Efficacy of a low-dose candidate malaria vaccine, R21 in adjuvant Matrix-M, with seasonal administration to children in Burkina Faso: a randomised controlled trial.

BACKGROUND: Stalled progress in controlling Plasmodium falciparum malaria highlights the need for an effective and deployable vaccine. RTS,S/AS01, the most effective malaria vaccine candidate to date, demonstrated 56% efficacy over 12 months in African children. We therefore assessed a new candidate vaccine for safety and efficacy. METHODS: In this double-blind, randomised, controlled, phase 2b trial, the low-dose circumsporozoite protein-based vaccine R21, with two different doses of adjuvant Matrix-M (MM), was given to children aged 5-17 months in Nanoro, Burkina Faso-a highly seasonal malaria transmission setting. Three vaccinations were administered at 4-week intervals before the malaria season, with a fourth dose 1 year later. All vaccines were administered intramuscularly into the thigh. Group 1 received 5 µg R21 plus 25 µg MM, group 2 received 5 µg R21 plus 50 µg MM, and group 3, the control group, received rabies vaccinations. Children were randomly assigned (1:1:1) to groups 1-3. An independent statistician generated a random allocation list, using block randomisation with variable block sizes, which was used to assign participants. Participants, their families, and the local study team were all masked to group allocation. Only the pharmacists preparing the vaccine were unmasked to group allocation. Vaccine safety, immunogenicity, and efficacy were evaluated over 1 year. The primary objective assessed protective efficacy of R21 plus MM (R21/MM) from 14 days after the third vaccination to 6 months. Primary analyses of vaccine efficacy were based on a modified intention-to-treat population, which included all participants who received three vaccinations, allowing for inclusion of participants who received the wrong vaccine at any timepoint. This trial is registered with ClinicalTrials.gov, NCT03896724. FINDINGS: From May 7 to June 13, 2019, 498 children aged 5-17 months were screened, and 48 were excluded. 450 children were enrolled and received at least one vaccination. 150 children were allocated to group 1, 150 children were allocated to group 2, and 150 children were allocated to group 3. The final vaccination of the primary series was administered on Aug 7, 2019. R21/MM had a favourable safety profile and was well tolerated. The majority of adverse events were mild, with the most common event being fever. None of the seven serious adverse events were attributed to the vaccine. At the 6-month primary efficacy analysis, 43 (29%) of 146 participants in group 1, 38 (26%) of 146 participants in group 2, and 105 (71%) of 147 participants in group 3 developed clinical malaria. Vaccine efficacy was 74% (95% CI 63-82) in group 1 and 77% (67-84) in group 2 at 6 months. At 1 year, vaccine efficacy remained high, at 77% (67-84) in group 1. Participants vaccinated with R21/MM showed high titres of malaria-specific anti-Asn-Ala-Asn-Pro (NANP) antibodies 28 days after the third vaccination, which were almost doubled with the higher adjuvant dose. Titres waned but were boosted to levels similar to peak titres after the primary series of vaccinations after a fourth dose administered 1 year later. INTERPRETATION: R21/MM appears safe and very immunogenic in African children, and shows promising high-level efficacy. FUNDING: The European & Developing Countries Clinical Trials Partnership, Wellcome Trust, and National Institute for Health Research Oxford Biomedical Research Centre.

Phenotypic Detection of Carbapenemase and AmpC-ß-Lactamase Production among Extended Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli and Klebsiella spp. Isolated from Clinical Specimens.

Introduction: Data on antimicrobial resistance (AMR) are sparse across numerous African countries, as microbiological analyses are not routinely conducted and surveillance data are not collected. Accordingly, clinical samples are not routinely tested for carbapenem-resistant bacteria and, therefore, the general understanding of their prevalence in the region remains limited. Methods: Between January 2020 and June 2022, we collected extended spectrum ß-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) isolates from five hospitals in Burkina Faso. After an initial culture on ESBL-selective media, the species were identified using API20E and isolates were tested against 13 antimicrobial agents using the disc diffusion method on Mueller-Hinton (MH) agar. ESBL production was confirmed via a double-disc synergy test. Production of carbapenemases and AmpC-ß-lactamases and phenotypic co-resistance were determined. Results: Among the 473 ESBL-PE, 356 were ESBL-E. coli (ESBL-Ec) and 117 were Klebsiella spp. (ESBL-K). Of these isolates, 5.3% were carbapenemase and 5.3% were AmpC-ß-lactamase-positive. Three types of carbapenemases were identified: 19 NDM, 3 OXA-48-like and 1 VIM. Two isolates produced both NDM and OXA-48-like carbapenemases. Carbapenemase producers were detected at all levels of healthcare. Co-resistance rates were up to 85% for aminoglycosides, 90% for sulfonamides, 95% for fluoroquinolones and 25% for chloramphenicol. Fosfomycin resistance was 6% for ESBL-Ec and 49% for ESBL-K (49%). Conclusions: Some of the ESBL-Ec and ESBL-K co-produced carbapenemases and/or AmpC-ß-lactamases at all healthcare levels and in various sample types with high co-resistance rates to non-betalactams. Carbapenem resistance is no longer rare, calling for testing in routine diagnostics, a comprehensive resistance surveillance system and infection control within healthcare.

