Performance of the new VITEK 2 GP card for identification of medically relevant gram-positive cocci in a routine clinical laboratory

The VITEK 2 gram-positive (GP) identification card (bioMerieux, Marcy l'Etoile, France) has been redesigned to achieve greater accuracy in the identification of gram-positive cocci. A total of 43 biochemical tests, including 17 enzymatic tests, are present in the card and interpreted in a kinetic mode, for up to 8 h. The VITEK 2 database, used in conjunction with the GP identification card, allows the identification of 115 different taxa. A total of 364 strains of GP cocci (217 Streptococcaceae strains and 147 Micrococcaceae strains) belonging to 31 taxa were tested with the new VITEK 2 GP identification card. Of the 364 strains, 105 were taken from routine primary plating media. A total of 344 strains (94.5%) were correctly identified to the species level and 17 strains (4.7%) were identified with low discrimination, requiring additional tests, whereas 1 strain (0.3%) was incorrectly identified and 2 strains (0.5%) remained unidentified. Within 7 h of the start of incubation, more than 90% of all strains were identified. Of the 105 primary cultures, 97% were correctly identified to the species level, 2% were identified with low discrimination, and 1% remained unidentified. Identification performance data were independent of each of the three plating media used. It is concluded that the new VITEK 2 GP identification card provides reliable results for the identification of GP cocci under routine laboratory conditions.     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

Use of the BD PHOENIX Automated Microbiology System for direct identification and susceptibility testing of gram-negative rods from positive blood cultures in a three-phase trial

The present study describes the use of the automated BACTEC 9240 blood culture system, the Serum Separator Tube (SST), and the BD PHOENIX Automated Microbiology System in combination for the direct identification and antimicrobial susceptibility testing (AST) of gram-negative rods (GNRs) from positive blood cultures (BCs) without subculture. The study was conducted in three phases: (i) the recovery yield of Escherichia coli ATCC 25922 was determined with the SST between 0 and 8 h after spiked BC bottles turned positive; (ii) the identifications and susceptibility testing results obtained with the PHOENIX system for nine American Type Culture Collection strains of GNRs processed by the SST procedure and for colonies from agar medium were compared; and (iii) the procedure with the BACTEC system, SSTs, and the PHOENIX system was applied to positive cultures of blood from 309 patients during a 3-month period. The SST procedure with E. coli yielded sufficient numbers of cells to perform direct inoculation at any time between 0 and 8 h after a BC bottle turned positive. By using the identities obtained from pure cultures with the PHOENIX system and other biochemical identification systems as reference methods, the agreement between the reference methods and the PHOENIX system tested directly by using cultures of blood from patients was 92.9%. The 7.1% discrepant results were due to 6.5% incorrect identifications with the PHOENIX system with BC samples and 0.6% incorrect identifications with the PHOENIX system with samples from agar cultures. By AST the overall categorical accuracy was 99.0%, with 0.1% very major errors, 0.1% major errors, and 0.8% minor errors. In conclusion, use of the combination of the BACTEC system, SSTs, and the PHOENIX system has the potential to allow the agar isolation step to be skipped and the procedures for rapid direct identification and susceptibility testing of GNRs from positive BCs to be improved both in hospital-based and in central non-hospital-based laboratories.     

Compilation of published comparative genomic hybridization studies

The power of comparative genomic hybridization (CGH) has been clearly proven since the first paper appeared in 1992 as a tool to characterize chromosomal imbalances in neoplasias. This review summarizes the chromosomal imbalances detected by CGH in solid tumors and in hemopathies. In May of 2001, we took a census of 430 articles providing information on 11,984 cases of human solid tumors or hematologic malignancies. Comparative generic hybridization has detected a number of recurrent regions of amplification or deletion that allows for identification of new chromosomal loci (oncogenes, tumor suppressor genes, or other genes) involved in the development, progression, and clonal evolution of tumors. When CGH data from different studies are combined, a pattern of nonrandom genetic aberrations appears. As expected, some of these gains and losses are common to different types of pathologies, while others are more tumor-specific.     

Outer membrane protein A renders dendritic cells and macrophages responsive to CCL21 and triggers dendritic cell migration to secondary lymphoid organs

Outer membrane protein A (OmpA) is a class of bacterial cell wall protein that is immunogenic without adjuvant. As specific immune responses are initiated in the lymph nodes (LN, we analyzed the effect of the OmpA from Klebsiella pneumoniae (KpOmpA) onchemokine/ chemokine receptor expression by APC and on cell migration to the LN. Upon contact with KpOmpA, human immature DC and macrophages acquire CCR7 expression and responsiveness to CCL21. In parallel, CCR1 and CCR5 expression is down-regulated and CXCL8, CCL2, CCL3 and CCL5 production is up-regulated. Mice injected subcutaneously with KpOmpA present a transient inflammatory reaction at the site of injection accompanied by an enlargement of the draining LN with a higher proportion of DC and macrophages. Lastly, when exposed to KpOmpA prior injection, DC but not macrophages migrate to the draining LN. In conclusion, KpOmpA confers a migratory phenotype to DC and triggers their migration to the regional LN. This property contributes to explain how innate cells initiate adaptive immune response upon recognition of conserved bacterial components and also why OmpA is immunogenic in the absence of adjuvant.     

