A murine Staphylococcus aureus fracture-related infection model characterised by fracture non-union, staphylococcal abscess communities and myeloid-derived suppressor cells.

A fracture-related infection (FRI) is a serious complication that can occur after surgical fixation of bone fractures. Affected patients may encounter delayed healing and functional limitations. Although it is well established that Staphylococcus aureus (S. aureus) is the main causative pathogen of an FRI, the pathophysiology of an S. aureus-induced FRI is not well characterised over time. Therefore, an experimental study in mice comparing S. aureus-inoculated and non-inoculated groups was performed that particularly focused on staphylococcal abscess communities (SACs) and host cellular response. C57Bl/6N female mice received a double osteotomy of the femur, which was stabilised using a titanium 6-hole MouseFix locking plate and four screws. Animals were either S. aureus-inoculated or non-inoculated and euthanised between 1 and 28 d post-surgery. Histopathological evaluation showed normal bone healing for non-inoculated mice, whereas inoculated mice had no fracture consolidation and severe osteolysis. Within the bone marrow of inoculated mice, SACs were observed from 7 d, which increased in size and number over time. A fibrin pseudocapsule enclosed the SACs, which were surrounded by many Ly6G+ neutrophils with some Ly6C+ monocytes and F4/80+ macrophages, the majority of which were viable. The abscesses were encapsulated by fibrin(ogen), collagen and myofibroblasts, with regulatory T cells and M2 macrophages at the periphery. Only bone marrow monocytes and neutrophils of inoculated mice displayed functional suppression of T cells, indicative of myeloid-derived suppressor cells. The present study revealed that an FRI in mice is persistent over time and associated with osteolysis, SAC formation and an immunosuppressive environment.

Environmental tobacco smoke (ETS) exposure and respiratory morbidity in school age children.

INTRODUCTION: Tobacco smoke is a risk factor for Chronic Obstructive Pulmonary Disease and a major public health problem. Prenatal maternal smoking and post-natal environmental tobacco smoke (ETS) lead to dose-dependent decrease in lung function and respiratory morbidity. Influence of different socioeconomic indicators and ETS in the home has also been suggested. METHODS: Data on 313 children (52 % male) from 4 public schools in Lisbon was analyzed [1st (46 %) and 4th graders]. ETS assessment and respiratory symptoms were based on a self-answered questionnaire. All children performed standard spirometry in the school setting and 54 % were acceptable according to ATS/ERS criteria. Descriptive and bivariate analysis of the most relevant variables was done, followed by multiple logistic regression analysis adjusted to the variables with clinical/statistical relevance. RESULTS: ETS in the home was found in 41 % (maternal smoking during pregnancy 18 %, smoking mother 32 %, smoking father 38 %). Smoking fathers had lower education and less qualified occupation. Cough was more frequent in children with a smoking mother (adjusted OR = 2.1 95CI 1.1-4.0) and wheezing in children with maternal smoking during pregnancy and smoking parents. All differences were significant (p < 0.05). No association was found between parental education and cough/wheeze or ETS and respiratory infections/asthma/decreased spirometric values. CONCLUSIONS: Children in Lisbon are frequently exposed to ETS which results in significant respiratory morbidity. Targeted interventions must have social conditions in consideration. In this study, field spirometry was not helpful in early detection of lung function disability in children associated with ETS.

Alpha4 integrin is expressed during peripheral nerve regeneration and enhances neurite outgrowth.

We have shown previously that repair in the peripheral nervous system is associated with a reversion to an embryonic pattern of alternative splicing of the extracellular matrix molecule fibronectin. One of the consequent changes is a relative increase in the number of fibronectins expressing the binding site for alpha4 integrins. Here we show that alpha4 integrins are expressed on dorsal root ganglion neuron cell bodies and growth cones in the sciatic nerve during regeneration and that the interaction of alpha4 integrin with alternatively spliced isoforms of recombinant fibronectins containing the alpha4 binding site enhances neurite outgrowth in dorsal root ganglion neurons. The pheochromocytoma (PC12) neuronal cell line, which normally extends neurites poorly on fibronectin, does so efficiently when alpha4 is expressed in the cells. Experiments using chimeric integrins expressed in PC12 cells show that the alpha4 cytoplasmic domain is necessary and sufficient for this enhanced neurite outgrowth. In both dorsal root ganglion neurons and PC12 cells the alpha4 cytoplasmic domain is tightly linked to the intracellular adapter protein paxillin. These experiments suggest an important role for alpha4 integrin and paxillin in peripheral nerve regeneration and show how alternative splicing of fibronectin may provide a mechanism to enhance repair after injury.