[Biological dental implant: myth or reality?]. / L'implant dentaire biologique : mythe ou réalité ?

The currently available options for tooth-loss are prostheses, implants, or surgery (auto-transplantation). They all have their limitations. The emergence of tissue engineering, 15 years ago, was made possible by a better knowledge of the various stages of dental development, and the mastery of stem cell differentiation. It opened a new alternative approach for tooth regeneration. Even if animal experiments have demonstrated that it was possible to obtain a biological tooth from stem cells, two major issues remain to be discussed. Is it possible to use induced pluripotent stem cells instead of embryonic stem cells, which raise an ethical problem? Is it possible to reproduce a dental crown with an adapted shape and colour? Or should we consider the simpler creation of a biological root secondarily covered by a ceramic prosthesis? Our study mentions the main landmarks and the key cells involved in the embryological development of the tooth, establishes a mapping and a list of the various types of stem cells. It details the various methods used to create a biological implant.

HUGO (FNDC3A): a new gene overexpressed in human odontoblasts.

Previously, we established a subtractive cDNA library enriched in odontoblast-specific genes and hypothesized that new, previously unidentified, markers would be present, associated with the odontoblast phenotype. In this paper, we report the first characterization of a new gene we have named HUGO, and its associated deduced protein sequence. This gene expression is under the control of two alternative promoters, resulting in the synthesis of two proteins, one of which, HUGO2, is included in the other, HUGO1. HUGO proteins are mainly composed of a proline-rich region at the N-terminus, 8 type III-fibronectin modules, and a transmembranous helix at the C-terminus. In odontoblasts, the proteins are located in Golgi vesicles. However, they display a broader expression pattern, since they are also expressed by nerve fibers in the dental pulp and other tissues (e.g., trachea, brain, kidney), as demonstrated by immunohistochemistry and qPCR, respectively. Their location in odontoblasts suggests a role in collagen and glycosaminoglycan synthesis.

Cloning, sequencing, and expression of the amelogenin gene in two scincid lizards.

Our knowledge of the gene coding for amelogenin, the major enamel protein, is mainly based on mammalian sequences. Only two sequences are available in reptiles. To know whether the snake sequence is representative of the amelogenin condition in squamates, we have studied amelogenin in two scincid lizards. Lizard amelogenin possesses numerous conserved residues in the N- and C-terminal regions, but its central region is highly variable, even when compared with the snake sequence. This rapid evolution rate indicates that a single squamate sequence was not representative, and that comparative studies of reptilian amelogenins might be useful to detect the residues which are really important for amelogenin structure and function. Reptilian and mammalian enamel structure is roughly similar, but no data support amelogenin being similarly expressed during amelogenesis. By performing in situ hybridization using a specific probe, we showed that lizard ameloblasts express amelogenin as described during mammalian amelogenesis. However, we have not found amelogenin transcripts in odontoblasts. This indicates that full-length amelogenin is specific to enamel matrix, at least in this lizard.

[Clinical consequences of dioxins exposure during tooth development]. / Conséquences cliniques des effets des dioxines sur le développement dentaire.

Commonly designed by the term "dioxins", polychlorinated aromatic hydrocarbons are environmental pollutants leading to several toxic effects during development and growth in embryo and child. The general consequences of dioxin exposure are particularly well-documented whereas only few data are mentioned by the experts concerning tooth development. However, studies performed in rodents have shown many disruptions during odontogenesis and enamel mineralisation. Moreover, recent epidemiological studies have demonstrated in human the incidence of dioxin exposure on enamel hypomineralisation and hypodontia. The aim of this review is to report recent data about consequences of dioxin exposure on tooth development, tooth being considered as a biological marker of potential dioxin poisoning.

A substractive PCR-based cDNA library from human odontoblast cells: identification of novel genes expressed in tooth forming cells.

