Wear particles and surface topographies are modulators of osteoclastogenesis in vitro

Prosthetic and osteosynthetic implants from metal alloys will be indispensable in orthopedic surgery, as long as tissue engineering and biodegradable bone substitutes do not lead to products that will be applied in clinical routine for the repair of bone, cartilage, and joint defects. Therefore, the elucidation of the interactions between the periprosthetic tissues and the implant remains of clinical relevance and several factors are known to affect the longevity of implants. Within this study, the effects of metal particles and surface topography on the recruitment of osteoclasts was investigated in vitro in a coculture of osteoblasts and bone marrow cells. The cells were grown in the presence of particles of different sizes and chemical composition or on metal discs with polished or sandblasted surfaces, respectively. At the end of the culture, newly formed osteoclasts were counted. Osteoclastogenesis was reduced when particles were added directly to the coculture. The effect depended on the size of the particles, small particles exerting stronger effects than larger ones. The chemical composition of the particles, however, did not affect the development of osteoclasts. In cocultures grown on sandblasted surfaces, osteoclasts developed at higher rates than they did in cultures on polished surfaces. The data demonstrate that wear particles and implant surfaces affect osteoclastogenesis and thus may be involved in the induction of local bone resorption and the formation of osteolytic lesions, leading eventually to the loosening of orthopedic implants.

Reliable gene expression measurements from degraded RNA by quantitative real-time PCR depend on short amplicons and a proper normalization

Quantitative reverse transcriptase real-time PCR (QRT-PCR) is a robust method to quantitate RNA abundance. The procedure is highly sensitive and reproducible as long as the initial RNA is intact. However, breaks in the RNA due to chemical or enzymatic cleavage may reduce the number of RNA molecules that contain intact amplicons. As a consequence, the number of molecules available for amplification decreases. We determined the relation between RNA fragmentation and threshold values (Ct values) in subsequent QRT-PCR for four genes in an experimental model of intact and partially hydrolyzed RNA derived from a cell line and we describe the relation between RNA integrity, amplicon size and Ct values in this biologically homogenous system. We demonstrate that degradation-related shifts of Ct values can be compensated by calculating delta Ct values between test genes and the mean values of several control genes. These delta Ct values are less sensitive to fragmentation of the RNA and are unaffected by varying amounts of input RNA. The feasibility of the procedure was demonstrated by comparing Ct values from a larger panel of genes in intact and in partially degraded RNA. We compared Ct values from intact RNA derived from well-preserved tumor material and from fragmented RNA derived from formalin-fixed, paraffin-embedded (FFPE) samples of the same tumors. We demonstrate that the relative abundance of gene expression can be based on FFPE material even when the amount of RNA in the sample and the extent of fragmentation are not known.

Physiologic cold shock increases adherence of Moraxella catarrhalis to and secretion of interleukin 8 in human upper respiratory tract epithelial cells

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26 degrees C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26 degrees C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26 degrees C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26 degrees C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulence.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

TLR2 and TLR4 agonists induce production of the vasoactive peptide endothelin-1 by human dendritic cells

Endothelin-1 (ET-1) is mainly secreted by endothelial cells and acts as a potent vasoconstrictor. In addition ET-1 has also been shown to have pleiotropic effects on a variety of other systems including adaptive immunity. There are two main ET-1 receptors, ET(A) and ET(B), which have different tissue and functional distributions. Dendritic cells (DC) are pivotal antigen-presenting cells linking the innate with the adaptive immune system. DC are sentinels expressing pattern-recognition receptors, e.g. the toll-like receptors (TLR) for detecting danger signals released from pathogens or tissue injury. Here we show for the first time that stimulation of human monocyte-derived DC with exogenous as well as endogenous selective TLR4 and TLR2 agonists induces the production of ET-1 in a dose- and time-dependent manner. 'Alternative' activation of DC in the presence of 1alpha,25-dihydroxyvitamin D(3) results in a marked potentiation of the endothelin response, whereas prostaglandin E(2) or dexamethasone do not increase ET-1 production. Furthermore, chetomin, an inhibitor of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), prevents TLR-mediated secretion of ET-1. Surprisingly, stimulation of human monocytes with LPS does not lead to secretion of detectable amounts of ET-1. These results suggest a role of ET-1 as an important player in human DC biology and innate immunity in general.

