Effects of metal-induced oxidative stress on endothelial cells in vitro

Aseptic loosening of metal implants is mainly attributed to the formation of metal degradation products. These include particulate debris and corrosion products, such as metal ions (anodic half-reaction) and ROS (cathodic half-reaction). While numerous clinical studies describe various adverse effects of metal degradation products, detailed knowledge of metal-induced cellular reactions, which might be important for possible therapeutic intervention, is not comprehensive. Since endothelial cells are involved in inflammation and angiogenesis, two processes which are critical for wound healing and integration of metal implants, the effects of different metal alloys and their degradation products on these cells were investigated. Endothelial cells on Ti6Al4V alloy showed signs of oxidative stress, which was similar to the response of endothelial cells to cathodic partial reaction of corrosion induced directly on Ti6Al4V surfaces. Furthermore, oxidative stress on Ti6Al4V alloy reduced the pro-inflammatory stimulation of endothelial cells by TNF-α and LPS. Oxidative stress and other stress-related responses were observed in endothelial cells in contact with Co28Cr6Mo alloy. Importantly, these features could be reduced by coating Co28Cr6Mo with a TiO2 layer, thus favouring the use of such surface modification in the development of medical devices for orthopaedic surgery. The reaction of endothelial cells to Co28Cr6Mo alloy was partially similar to the effects exerted by Co2+, which is known to be released from metal implants. Co2+ also induced ROS formation and DNA damage in endothelial cells. This correlated with p53 and p21 up-regulation, indicating the possibility of cell cycle arrest. Since CoCl2 is used as an hypoxia-mimicking agent, HIF-1α-dependence of cellular responses to Co2+ was studied in comparison to anoxia-induced effects. Although important HIF-1α-dependent genes were identified, a more detailed analysis of microarray data will be required to provide additional information about the mechanisms of Co2+ action. All these reactions of endothelial cells to metal degradation products might play their role in the complex processes taking place in the body following metal device implantation. In the worst case this can lead to aseptic loosening of the implant and requirement for revision surgery. Knowledge of molecular mechanisms of metal-induced responses will hopefully provide the possibility to interfere with undesirable processes at the implant/tissue interface, thus extending the life-time of the implant and the overall success of metal implant applications.

Exploring the biofilm of Streptococcus agalactiae to identify virulence factors

Streptococcus agalactiae, also known as Group B Streptococcus (GBS) is the primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30-76% of the cases of neonatal meningitis. Biofilms are dense aggregates of surface-adherent microorganisms embedded in an exopolysaccharide matrix. Centers for Disease Control and Prevention estimate that 65% of human bacterial infections involve biofilms (Post et al., 2004). In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and/or virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low- and non- biofilm forming strains and reduce ambiguous data interpretation. This protocol was applied to screen the in vitro biofilm formation ability of more than 350 GBS clinical isolates from pregnant women and neonatal infections belonging to different serotype, in relation to media composition and pH. The results showed the enhancement of GBS biofilm formation in acidic condition and identified a subset of isolates belonging to serotypes III and V that forms strong biofilms in these conditions. Interestingly, the best biofilm formers belonged to the serotype III hypervirulent clone ST-17.It was also found that pH 5.0 induces down-regulation of the capsule but that this reduction is not enough by itself to ensure biofilm formation. Moreover, the ability of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm formation and contribute to the biofilm structural stability. Finally, a set of proteins potentially expressed during the GBS in vitro biofilm formation were identified by mass spectrometry.

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Exploring host-pathogen interactions through protein microarray. Large-scale protein microarray analysis revealed novel human receptors for the staphylococcal immune evasion protein FLIPr and for the neisserial adhesin NadA

Adhesion, immune evasion and invasion are key determinants during bacterial pathogenesis. Pathogenic bacteria possess a wide variety of surface exposed and secreted proteins which allow them to adhere to tissues, escape the immune system and spread throughout the human body. Therefore, extensive contacts between the human and the bacterial extracellular proteomes take place at the host-pathogen interface at the protein level. Recent researches emphasized the importance of a global and deeper understanding of the molecular mechanisms which underlie bacterial immune evasion and pathogenesis. Through the use of a large-scale, unbiased, protein microarray-based approach and of wide libraries of human and bacterial purified proteins, novel host-pathogen interactions were identified. This approach was first applied to Staphylococcus aureus, cause of a wide variety of diseases ranging from skin infections to endocarditis and sepsis. The screening led to the identification of several novel interactions between the human and the S. aureus extracellular proteomes. The interaction between the S. aureus immune evasion protein FLIPr (formyl-peptide receptor like-1 inhibitory protein) and the human complement component C1q, key players of the offense-defense fighting, was characterized using label-free techniques and functional assays. The same approach was also applied to Neisseria meningitidis, major cause of bacterial meningitis and fulminant sepsis worldwide. The screening led to the identification of several potential human receptors for the neisserial adhesin A (NadA), an important adhesion protein and key determinant of meningococcal interactions with the human host at various stages. The interaction between NadA and human LOX-1 (low-density oxidized lipoprotein receptor) was confirmed using label-free technologies and cell binding experiments in vitro. Taken together, these two examples provided concrete insights into S. aureus and N. meningitidis pathogenesis, and identified protein microarray coupled with appropriate validation methodologies as a powerful large scale tool for host-pathogen interactions studies.

Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

Development of in vitro methods to test acetabular prosthetic reconstructions (messa a punto di metodi in vitro per la caratterizzazione biomeccanica di ricostruzioni acetabolari)

The revision hip arthroplasty is a surgical procedure, consisting in the reconstruction of the hip joint through the replacement of the damaged hip prosthesis. Several factors may give raise to the failure of the artificial device: aseptic loosening, infection and dislocation represent the principal causes of failure worldwide. The main effect is the raise of bone defects in the region closest to the prosthesis that weaken the bone structure for the biological fixation of the new artificial hip. For this reason bone reconstruction is necessary before the surgical revision operation. This work is born by the necessity to test the effects of bone reconstruction due to particular bone defects in the acetabulum, after the hip prosthesis revision. In order to perform biomechanical in vitro tests on hip prosthesis implanted in human pelvis or hemipelvis a practical definition of a reference frame for these kind of bone specimens is required. The aim of the current study is to create a repeatable protocol to align hemipelvic samples in the testing machine, that relies on a reference system based on anatomical landmarks on the human pelvis. In chapter 1 a general overview of the human pelvic bone is presented: anatomy, bone structure, loads and the principal devices for hip joint replacement. The purpose of chapters 2 is to identify the most common causes of the revision hip arthroplasty, analysing data from the most reliable orthopaedic registries in the world. Chapter 3 presents an overview of the most used classifications for acetabular bone defects and fractures and the most common techniques for acetabular and bone reconstruction. After a critical review of the scientific literature about reference frames for human pelvis, in chapter 4, the definition of a new reference frame is proposed. Based on this reference frame, the alignment protocol for the human hemipelvis is presented as well as the statistical analysis that confirm the good repeatability of the method.

Immune reconstitution inflammatory syndrome in patients starting antiretroviral therapy for HIV infection: a systematic review and meta-analysis

In patients with HIV-1 infection who are starting combination antiretroviral therapy (ART), the incidence of immune reconstitution inflammatory syndrome (IRIS) is not well defined. We did a meta-analysis to establish the incidence and lethality of the syndrome in patients with a range of previously diagnosed opportunistic infections, and examined the relation between occurrence and the degree of immunodeficiency. Systematic review identified 54 cohort studies of 13 103 patients starting ART, of whom 1699 developed IRIS. We calculated pooled cumulative incidences with 95% credibility intervals (CrI) by Bayesian methods and did a random-effects metaregression to analyse the relation between CD4 cell count and incidence of IRIS. In patients with previously diagnosed AIDS-defining illnesses, IRIS developed in 37.7% (95% CrI 26.6-49.4) of those with cytomegalovirus retinitis, 19.5% (6.7-44.8) of those with cryptococcal meningitis, 15.7% (9.7-24.5) of those with tuberculosis, 16.7% (2.3-50.7) of those with progressive multifocal leukoencephalopathy, and 6.4% (1.2-24.7) of those with Kaposi's sarcoma, and 12.2% (6.8-19.6) of those with herpes zoster. 16.1% (11.1-22.9) of unselected patients starting ART developed any type of IRIS. 4.5% (2.1-8.6) of patients with any type of IRIS died, 3.2% (0.7-9.2) of those with tuberculosis-associated IRIS died, and 20.8% (5.0-52.7) of those with cryptococcal meningitis died. Metaregression analyses showed that the risk of IRIS is associated with CD4 cell count at the start of ART, with a high risk in patients with fewer than 50 cells per microL. Occurrence of IRIS might therefore be reduced by initiation of ART before immunodeficiency becomes advanced.

Reticulate eruptions. Part 1: Vascular networks and physiology

Reticulate pattern is one of the most important dermatological signs of a pathological process involving the superficial vascular networks. Vascular malformations, such as cutis marmorata congenita telangiectasia and benign forms of livedo reticularis, and sinister conditions, such as meningococcal meningitis or Sneddon's syndrome, can all present with a reticulate pattern. The clinical presentation and morphology is determined by the nature and extent of the underlying pathology and the involvement of a particular vascular network. This review has been divided into four instalments. In the present paper, we discuss the anatomy and physiology of the complex network of vascular structures that support the function of the skin and subcutis.