Odontoblasts are highly specialized cells aligned at the edge of the dental pulp. As a step towards understanding the complex mechanisms underlying their terminal differentiation, the gene expression pattern was examined in human cultured odontoblast cells. Suppression substractive hybridization (SSH) was used to establish a substracted cDNA library specific for human odontoblasts. For this purpose, cDNAs from human cultured fibroblastic pulp cells were substracted to cDNA from human cultured odontoblasts. The nucleotide sequence of 154 substracted cDNA clones was determined. We identified 130 preferentially expressed gene fragments in odontoblasts as compared with the fibroblastic pulp cells. Ten of them were already identified in odontoblasts such as DSPP, BSP, enamelysin and Col1A1. We confirmed their overexpression by RT-PCR on the cultured cells and in vivo by in situ hybridization on human molars. Another 64 clones corresponded to known genes. Among them, two clones were of particular interest: reelin, which was first detected in the brain and osteoadherin, which was first located in bone. Fifty-six clones were unknown genes even though 82% matched expressed sequence tags or genomic clones. A reverse Northern dot blot showed that 96% of them were overexpressed at different rates in cultured odontoblasts. These latest results indicate that there are still unknown genes that are associated with the control of the odontoblast phenotype. Thus, cloning of odontoblast differentiation-associated genes not only opens up new methods of elucidating the normal development but also the recruitment of odontoblasts when required to initiate repair of dentin.

Sequential expression of matrix protein genes in developing rat teeth.

Tooth organogenesis is dependent on reciprocal and sequential epithelial-mesenchymal interactions and is marked by the appearance of phenotypic matrix macromolecules in both dentin and enamel. The organic matrix of enamel is composed of amelogenins, ameloblastin/amelin, enamelins and tuftelin. Dentin is mainly composed of type I collagen, but its specificity arises from the nature of the non-collagenous proteins (NCPs) involved in mineralization, phosphophoryn (DPP), dentin sialoprotein (DSP), osteocalcin, bone sialoprotein and dentin matrix protein-1 (Dmp1). In this paper, we studied the pattern of expression of four mineralizing protein genes (type I collagen, amelogenin, DSPP and osteocalcin) during the development of rat teeth by in situ hybridization on serial sections. For this purpose, we used an easy and rapid procedure to prepare highly-specific labeled single-stranded DNA probes using asymmetric polymerase chain reaction (PCR). Our results show that type I collagen is primarily expressed in polarizing odontoblasts, followed by the osteocalcin gene expression in the same polarized cells. Concomitantly, polarized ameloblasts start to accumulate amelogenin mRNAs and transiently express the DSPP gene. This latter expression switches over to odontoblasts whereas mineralization occurs. At the same time, osteocalcin gene expression decreases in secretory odontoblasts. Osteocalcin may thus act as an inhibitor of mineralization whereas DSP/DPP would be involved in more advanced steps of mineralization. Amelogenin and type I collagen gene expression increases during dentin mineralization. Their expression is spatially and temporally controlled, in relation with the biological role of their cognate proteins in epithelial-mesenchymal interactions and mineralization.

Expression of the small leucine-rich proteoglycan osteoadherin/osteomodulin in human dental pulp and developing rat teeth.

Because the extracellular matrices of dentin and bone are composed mainly of type I collagen, their characteristics are determined by the nature of noncollagenous proteins (NCPs). Among these NCPs, some proteoglycans (PGs) belong to the small leucine-rich proteoglycans (SLRPs). Recently, osteoadherin (OSAD) has been described as a new member of this family, that is expressed by mature bovine osteoblasts. Here, we report the expression of OSAD messenger RNA (mRNA) in human dental tissues and during the development of rat molars, using in situ hybridization. For this purpose, we constructed a probe for OSAD mRNA transcripts from human odontoblast cells cultured in vitro. Our results indicate that the mature human odontoblasts overexpress the OSAD gene as compared with cells present in the pulp core. In rat developing molars, mRNA transcripts were first detected in alveolar bone in 19-day-old embryos. At the same age, no signal was detected in any cell of the first molar. In more mature teeth (newborn and 2-day-old rats), OSAD expression starts in the polarized odontoblasts and increases in the secretory and mature odontoblasts, respectively. Interestingly, a similar pattern of expression was observed in the ameloblast layer responsible for the deposition of enamel mineralized matrix. Together, these results lead us to speculate that OSAD may be implicated in biomineralization processes.

Evaluation of the biocompatibility of a Ni-Cr-Mo dental alloy with human gingival explant culture in vitro: morphological study, immunodetection of fibronectin, and collagen production.

