Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1

 Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation. Accepted: 19 December 1998     

Characterizing Tropical Tree Species Growth Strategies: Learning from Inter-Individual Variability and Scale Invariance

Understanding how tropical tree species differ in their growth strategies is critical to predict forest dynamics and assess species coexistence. Although tree growth is highly variable in tropical forests, species maximum growth is often considered as a major axis synthesizing species strategies, with fast-growing pioneer and slow-growing shade tolerant species as emblematic representatives. We used a hierarchical linear mixed model and 21-years long tree diameter increment series in a monsoon forest of the Western Ghats, India, to characterize species growth strategies and question whether maximum growth summarizes these strategies. We quantified both species responses to biotic and abiotic factors and individual tree effects unexplained by these factors. Growth responses to competition and tree size appeared highly variable among species which led to reversals in performance ranking along those two gradients. However, species-specific responses largely overlapped due to large unexplained variability resulting mostly from inter-individual growth differences consistent over time. On average one-third of the variability captured by our model was explained by covariates. This emphasizes the high dimensionality of the tree growth process, i.e. the fact that trees differ in many dimensions (genetics, life history) influencing their growth response to environmental gradients, some being unmeasured or unmeasurable. In addition, intraspecific variability increased as a power function of species maximum growth partly as a result of higher absolute responses of fast-growing species to competition and tree size. However, covariates explained on average the same proportion of intraspecific variability for slow- and fast-growing species, which showed the same range of relative responses to competition and tree size. These results reflect a scale invariance of the growth process, underlining that slow- and fast-growing species exhibit the same range of growth strategies.     

The Spread of Aedes albopictus in Metropolitan France: Contribution of Environmental Drivers and Human Activities and Predictions for a Near Future

Invasion of new territories by insect vector species that can transmit pathogens is one of the most important threats for human health. The spread of the mosquito Aedes albopictus in Europe is emblematic, because of its major role in the emergence and transmission of arboviruses such as dengue or chikungunya. Here, we modeled the spread of this mosquito species in France through a statistical framework taking advantage of a long-term surveillance dataset going back to the first observation of Ae. albopictus in the Metropolitan area. After validating the model, we show that human activities are especially important for mosquito dispersion while land use is a major factor for mosquito establishment. More importantly, we show that Ae. albopictus invasion is accelerating through time in this area, resulting in a geographic range extending further and further year after year. We also show that sporadic “jump” of Ae. albopictus in a new location far from the colonized area did not succeed in starting a new invasion front so far. Finally, we discuss on a potential adaptation to cooler climate and the risk of invasion into Northern latitudes.     

Highly Overlapping Winter Diet in Two Sympatric Lemming Species Revealed by DNA Metabarcoding

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.     

Discovery and Targeted Proteomics on Cutaneous Biopsies Infected by Borrelia to Investigate Lyme Disease