Involvement of E-cadherin and beta-catenin in germ cell tumours and in normal male fetal germ cell development

Intercellular contacts, mediated by E-cadherin, are essential for germ cell migration and maturation. Furthermore, it has been suggested that decrease or loss of E-cadherin correlates with tumour progression and invasive behaviour. beta-catenin is involved in a number of different processes, including cell--cell interaction when bound to cadherins, and determination of cell fate in pluripotent cells when activated via the Wnt signal-transduction pathway. To shed more light on the role of these factors in normal fetal germ cell development and the pathogenesis of germ cell tumours (GCTs), the present study investigated the presence and localization of E-cadherin and beta-catenin by immunohistochemistry. E-cadherin was only weakly expressed in or absent from fetal germ cells of the second and third trimesters, and was not expressed in carcinoma in situ/intratubular germ cell neoplasia unclassified (CIS/ITGCNU) and gonadoblastoma, the precursor of an invasive GCT in dysgenetic gonads. In GCTs, it was generally not expressed in seminoma and dysgerminoma, but was found in the vast majority of non-seminoma cells. beta-catenin was found in the cytoplasm of fetal germ cells at all gestational ages and in spermatogenesis in post-pubertal testes. It was also present in CIS/ITGCNU and gonadoblastoma. Whereas seminomas and dysgerminoma were negative, non-seminoma cells were frequently found to express beta-catenin. Expression of both factors therefore reflects the degree of differentiation of these tumours. No differences for either E-cadherin or beta-catenin were observed between samples of tumours resistant or sensitive to chemotherapy, and E-cadherin expression did not correlate with vascular invasion. E-cadherin and beta-catenin therefore play a role in both normal and malignant germ cell development and differentiation that warrants further investigation, but they seem to be of limited value as predictive or prognostic factors in GCTs.     

Distribution and migration of angiogenic cells from grafted avascular intraembryonic mesoderm

Summary The hemangiogenic potencies of initially avascular intra-embryonic mesoderm were studied in chick and quail embryos and in chick-quail chimeras. The prechordal mesoderm, primitive streak and primitive node of quail embryos were heterospecifically grafted into limb buds of chick embryos. Hemangiopoietic quail cells in the host limb were detected by immunohistological staining with the monoclonal anti-MB-1 antibody after 3–6 days of re-incubation. The antibody is specifically directed against quail hemangiopoietic cells and their derivatives. Quail endothelial cells were found in pure quail and in chimeric vessels, inside as well as outside the graft. The main artery of the limb and the vessels inside the graft were connected by chimeric arteries. Proximal to the graft, quail endothelial cells were located predominantly within the lining of the main artery, while distally they were found mainly in the veins and the marginal sinus. The results show that, as early as stage 3 (according to Hamburger and Hamilton 1951, HH) all parts of the avascular intraembryonic mesoderm tested, give rise to endothelial cells. Both mechanisms, angiogenesis and vasculogenesis, contribute to the vascularization of the limb. Immunocytological and scanning electron microscopic studies indicate that centrifugal and centripetal migration of angiogenic cells occurs outside the vessels as well as on the inner surface of the endothelium.Supported by the Deutsche Forschungsgemeinschaft (CH 44/9-1)     

Sequential Measurements of Chemokines in Urosepsis and Experimental Endotoxemia

Chemokines are a superfamily of small chemotactic proteins. While increased levels of interleukin-8 have been measured in serum and urine during urinary tract infection, little is known about other chemokines in this condition. Monocyte chemoattractant protein (MCP)–1, macrophage inflammatory protein (MIP)–1, MIP-1 and interferon- inducible protein (IP)–10 were measured in 30 patients with culture-proven urosepsis during a 3-day follow-up and in 11 healthy humans after intravenous injection of endotoxin (4 ng/kg). Urine and serum levels of MCP-1, MIP-1, and IP-10, but not of MIP-1, were elevated in patients on admission, and decreased after initiation of antibiotic treatment. Endotoxin administration to healthy subjects induced increases in plasma and urine concentrations of all four chemokines. These data indicate that clinical and experimental gram-negative infection in humans is associated with enhanced production of chemokines that act mainly on mononuclear cells and that these chemokines are at least in part locally produced.     

Novel FARS2 variants in patients with early onset encephalopathy with or without epilepsy associated with long survival

Mitochondrial translation is essential for the biogenesis of the mitochondrial oxidative phosphorylation system (OXPHOS) that synthesizes the bulk of ATP for the cell. Hypomorphic and loss-of-function variants in either mitochondrial DNA or in nuclear genes that encode mitochondrial translation factors can result in impaired OXPHOS biogenesis and mitochondrial diseases with variable clinical presentations. Compound heterozygous or homozygous missense and frameshift variants in the FARS2 gene, that encodes the mitochondrial phenylalanyl-tRNA synthetase, are commonly linked to either early-onset epileptic mitochondrial encephalopathy or spastic paraplegia. Here, we expand the genetic spectrum of FARS2-linked disease with three patients carrying novel compound heterozygous variants in the FARS2 gene and presenting with spastic tetraparesis, axial hypotonia and myoclonic epilepsy in two cases.Subject terms: Metabolic disorders, Mutation