Immunization Strategies Against Clostridioides difficile.

Clostridioides difficile (C. difficile) infection (CDI) is an important healthcare but also a community-associated disease. CDI is considered a public health threat and an economic burden. A major problem is the high rate of recurrences. Besides classical antibiotic treatments, new therapeutic strategies are needed to prevent infection, to treat patients, and to prevent recurrences. If fecal transplantation has been recommended to treat recurrences, another key approach is to elicit immunity against C. difficile and its virulence factors. Here, after a summary concerning the virulence factors, the host immune response against C. difficile, and its role in the outcome of disease, we review the different approaches of passive immunotherapies and vaccines developed against CDI. Passive immunization strategies are designed in function of the target antigen, the antibody-based product, and its administration route. Similarly, for active immunization strategies, vaccine antigens can target toxins or surface proteins, and immunization can be performed by parenteral or mucosal routes. For passive immunization and vaccination as well, we first present immunization assays performed in animal models and second in humans and associated clinical trials. The different studies are presented according to the mode of administration either parenteral or mucosal and the target antigens and either toxins or colonization factors.

Mouse Wnt1-CRE-RosaTomato Dental Pulp Stem Cells Directly Contribute to the Calvarial Bone Regeneration Process.

Stem cells endowed with skeletogenic potentials seeded in specific scaffolds are considered attractive tissue engineering strategies for treating large bone defects. In the context of craniofacial bone, mesenchymal stromal/stem cells derived from the dental pulp (DPSCs) have demonstrated significant osteogenic properties. Their neural crest embryonic origin further makes them a potential accessible therapeutic tool to repair craniofacial bone. The stem cells' direct involvement in the repair process versus a paracrine effect is however still discussed. To clarify this question, we have followed the fate of fluorescent murine DPSCs derived from PN3 Wnt1-CRE- RosaTomato mouse molar (T-mDPSCs) during the repair process of calvaria bone defects. Two symmetrical critical defects created on each parietal region were filled with (a) dense collagen scaffolds seeded with T-mDPSCs, (b) noncellularized scaffolds, or (c) no scaffold. Mice were imaged over a 3-month period by microcomputed tomography to evaluate the extent of repair and by biphotonic microscopy to track T-mDPSCs. Histological and immunocytochemical analyses were performed in parallel to characterize the nature of the repaired tissue. We show that T-mDPSCs are present up to 3 months postimplantation in the healing defect and that they rapidly differentiate in chondrocyte-like cells expressing all the expected characteristic markers. T-mDPSCs further maturate into hypertrophic chondrocytes and likely signal to host progenitors that form new bone tissue. This demonstrates that implanted T-mDPSCs are able to survive in the defect microenvironment and to participate directly in repair via an endochondral bone ossification-like process. Stem Cells 2019;37:701-711.

Variable spectrum of disease and risk factors of peripartum Clostridium difficile infection: report of 14 cases from French hospitals and literature review.

Our aim was to study Clostridium difficile infection (CDI) in peripartum women in France and compare them to cases published in the literature. We characterize these infections regarding clinico-biological features and specific risk factors in order to raise awareness for obstetricians and midwifes. Eight antepartum and six post-partum CDI cases were retrospectively studied in 6 French centers during the period between 2008 and 2013. In addition, 59 literature cases were reviewed. Cases were identified with CDI clinical symptoms associated to characteristic imagery or detection of C. difficile toxins. The key risk factors of CDI (antibiotherapy, hospitalization) and other risk factors (cesarean section, obstetric complications, corticotherapy, and underlying disease) were retrospectively collected. Most of the cases were exposed to at least one key risk factor of CDI: previous exposure to antibiotics and/or hospitalization. The post-partum cases often had cesarean section: 67% (4/6) in French cases and 89% (31/35) in literature cases. Metronidazole was the most used antibiotic. Relapses occurred in two French cases and in nine published cases. Two French cases and 15 literature cases were reported to have complications (pseudomembranous colitis, toxic megacolon, death…). Diverse C. difficile PCR ribotypes were involved, but the BI/NAP1/027 strain was not detected in the French case series contrary to the literature cases. The delay for diagnosis CDI could be long and peripartum CDI could be severe. In case of unexplained diarrhea in pregnant women, clinicians need to consider CDI and ask for research of C. difficile and its toxins in stool.