This study was undertaken to evaluate the biocompatibility of a Ni-Cr-Mo dental casting alloy by an in vitro explant culture method. Through ultrastructural features, the immunolabelling of fibronectin, and the immunodetection of type I and III collagen production, we investigated the behaviour of gingival cells (both fibroblasts and epithelial cells) in contact with the alloy in comparison with control cultures. Our results indicate that cultured gingival cells present a well preserved ultrastructure and synthesized fibronectin (the main glycoprotein involved in adhesion to substrates). The alteration of collagen production concerned only type III collagen which decreased significantly in the cultures on the dental alloy.

Mechanical properties and biocompatibility of two polyepoxy matrices: DGEBA-DDM and DGEBA-IPD.

The aim of this paper was to study the biocompatibility and mechanical properties of materials for orthopaedic and odontologic surgical use. The products used were obtained by polycondensation of a diepoxy resin (DGEBA) with two curing agents (DDM or IPD). The materials present a slight swelling in liquid medium and their thermomechanical properties are hardly affected after 12 month implantation. The absence of molecular desorption in isotonic liquid and human serum confirms their hydrolytic stability and thus their inertia. These materials do not give rise to an intolerance reaction by neighbouring tissues during implantation time (1 d to 12 month).

An in vitro model of human dental pulp repair.

Pulp tissue responds to dentin injury by laying down reactionary dentin secreted by existing odontoblasts or reparative dentin elaborated by odontoblast-like cells that differentiated from precursor cells in the absence of inner dental epithelium and basement membrane. Furthermore, growth factors or active dentin matrix components are fundamental signals involved in odontoblast differentiation. In vitro, dental pulp cells cultured under various conditions are able to express typical markers of differentiation, but no culture system can re-create pulp response to dentin drilling. This paper reports the behavior of thick slices from human teeth drilled immediately after extraction and cultured from 3 days to 1 month. Results show that the damaged pulp beneath the cavity is able to develop, in vitro, some typical aspects correlated to tissue healing, evidenced by cell proliferation (BrdU-positive cells), neovascularization (positive with antitype-IV collagen antibodies), and the presence of functional (3H proline-positive) cuboidal cells close to the injured area. After 30 days of culture, elongated spindle-shaped cells can be seen aligned along the edges of the relevant dentin walls, whereas sound functional odontoblasts are well-preserved beneath healthy areas. This tissue recovery leads us to believe that such a culture model will be a useful system for testing factors regulating pulp repair.

Expression and localization of TREK-1 K+ channels in human odontoblasts.

During tooth development, odontoblasts are the cells that form dentin and possibly mediate early stages of sensory processing in teeth. It is suggested that ion channels assist in these events. Indeed, mechanosensitive potassium currents, transducing mechanical stimuli into electrical cell signals, have been previously recorded in the human odontoblast cell membrane. Here, we show by RT-PCR that the mechanosensitive potassium channel TREK-1 (a member of the two-pore-domain potassium channel family) is overexpressed in these cultured cells compared with pulp cells in vitro. In situ hybridization showed that transcripts are detected in the odontoblast layer in vivo. The use of antibodies shows that TREK-1 is strongly expressed in the membrane of coronal odontoblasts and absent in the root. This distribution is related to the spatial distribution of nerve endings identified by labeling of the low-affinity nerve growth factor (NGF) receptor (p75(NTR)). These results demonstrate the expression of TREK-1 in human odontoblasts in vitro and in vivo.

Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices.

Transforming growth factor-beta1 (TGF beta1) is a potent modulator of tissue repair in various tissues. To analyze its role during human dental repair, we used thick-sliced teeth cultured as described previously (Magloire et al., 1996). The supply of TGF beta1 to the pulp tissue was accomplished by means of a small tube glued onto the dentin. We show that this device allowed the growth factor to diffuse locally through dentinal tubules and to bind to the cells present in the coronal pulp opposite the TGF beta1-delivery tube. The tube was filled with 20 ng/mL TGF beta1, and slices were cultured for 4 days. Results show a preferential accumulation of cells in the odontoblastic and subodontoblastic layers in the vicinity of the tube. Cell proliferation increased in the subodontoblastic layer and in the underlying pulp, and BrdU-positive cells were abundant around the blood vessels. TGF beta1 induced type I collagen production by the odontoblastic/subodontoblastic/pulp cells in the stimulated zone, as demonstrated by in situ hybridization. These results suggest that TGF beta1 could be directly involved in the regulation of cell proliferation, migration, and extracellular matrix production in the human dental pulp and eventually in the repair process occurring after tooth injury.