Lyme disease is the most important vector-borne disease in the Northern hemisphere and represents a major public health challenge with insufficient means of reliable diagnosis. Skin is rarely investigated in proteomics but constitutes in the case of Lyme disease the key interface where the pathogens can enter, persist, and multiply. Therefore, we investigated proteomics on skin samples to detect Borrelia proteins directly in cutaneous biopsies in a robust and specific way. We first set up a discovery gel prefractionation-LC-MS/MS approach on a murine model infected by Borrelia burgdorferi sensu stricto that allowed the identification of 25 Borrelia proteins among more than 1300 mouse proteins. Then we developed a targeted gel prefractionation-LC-selected reaction monitoring (SRM) assay to detect 9/33 Borrelia proteins/peptides in mouse skin tissue samples using heavy labeled synthetic peptides. We successfully transferred this assay from the mouse model to human skin biopsies (naturally infected by Borrelia), and we were able to detect two Borrelia proteins: OspC and flagellin. Considering the extreme variability of OspC, we developed an extended SRM assay to target a large set of variants. This assay afforded the detection of nine peptides belonging to either OspC or flagellin in human skin biopsies. We further shortened the sample preparation and showed that Borrelia is detectable in mouse and human skin biopsies by directly using a liquid digestion followed by LC-SRM analysis without any prefractionation. This study thus shows that a targeted SRM approach is a promising tool for the early direct diagnosis of Lyme disease with high sensitivity (<10 fmol of OspC/mg of human skin biopsy).Lyme borreliosis is an arthropod-borne disease transmitted by hard ticks (Ixodes spp.). The causative agents are bacteria belonging to the Borrelia burgdorferi sensu lato group. In the United States, more than 30,000 cases have been reported to the Centers for Disease Control and Prevention in 2012. There, the unique pathogenic species of Borrelia is B. burgdorferi sensu stricto (s.s.). In Europe, between 65,000 and 85,000 cases are reported depending on the epidemiological study (1, 2), and the three most prevalent pathogenic species of Borrelia are Borrelia afzelii, Borrelia garinii, and B. burgdorferi s.s. The disease in both Europe and the United States is first characterized in most patients by an inflammatory skin lesion, erythema migrans (EM),¹ which is the most frequent manifestation of the disease. Dissemination to other sites occurs secondarily and can involve among others articulation, nervous system, heart, and skin at other sites (3, 4). The diagnosis can be a real challenge because of the proteiform clinical manifestations. When an EM is present, which is the case for 80% of patients (3), early diagnosis is facilitated. However, EM presentation can be clinically atypical, making the recognition of this manifestation of Lyme borreliosis difficult (5). Later on, when Borrelia has disseminated to the target organs, biological diagnosis is based either on the direct detection of the pathogen in different patient body fluids and biopsies by means of culture and/or PCR or on the indirect demonstration of presence of Borrelia by detection of anti-pathogen-directed IgM and IgG antibodies (enzyme-linked immunosorbent assay (ELISA) and Western blot) (6).Concerning the direct detection of Borrelia, culture of the bacteria has allowed the spirochete isolation since the 80s in different specific Barbour-Stoenner-Kelly-based media by using skin biopsies or biological fluids such as blood or cerebrospinal fluid (7, 8). However, Borrelia culture is not routinely used as a diagnostic test because the bacterial growth takes several weeks and does not yield timely results. Indeed, it requires the use of the specific and expensive Barbour-Stoenner-Kelly medium and a dark field microscope to detect, frequently after at least 2 weeks of incubation, the presence of Borrelia in tissues or biological fluids. When performed from patients with EM, only 40–80% of the cultures are positive (6). In addition, the success of culture varies greatly according to the Borrelia species. PCR is quicker and generally more sensitive than culture with a range of 36–88%, although the success of bacterial detection varies with the gene selected for the assay (6). PCR is efficient for Borrelia detection in synovial liquid (60–85% of the cases) in the case of arthritis (9, 10) but less sensitive in cases of neuroborreliosis in cerebrospinal fluid (<20–40% of the cases) (9, 11). Moreover, PCR detects DNA and not proteins and therefore prevents the detection of active infection. As far as the skin biopsies are concerned, the sensitivity of detection is variable in cases of EM or acrodermatitis chronica atrophicans (12). Conversely, indirect detection using serological tests is not adapted to the early diagnosis as it relies on antibodies only detectable after at least 4–6 weeks after the infectious tick bite. These tests also suffer from lack of specificity (13). New diagnostic approaches are therefore required. Selected reaction monitoring (SRM) has been recognized as an efficient mass spectrometry-based technique for the biomarker verification and validation in several biological fluids (blood, plasma, and urine) (14 –18). The demonstrated specificity, selectivity, and high sensitivity (low attomole range) of the technique (19) makes it promising for the development of an SRM-based method for early diagnosis of Lyme disease. To our knowledge, this strategy has only rarely been used on skin tissue (20). It would allow the direct and rapid detection of Borrelia proteins in the skin, demonstrating the presence of an active infection very early after the tick transmission.In the present study, we set up a workflow to develop a robust and sensitive SRM assay to detect Borrelia in human skin samples (Fig. 1). First, we looked for Borrelia proteins in infected mouse skin samples by using a classical shotgun/discovery strategy. This experiment afforded a list of bacterial proteins that are expressed in vivo in the skin of an infected mammalian host. Then, we selected protein targets and optimized a Ge-LC-SRM assay to specifically detect and quantify these proteins in mouse skin samples. We demonstrated the transferability of the SRM assay for the detection of the targeted proteins in human skin samples naturally infected with Borrelia. Finally, we improved the experimental protocol to avoid gel prefractionation.Open in a separate windowFig. 1.Summary of the experimental workflow. Experimentally infected mouse skin biopsies were analyzed by a shotgun Ge-LC-MS/MS strategy to identify Borrelia target proteins. Then we developed targeted LC-SRM assays with or without gel prefractionation. Finally, these targeted methods were transferred on tick-infected human skin samples.     