Developmental regulation of alphav integrins produces functional changes in astrocyte behavior.

To examine the role of the extracellular matrix in regulating astrocyte behavior we previously characterized alphav integrin expression on postnatal astrocytes in vitro and found that they express alphavbeta5 and alphavbeta8. Here we show that differentiation of immature cells into astrocytes is accompanied by developmental regulation of alphav integrins, downregulation of alphavbeta1 and alphavbeta8, and upregulation of alphavbeta5. In addition, using two previously described astrocyte cell lines, we found that the neurite-permissive A7 cell line expressed high levels of alphavbeta1 in addition to alphavbeta5 and alphavbeta8, while the neurite-inhibitory Neu7 cell line expressed only alphavbeta5. To examine integrin function we generated clones of the Neu7 cell line expressing alphavbeta1 or alphavbeta3 in addition to alphavbeta5. This showed that the parent Neu7 cells migrated more slowly than the A7 cells on fibronectin and vitronectin, but that Neu7 cells expressing alphavbeta1 or alphavbeta3 integrins showed enhanced migration on fibronectin and vitronectin, respectively. These results show that alphav integrin expression is regulated during astrocyte development and confirm an instructive role in cell migration for alphavbeta1 in embryonic cells and alphavbeta3 in astroglial tumors.

Association between integrin-dependent migration capacity of neural stem cells in vitro and anatomical repair following transplantation.

In previous transplantation studies using neural stem cell lines immortalized by the temperature-sensitive SV40 large T-antigen, we have shown that animals with experimental hippocampal lesions resulting from four vessel occlusion recover spatial memory functions more effectively when grafted with the MHP36 cell line than with the MHP15 cell line [Gray et al. (1999). Philos. Trans. R. Soc. London Biol. Sci. 354:1407-1421]. In the present study, we have investigated the cellular and molecular basis of these differences in repair capacity both in vivo and in vitro. Using the same model of hippocampal damage we have shown that following transplantation MHP36 cells migrate and align within the damaged CA1 of the ipsilateral hippocampus. MHP15 cells, in contrast, migrate in a more indiscriminate pattern that does not reflect the anatomy of the region. To analyze the migratory properties of these two cell lines in more detail, we performed migration assays at a nonpermissive temperature on the extracellular matrix substrates laminin, fibronectin, and vitronectin. These showed that MHP36 cells have a greater migration potential than the MHP15 cells. While the pattern of cell surface extracellular matrix receptors of the integrin family was identical in both cell lines, the different degrees of migration on vitronectin were both blocked by inhibitors of alphaV integrins. Differences in integrin signaling therefore contribute to the greater migration potential of the repairing MHP36 cell line.

A novel mechanism for induction of increased cortical porosity in cases of intracapsular hip fracture.

It has been suggested that, in hip fracture, the cortex on the inferoanterior (IA) to superoposterior (SP) axis is thinned and shows increased porosity. This is dependent on the presence of giant canals (i.e., diameter >385 microm), which are related to clusters of remodeling osteons. To investigate further the relationship between remodeling and bone loss, osteonal diameter (On.Dm), wall thickness (W.Th), osteoid width (O.Wi), and extent (OS) were measured in femoral neck biopsies from 12 female intracapsular hip fracture cases and 11 age- and gender-matched controls. Over 83% of giant canals were "composite" osteonal systems in which a single canal was surrounded by multiple packets of osteonal bone. Among smaller canals, over 80% of systems had a canal encircled by a single cement line containing one packet of bone ("simple"). Composites were nearly twice as prevalent in fractures (fracture cases 9.8 +/- 0.7/25 mm(2), controls 5.3 +/- 0.4/25 mm(2), p < 0. 0001), and were dependent (R(2) = 0.52) on femoral neck region (p = 0.0008) and the regional distribution of clusters of remodeling osteons (p = 0.0045). Both the inferior (I) and anterior (A) regions had an elevated number of composites (I: 263% of control values, p = 0.0054; A: 202% of control values, p = 0.0092). On.Dm was similar in fracture cases and controls (simple: fracture cases 183 +/- 3 microm, controls 191 +/- 4 microm; composites: fracture cases 446 +/- 13 microm, controls 460 +/- 13 microm). W.Th in simples was similar in fracture cases and controls (fracture cases 51 +/- 0.8 microm, controls 49 +/- 0.7 microm), but composites had significantly (p < 0. 0001) thinner walls, with the reduction in fracture cases (31%) being twice that of controls (12%, p < 0.0001). There were no differences in O.Wi. It was unusual for osteoid to fully surround the composite canal surface; OS was 38% lower in composite than simple canals (p < 0.0001). This study indicates that, in the femoral neck cortex, the principal remodeling deficit in hip fracture is specific to composite osteons. Hip fracture cases had zonal increases in composite osteon density with reduced bone formation. The data suggest that generation of composite osteons is a plausible mechanism leading to increasing porosity and trabecularization of the cortex, thus weakening the cortex in regions maximally loaded on fall impact.