Combined bone and soft-tissue augmentation surgery in temporo-orbital contour reconstruction

Temporal hollowing due to temporal muscle atrophy after standard skull base surgery is common. Various techniques have been previously described to correct the disfiguring defect. Most often reconstruction is performed using freehand molded polymethylmethacrylate cement. This method and material are insufficient in terms of aesthetic results and implant characteristics. We herein propose reconstruction of such defects with a polyetheretherketone (PEEK)-based patient-specific implant (PSI) including soft-tissue augmentation to preserve normal facial topography. We describe a patient who presented with a large temporo-orbital hemangioma that had been repaired with polymethylmethacrylate 25 years earlier. Because of a toxic skin atrophy fistula, followed by infection and meningitis, this initial implant had to be removed. The large, disfiguring temporo-orbital defect was reconstructed with a PEEK-based PSI. The lateral orbital wall and the temporal muscle atrophy were augmented with computer-aided design and surface modeling techniques. The operative procedure to implant and adopt the reconstructed PEEK-based PSI was simple, and an excellent cosmetic outcome was achieved. The postoperative clinical course was uneventful over a 5-year follow-up period. Polyetheretherketone-based combined bony and soft contour remodeling is a feasible and effective method for cranioplasty including combined bone and soft-tissue reconstruction of temporo-orbital defects. Manual reconstruction of this cosmetically delicate area carries an exceptional risk of disfiguring results. Augmentation surgery in this anatomic location needs accurate PSIs to achieve satisfactory cosmetic results. The cosmetic outcome achieved in this case is superior compared with previously reported techniques.

Total hip arthroplasty: leg length inequality impairs functional outcomes and patient satisfaction

Background Leg length inequality (LLI) was identified as a problem of total hip arthroplasty soon after its introduction. Leg lengthening is the most common form of LLI. Possible consequences are limping, neuronal dysfunction and aseptic component loosening. LLI can result in an increased strain both on the contralateral hip joint and on the abductor muscles. We assessed the influence of leg lengthening and shortening on walking capacity, hip pain, limping and patient satisfaction at 2-year follow-up. Methods 478 cases with postoperative lengthening and 275 with shortening were identified, and matched with three controls each. Rigorous adjustment for potential differences in baseline patient characteristics was performed by propensity-score matching of covariates. The arbitrarily defined desired outcomes were a walking capacity >60 minutes, no hip pain, no limping, and excellent patient satisfaction. Differences in not achieving the desired outcomes between the groups were expressed as odds ratios. Results In the lengthened case group, the odds ratio for not being able to walk for an hour was 1.70 (95% CI 1.28-2.26) for cases compared to controls, and the odds ratio for having hip pain at follow-up was 1.13 (95% CI 0.78-1.64). The odds ratio for limping was 2.08 (95% CI 1.55-2.80). The odds ratio for not achieving excellent patient satisfaction was 1.67 (95% CI 1.23-2.28). In the shortening case group, the odds ratio for not being able to walk for an hour was 1.23 (95% CI 0.84-1.81), and the odds ratio for having hip pain at follow-up was 1.60 (95% CI 1.05-2.44). The odds ratio for limping for cases was 2.61 (95% CI 1.78-3.21). The odds ratio for not achieving excellent patient satisfaction was 2.15 (95% CI 1.44-3.21). Conclusions Walking capacity, limping and patient satisfaction were all significantly associated with leg lengthening, whereas pain alleviation was not. In contrast, hip pain, limping and patient satisfaction were all significantly associated with leg shortening, whereas walking capacity was not.

Multiscale Characterization of Acrylic Bone Cement Modified with Functionalized Mesoporous Silica Nanoparticles

Acrylic bone cement is widely used to anchor orthopedic implants to bone and mechanical failure of the cement mantle surrounding an implant can contribute to aseptic loosening. In an effort to enhance the mechanical properties of bone cement, a variety of nanoparticles and fibers can be incorporated into the cement matrix. Mesoporous silica nanoparticles (MSNs) are a class of particles that display high potential for use as reinforcement within bone cement. Therefore, the purpose of this study was to quantify the impact of modifying an acrylic cement with various low-loadings of mesoporous silica. Three types of MSNs (one plain variety and two modified with functional groups) at two loading ratios (0.1 and 0.2 wt/wt) were incorporated into a commercially available bone cement. The mechanical properties were characterized using four-point bending, microindentation and nanoindentation (static, stress relaxation, and creep) while material properties were assessed through dynamic mechanical analysis, differential scanning calorimetry, thermogravimetric analysis, FTIR spectroscopy, and scanning electron microscopy. Four-point flexural testing and nanoindentation revealed minimal impact on the properties of the cements, except for several changes in the nano-level static mechanical properties. Conversely, microindentation testing demonstrated that the addition of MSNs significantly increased the microhardness. The stress relaxation and creep properties of the cements measured with nanoindentation displayed no effect resulting from the addition of MSNs. The measured material properties were consistent among all cements. Analysis of scanning electron micrographs images revealed that surface functionalization enhanced particle dispersion within the cement matrix and resulted in fewer particle agglomerates. These results suggest that the loading ratios of mesoporous silica used in this study were not an effective reinforcement material. Future work should be conducted to determine the impact of higher MSN loading ratios and alternative functional groups. (C) 2014 Elsevier Ltd. All rights reserved.