Immunization Strategies Against Clostridium difficile.

C. difficile infection (CDI) is an important healthcare- but also community-associated disease. CDI is considered a public health threat and an economic burden. A major problem is the high rate of recurrences. Besides classical antibiotic treatments, new therapeutic strategies are needed to prevent infection, to treat patients and prevent recurrences. If fecal transplantation has been recommended to treat recurrences, another key approach is to restore immunity against C. difficile and its virulence factors. Here, after a summary concerning the virulence factors, the host immune response against C. difficile and its role in the outcome of disease, we review the different approaches of passive immunotherapies and vaccines developed against CDI. Passive immunization strategies are designed in function of the target antigen, the antibody-based product and its administration route. Similarly, for active immunization strategies, vaccine antigens can target toxins or surface proteins and immunization can be performed by parenteral or mucosal routes. For passive immunization and vaccination as well, we first present immunization assays performed in animal models and second in humans and associated clinical trials. The different studies are presented according to the mode of administration either parenteral or mucosal and the target antigens, either toxins or colonization factors.

Immune responses induced by Clostridium difficile.

The spectrum of Clostridium difficile infections is highly variable, ranging from asymptomatic carriage to fatal colitis depending on the strain virulence and on the host, its gut microbiota and its immune response. After disruption of the gut microbiota, C. difficile pathogenesis can be divided into three steps: 1) contamination by spores and their germination; 2) multiplication of vegetative cells and intestinal colonization using colonization factors; 3) production of the toxins TcdA and TcdB, and for some strains, the binary toxin, which are responsible for the clinical signs. Three lines of defense counteract C. difficile. The first line is the epithelial barrier, which is breached by the toxins. Then, a rapid innate immune response follows, which forms the second line of defense. It provides very quick defense reactions against C. difficile but is non-specific and does not confer memory. C. difficile and its virulence factors, the toxins and colonization factors, induce a highly pro-inflammatory response, which can be either beneficial or harmful, but triggers the adaptive immunity as the third line of defense required to control the infectious process. Adaptive immunity provides a highly specific immune response against C. difficile with memory and long lasting immunity. The innate and adaptive immune responses against the toxins and surface components are analyzed as well as their role in disease susceptibility, severity and recurrences.

Clostridium difficile flagella predominantly activate TLR5-linked NF-κB pathway in epithelial cells.

Clostridium difficile has become the most common enteropathogen responsible for intestinal nosocomial post-antibiotic infections. This has coincided with the appearance of serious cases related to the emergence of hypervirulent strains. The toxins are the main virulence factors and elicit an inflammatory response during C. difficile infection. However, other bacterial components appear to be involved in the inflammatory process. In some pathogens, flagella play a role in pathogenesis through abnormal stimulation of the TLR5-mediated host immune response. To date, few studies have addressed this role for C. difficile flagella. In the current study, we confirm in two different epithelial cell models that C. difficile thanks to its FliC flagellin interacts with TLR5. In addition, thanks to inhibition and transcriptomic studies we demonstrate that the interaction of flagellin and TLR5 predominantly activates the NF-κB and, in a lesser degree, the MAPK pathways, via TLR5, leading to up-regulation of pro-inflammatory gene expression and synthesis of pro-inflammatory mediators. These results suggest a role for C. difficile flagella in contributing to inflammatory response in host intestinal cells.

Passive and active immunization strategies against Clostridium difficile infections: state of the art.

The pathophysiology of Clostridium difficile infections (CDI) could be considered as a three-step process that takes place after disruption of the digestive microbiota by antibiotics: 1) germination of spores; 2) multiplication and persistence of C. difficile in the colonic niche thanks to colonization factors; 3) production of the two toxins TcdA and TcdB and for some strains an additional toxin, the binary toxin CDT. Different immunization strategies against C. difficile have been developed, first regarding the toxins. Immunization assays with colonization factors have followed, and allowed accumulation of new data concerning theirs functions and immunogenicity. Here, we present the toxins, the colonization factors and their use in passive and active immunizations to treat and/or to prevent C. difficile infections. The various experiments performed in animal models and the first clinical trials in humans are reported.

Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics.