A case of discordant related abnormal karyotypes from chorionic villi and amniocytes.

A case of three discordant cell lines in prenatal diagnosis is described, of which two were abnormal related structural abnormalities of chromosome 11. One of the abnormal cell lines was seen in all metaphases examined from direct preparations of chorionic villi, the cultured preparations showing an apparently normal male karyotype; the other abnormal cell line was seen in conjunction with a normal cell line in cultured amniocytes. Prenatal diagnosis offered solely on chorionic villus sampling would have yielded a mistakenly normal result on the basis of established criteria for distinguishing true mosaicism from confined placental mosaicism.

Diagnostic and therapeutic impact of MRI and arthrography in the investigation of full-thickness rotator cuff tears.

Investigation of shoulder pain is important before surgical treatment. The presence or absence of a full-thickness rotator cuff tear (FTRCT) may determine the type of surgical treatment. Both MRI and conventional arthrography can be used, but little is known about their relative diagnostic and therapeutic impact. We performed a prospective trial assessing: (a) the influence of MRI and arthrography results on the clinician's diagnostic thinking (diagnostic impact); (b) the influence of the results on the clinician's therapeutic thinking (therapeutic impact); and (c) the diagnostic performance of the two techniques in patients with surgical confirmation. A total of 104 consecutive patients with shoulder problems referred to a specialist orthopaedic shoulder clinic underwent pre-operative investigation with MRI and arthrography. The surgeon's diagnosis, diagnostic confidence and planned treatment were measured before the investigation, and then again after the results of each investigation. Before the presentation of the investigation, results, the patients were randomised into two groups. In one group MRI was presented first; in the other group, arthrography. The MRI results led to fewer changes in diagnostic category (14 of 46, 30%) than arthrography (20 of 54, 37%), but the difference was not significant (P > 0.5). Magnetic resonance imaging led to slightly more changes in planned management (17 of 47, 36%) than arthrography (14 of 55, 25%), but again the difference was not statistically significant (P > 0.3). The results of the second investigation always had less diagnostic and therapeutic impact than the first. The accuracy of MRI for FTRCT in 38 patients with surgical confirmation was 79%, sensitivity 81% and specificity 78%; the accuracy of arthrography was 82%, sensitivity 50% and specificity 96%. The clinical diagnosis and management plan can be adequately defined by a single radiological investigation. Magnetic resonance imaging and arthrography had fairly similar diagnostic and therapeutic impact and comparable accuracy, although MRI was more sensitive and less specific. Magnetic resonance imaging may be the preferred investigation because of its better demonstration of soft tissue anatomy.

Expression of the rat homologue of the Drosophila fat tumour suppressor gene.

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the b-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.

Magnetic resonance imaging of transplanted oligodendrocyte precursors in the rat brain.

The lack of any markers for oligodendrocyte precursors that can be visualized within the intact CNS is a significant barrier to trials of transplantation of these cells which aim to enhance remyelination in multiple sclerosis. We have therefore asked whether dextran-coated superparamagnetic iron oxide (SPIO) can be used to label cells prior to transplantation and then visualized within the brain using MRI. We have shown that an oligodendrocyte precursor cell line CG-4 will take up dextran-coated SPIO particles in vitro. The label remains within the cells after transplantation into adult rat brain, as assessed by electron microscopy, and is visible by MRI as a reduction in signal intensity at the transplant site at both 1 and 7 days after surgery. We conclude that MRI detection of SPIO-labelled cells represents a promising and novel approach to the analysis of oligodendroglial cell behaviour following transplantation that has very significant advantages over currently available methods.

Imaging of the rotator cuff: an arthrographic pitfall.

We present a case where MRI and arthrography of the shoulder reports provided seemingly conflicting data. The subsequent findings at arthroscopy revealed a potential pitfall in arthrographic interpretation.