Neoadjuvant chemotherapy and radiotherapy followed by surgery in selected patients with stage IIIB non-small-cell lung cancer: a multicentre phase II trial

BACKGROUND: Stage IIIB non-small-cell lung cancer (NSCLC) is usually thought to be unresectable, and is managed with chemotherapy with or without radiotherapy. However, selected patients might benefit from surgical resection after neoadjuvant chemotherapy and radiotherapy. The aim of this multicentre, phase II trial was to assess the efficacy and toxicity of a neoadjuvant chemotherapy and radiotherapy followed by surgery in patients with technically operable stage IIIB NSCLC. METHODS: Between September, 2001, and May, 2006, patients with pathologically proven and technically resectable stage IIIB NSCLC were sequentially treated with three cycles of neoadjuvant chemotherapy (cisplatin with docetaxel), immediately followed by accelerated concomitant boost radiotherapy (44 Gy in 22 fractions) and definitive surgery. The primary endpoint was event-free survival at 12 months. Efficacy analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00030810. FINDINGS: 46 patients were enrolled, with a median age of 60 years (range 28-70). 13 (28%) patients had N3 disease, 36 (78%) had T4 disease. All patients received chemotherapy; 35 (76%) patients received radiotherapy. The main toxicities during chemotherapy were neutropenia (25 patients [54%] at grade 3 or 4) and febrile neutropenia (nine [20%]); the main toxicity after radiotherapy was oesophagitis (ten patients [29%]; nine grade 2, one grade 3). 35 patients (76%) underwent surgery, with pneumonectomy in 17 patients. A complete (R0) resection was achieved in 27 patients. Peri-operative complications occurred in 14 patients, including two deaths (30-day mortality 5.7%). Seven patients required a second surgical intervention. Pathological mediastinal downstaging was seen in 11 of the 28 patients who had lymph-node involvement at enrolment, a complete pathological response was seen in six patients. Event-free survival at 12 months was 54% (95% CI 39-67). After a median follow-up of 58 months, the median overall survival was 29 months (95% CI 16.1-NA), with survival at 1, 3, and 5 years of 67% (95% CI 52-79), 47% (32-61), and 40% (24-55). INTERPRETATION: A treatment strategy of neoadjuvant chemotherapy and radiotherapy followed by surgery is feasible in selected patients. Toxicity is considerable, but manageable. Survival compares favourably with historical results of combined treatment for less advanced stage IIIA disease. FUNDING: Swiss Group for Clinical Cancer Research (SAKK) and an unrestricted educational grant by Sanofi-Aventis (Switzerland).

Selecting control genes for RT-QPCR using public microarray data

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. RESULTS: We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at http://www.isrec.isb-sib.ch/~vpopovic/research/ CONCLUSION: We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable.

Pharmacokinetics and safety profile of the human anti-Pseudomonas aeruginosa serotype O11 immunoglobulin M monoclonal antibody KBPA-101 in healthy volunteers

KBPA-101 is a human monoclonal antibody of the immunoglobulin M isotype, which is directed against the O-polysaccharide moiety of Pseudomonas aeruginosa serotype O11. This double-blind, dose escalation study evaluated the safety and pharmacokinetics of KBPA-101 in 32 healthy volunteers aged 19 to 46 years. Each subject received a single intravenous infusion of KBPA-101 at a dose of 0.1, 0.4, 1.2, or 4 mg/kg of body weight or placebo infused over 2 h. Plasma samples for pharmacokinetic assessments were taken before infusion as well as 0.25, 0.5, 1, 2, 2.5, 4, 6, 8, 12, 24, 36, and 48 h and 4, 7, 10, and 14 days after start of dosing. Plasma concentrations of KBPA-101 were detected with mean maximum concentrations of drug in plasma of 1,877, 7,571, 24,923, and 83,197 ng/ml following doses of 0.1, 0.4, 1.2, and 4.0 mg/kg body weight, respectively. The mean elimination half-life was between 70 and 95 h. The mean volume of distribution was between 4.76 and 5.47 liters. Clearance ranged between 0.039 and 0.120 liters/h. At the highest dose of 4.0 mg/kg, plasma KBPA-101 levels were greater than 5,000 ng/ml for 14 days. KBPA-101 exhibited linear kinetics across all doses. No anti-KBPA-101 antibodies were detected after dosing in any subject. Overall, the human monoclonal antibody KBPA-101 was well tolerated over the entire dose range in healthy volunteers, and no serious adverse events have been reported.