Chemotaxis of T-cells after infection of human choroid plexus papilloma cells with Echovirus 30 in an in vitro model of the blood-cerebrospinal fluid barrier

Enterovirus is the most common pathogen causing viral meningitis especially in children. Besides the blood-brain barrier (BBB) the choroid plexus, which forms the blood-cerebrospinal-fluid (CSF) barrier (BCSFB), was shown to be involved in the pathogenesis of enteroviral meningitis. In a human in vitro model of the BCSFB consisting of human choroid plexus papilloma cells (HIBCPP), the permissiveness of plexus epithelial cells for Echovirus 30 (EV30) was analyzed by immunoblotting and quantitative real-time PCR (Q-PCR). HIBCPP could be directly infected by EV30 from the apical as well as from the physiological relevant basolateral side. During an infection period of 5h no alterations of barrier function and cell viability could be observed. Analysis of the cytokine/chemokine-profile following enteroviral infection with a cytometric bead array (CBA) and Q-PCR revealed an enhanced secretion of PanGRO (CXCL1, CXCL2 and CXCL3), IL8 and CCL5. Q-PCR showed a significant upregulation of CXCL1, CXCL2 and CXCL3 in a time dependant manner. However, there was only a minor effect of HIBCPP-infection with EV30 on transepithelial T lymphocyte migration with or without the chemoattractant CXCL12. Moreover, CXCL3 did not significantly enhance T cell migrations. Therefore additional factors must be involved for the in vivo reported enhanced T cell migration into the CNS in the context of enteroviral meningitis. As HIBCPP are permissive for infection with EV30, they constitute a valuable human in vitro model to study viral infection at the BCSFB.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.

Evidence that N-acetylcysteine inhibits TNF-alpha-induced cerebrovascular endothelin-1 upregulation via inhibition of mitogen- and stress-activated protein kinase

N-acetylcysteine (NAC) is neuroprotective in animal models of acute brain injury such as caused by bacterial meningitis. However, the mechanism(s) by which NAC exerts neuroprotection is unclear. Gene expression of endothelin-1 (ET-1), which contributes to cerebral blood flow decline in acute brain injury, is partially regulated by reactive oxygen species, and thus a potential target of NAC. We therefore examined the effect of NAC on tumor necrosis factor (TNF)-alpha-induced ET-1 production in cerebrovascular endothelial cells. NAC dose dependently inhibited TNF-alpha-induced preproET-1 mRNA upregulation and ET-1 protein secretion, while upregulation of inducible nitric oxide synthase (iNOS) was unaffected. Intriguingly, NAC had no effect on the initial activation (i.e., IkappaB degradation, nuclear p65 translocation, and Ser536 phosphorylation) of NF-kappaB by TNF-alpha. However, transient inhibition of NF-kappaB DNA binding suggested that NAC may inhibit ET-1 upregulation by inhibiting (a) parallel pathway(s) necessary for full transcriptional activation of NF-kappaB-mediated ET-1 gene expression. Similar to NAC, the MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and the protein kinase inhibitor H-89 selectively inhibited ET-1 upregulation without affecting nuclear p65 translocation, suggesting that NAC inhibits ET-1 upregulation via inhibition of mitogen- and stress-activated protein kinase (MSK). Supporting this notion, cotreatment with NAC inhibited the TNF-alpha-induced rise in MSK1 and MSK2 kinase activity, while siRNA knock-down experiments showed that MSK2 is the predominant isoform involved in TNF-alpha-induced ET-1 upregulation.

[Invasive meningococcal infections: two cases that demonstrate the broad spectrum in clinical manifestation and outcome]

Invasive meningococcal infections show a broad clinical picture including sepsis and meningitis. Here we report on a case of sepsis and a case of meningitis, two clinical manifestations of meningococcal infections with striking differences in the clinical presentation and outcome. Meningococcal sepsis is characterized by a systemic release of endotoxins, that triggers an intense cytokine response of the host that can lead to shock and multi organ failure and death within hours. Meningococcal meningitis occurs when bacteria breach into the subarachnoidal and ventricular space during bacteremia and mortality is much lower that in sepsis. Thus meningitis may be seen as a consequence of lower pathogenicity and/or more efficient host control of the meningococci compared to sepsis.