to short-term response to ECT. RESULTS: When a reduction of at least 50% on the Hamilton Rating Scale for Depression (HRSD) post-ECT compared to pre-ECT (partial remission) is used as response criterion, medication resistant patients and patients without established medication resistance were equally likely to respond to subsequent ECT. When a post-ECT HRSD score < or = 7 (full remission) is used as response criterion, medication resistant patients were less likely to respond to subsequent ECT (8/29=27.6%) than patients who did not receive adequate antidepressant pharmacotherapy prior to ECT (6/12=50.0%), although the difference in response rate was not statistically significant. LIMITATIONS: This study has a retrospective nature and a relatively small sample size. CONCLUSION: Antidepressant medication resistance does not seem to have an influence on the short-term response to subsequent ECT. However, when the number of patients achieving full remission is concerned, a substantial percentage of antidepressant medication resistant patients respond to ECT, although their response rate was nearly half compared to that of patients without prior adequate treatment with antidepressants. This difference in response rate was not statistically significant. ECT seems to be an effective treatment for both patients with and without prior adequate treatment with antidepressants in this Dutch population.     

Mutations of FBN1 and genotype-phenotype correlations in Marfan syndrome and related fibrillinopathies

The Marfan syndrome (MFS) is a pleiotropic, autosomal dominant disorder of connective tissue with highly variable clinical manifestations including aortic dilatation and dissection, ectopia lentis, and a series of skeletal anomalies. Mutations in the gene for fibrillin-1 (FBN1) cause MFS, and at least 337 mainly unique mutations have been published to date. FBN1 mutations have been found not only in MFS but also in a range of connective tissue disorders collectively termed fibrillinopathies ranging from mild phenotypes, such as isolated ectopia lentis, to severe disorders including neonatal MFS, which generally leads to death within the first two years of life. The present article intends to provide an overview of mutations found in MFS and related disorders and to discuss potential genotype-phenotype correlations in MFS.     

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.