A Computational Framework for 3D Mechanical Modeling of Plant Morphogenesis with Cellular Resolution

The link between genetic regulation and the definition of form and size during morphogenesis remains largely an open question in both plant and animal biology. This is partially due to the complexity of the process, involving extensive molecular networks, multiple feedbacks between different scales of organization and physical forces operating at multiple levels. Here we present a conceptual and modeling framework aimed at generating an integrated understanding of morphogenesis in plants. This framework is based on the biophysical properties of plant cells, which are under high internal turgor pressure, and are prevented from bursting because of the presence of a rigid cell wall. To control cell growth, the underlying molecular networks must interfere locally with the elastic and/or plastic extensibility of this cell wall. We present a model in the form of a three dimensional (3D) virtual tissue, where growth depends on the local modulation of wall mechanical properties and turgor pressure. The model shows how forces generated by turgor-pressure can act both cell autonomously and non-cell autonomously to drive growth in different directions. We use simulations to explore lateral organ formation at the shoot apical meristem. Although different scenarios lead to similar shape changes, they are not equivalent and lead to different, testable predictions regarding the mechanical and geometrical properties of the growing lateral organs. Using flower development as an example, we further show how a limited number of gene activities can explain the complex shape changes that accompany organ outgrowth.     

Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus

In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.     

The Day-Hospital of the University Hospital,Bobo Dioulasso: An Example of Optimized HIV Management in Southern Burkina Faso

ObjectivesTo evaluate the epidemiological evolution of patients with HIV (PtHIV), between 2002 and 2012, in a day-hospital that became an HIV reference centre for south-west Burkina Faso.ResultsA total of 7320 patients have been treated at the centre since 2002; the active file of patients increased from 147 in 2002 to 3684 patients in 2012. Mean age was stable at 38.4 years and the majority were female (71%). The delay to initiation of antiretroviral (ARV) treatment after HIV diagnosis decreased from 12.9 months in 2002 to 7.2 months in 2012. The percentage of PtHIV lost to follow-up, untreated for HIV and deaths all decreased after 2005. Voluntary anonymous screening and/or an evocative clinical picture were the main reasons for HIV diagnosis, usually at a late stage (41.1% at WHO stage 3). Virological success increased due to a decrease in time to initiation of ARV treatment and an increase in percentage of patients treated (90.5% in 2012, mainly with 1^st line drugs). However, there was also a slight increase in the rate of therapeutic failures and the percentage of patients who progressed to 2^nd or 3^rd line-ARVs.ConclusionOur day-hospital is a good example of the implementation of a specialist centre for the management of PtHIV in a resource-limited country (Burkina Faso).     

Combining the Sterile Insect Technique with Wolbachia-Based Approaches: II- A Safer Approach to Aedes albopictus Population Suppression Programmes,Designed to Minimize the Consequences of Inadvertent Female Release

Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple-infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F₁ and F₂ progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population, released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.