TGF-beta1 induces accumulation of dendritic cells in the odontoblast layer.

TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents is suspected to influence dental pulp response, including the modulation of cell migration. To determine the consequences of TGF-beta1 action on pulp immune cells, we analyzed, by immunohistochemistry, the effect of transdentinally diffusing TGF-beta1 on their localization in a human tooth slice culture model. TGF-beta1 induced an accumulation of HLA-DR-positive cells in both odontoblast and subodontoblast layers of the stimulated zone. Together with HLA-DR, these cells co-expressed Factor XIIIa and CD68, two features of immature antigen-presenting dendritic cells (DC), as well as the TGF-beta1 specific receptor TbetaRII. In contrast, no effect could be detected on the localization of either mature DC-LAMP-positive DC or of T- and B-lymphocytes. Analysis of these data suggests that TGF-beta1 released from dentin degraded by bacterial or iatrogenic agents could be involved in the immune response of the dental pulp resulting from tooth injury.

Alpha v beta 3 integrin expression in human odontoblasts and co-localization with osteoadherin.

Integrins are heterodimeric transmembrane receptors which promote cell adhesion, thus contributing to the maintenance of tissue organization in both normal and pathological conditions. To characterize the way odontoblasts may interact with other cells and the extracellular matrix in human teeth, we studied expression of alpha v beta 3 integrin, a putative receptor for osteoadherin. We showed that alpha v beta 3 integrin expression was restricted to odontoblasts, blood vessels, and small rounded cells in sound and carious pulp. Odontoblast staining intensity increased from the apical to the cusp region. Osteoadherin staining was strong in the whole odontoblast layer (with a slight decrease in the cusp region) and in predentin. Odontoblasts differentiating in vitro were stained with the anti-alpha v beta 3 integrin antibody, first at the level of intercellular contacts, then throughout the cell membrane. These results suggest that the alpha v beta 3 integrin could play a role in interodontoblast adhesion and odontoblast binding to the surrounding predentin/dentin/pulp matrix, possibly through osteoadherin.

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

Intercellular junctions in human tooth-pulp cells in culture in vitro revealed by freeze-fracture, lanthanum impregnation and filipin treatment.

Three kinds of intercellular junctions were detected between human dental pulp cells in explant culture with electron microscopy included filipin detection for cholesterol; desmosome-like junctions observed on ultrathin sections probably contribute to the cohesiveness between cells in culture. Gap junctions, responsible for intercellular communication, exhibited two morphologies on freeze-fracture replicas: a conventional arrangement of their intramembranous particles and a crystalline array corresponding to the formation stage of junctions. Primitive tight junctions were detected on freeze-fracture replicas but not on ultrathin sections. It is likely that they contribute to the cell-to-cell adhesion under culture conditions.

In vitro mineralization of a three-dimensional collagen matrix by human dental pulp cells in the presence of chondroitin sulphate.

These matrices were used as cell culture substrates to investigate the influence of extracellular molecules on mineralization. Pulp cells seeded in type I collagen or type I collagen-chondroitin-4-sulphate sponges were able to grow and were morphologically similar to cells responsible for reparative dentine formation in vivo. In sponges consisting of collagen only, the cells elaborated an abundant new matrix which became organized with time and consisted of collagen fibres surrounded by fibrillar material, but no mineralization was observed. In collagen-chondroitin sulphate sponges, cells deposited less and poorly organized matrix; in these, calcification occurred, increasing with time, and at the ultrastructural level, small needle-like crystals containing calcium and phosphorus were scattered throughout the sponge fibres. These observations suggest that chondroitin sulphate might influence in vitro calcification induced by pulp cells.

Immunohistochemical localization of L-type calcium channels in the developing first molar of the rat during odontoblast differentiation.

To study the presence of L-type calcium channels during the different steps of odontoblast differentiation, a specific monoclonal antibody against 1,4-dihydropyridine receptors was used in combination with avidin-biotin-peroxidase complex labelling. Staining was seen on the cell bodies of pre-odontoblasts, concentrated at the apical pole (distal portion) of functional odontoblasts and localized on cell bodies and processes of mature odontoblasts. Thus calcium channels were expressed at the onset of differentiation and maintained in the differentiated cells but with some changes of localization. It is suggested that these channels may facilitate the entry of calcium to act as a second messenger for cellular polarization or be involved in the transcellular transport of calcium to the mineralizing front.