Clostridium difficile is currently the major cause of nosocomial intestinal diseases associated with antibiotic therapy in adults. In order to improve our knowledge of C. difficile-host interactions, we analyzed the genome-wide temporal expression of C. difficile 630 genes during the first 38 h of mouse colonization to identify genes whose expression is modulated in vivo, suggesting that they may play a role in facilitating the colonization process. In the ceca of the C. difficile-monoassociated mice, 549 genes of the C. difficile genome were differentially expressed compared to their expression during in vitro growth, and they were distributed in several functional categories. Overall, our results emphasize the roles of genes involved in host adaptation. Colonization results in a metabolic shift, with genes responsible for the fermentation as well as several other metabolic pathways being regulated inversely to those involved in carbon metabolism. In addition, several genes involved in stress responses, such as ferrous iron uptake or the response to oxidative stress, were regulated in vivo. Interestingly, many genes encoding conserved hypothetical proteins (CHP) were highly and specifically upregulated in vivo. Moreover, genes for all stages of sporulation were quickly induced in vivo, highlighting the observation that sporulation is central to the persistence of C. difficile in the gut and to its ability to spread in the environment. Finally, we inactivated two genes that were differentially expressed in vivo and evaluated the relative colonization fitness of the wild-type and mutant strains in coinfection experiments. We identified a CHP as a putative colonization factor, supporting the suggestion that the in vivo transcriptomic approach can unravel new C. difficile virulence genes.

Performance of rapid tests for detection of HBsAg and anti-HBsAb in a large cohort, France.

BACKGROUND & AIMS: The systematic use of rapid tests performed at points-of-care may facilitate hepatitis B virus (HBV) screening and substantially increase HBV infection awareness. The aim of this study was to evaluate the effectiveness of such tests for HBsAg and anti-HBsAb detection among individuals visiting a variety of healthcare centers located in a low HBV-prevalent area. METHODS: Three rapid tests for hepatitis B surface antigen (HBsAg) detection (VIKIA, Determine and Quick Profile) and one test for anti-hepatitis B surface antibody (anti-HBsAb) detection (Quick Profile) were evaluated in comparison to ELISA serology. Sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV, respectively) and area under the ROC curve were used to estimate test performance. Non-inferiority criteria of the joint Se, Sp were set at 0.80, 0.95. RESULTS: Among the 3956 subjects screened, 85 (2.1%) were HBsAg-positive and 2225 (56.5%) had a protective anti-HBsAb titer. Test Se and Sp (lower bound of 97.5% CI) were as follows: 96.5% (89.0%), 99.9% (99.8%) for Vikia; 93.6% (80.7%), 100.0% (99.8%) for Determine; and 90.5% (80.8%), 99.7% (99.5%) for Quick Profile; with all three tests achieving minimal non-inferiority criteria. False negatives were typically observed in inactive HBsAg carriers. The anti-HBsAb Quick Profile test had excellent specificity (97.8%) and PPV (97.8%) albeit low sensitivity (58.3%), thus failing to establish non-inferiority. CONCLUSIONS: All three HBsAg rapid tests could be considered ideal for HBV screening in low HBV-prevalent countries, given the ease of use, rapidity, and high classification probabilities. The anti-HBsAb Quick Profile could be considered reliable only for positive tests.

Influence of environmental conditions on the expression and the maturation process of the Clostridium difficile surface associated protease Cwp84.

Expression of the Clostridium difficile protease gene, cwp84, was moderately up-regulated by decreasing pH due to glucose metabolism. Purification under different pH conditions influenced the proteolytic process of Cwp84. Given this, acidic pH could favor the appearance of different forms of Cwp84 that may have different roles during the infection.

Schistosoma haematobium urinary tract complications in African migrants attending primary care facilities in Paris, France: A retrospective cohort study (2004-2018).

OBJECTIVES: Little is known about the burden of urogenital schistosomiasis (UGS) outside endemic areas. This study was aimed at describing urinary complications of UGS detected among African migrants in French primary care facilities. PATIENTS AND METHODS: A retrospective cohort study included patients with UGS diagnosed from 2004 to 2018 in 5 primary health centers in Paris. Cases were defined by the presence of typical Schistosoma haematobium eggs at urine microscopy. Demographic, clinical, biological and imaging data were collected. Ultrasonography (U-S) findings were classified in accordance with the WHO guidelines. RESULTS: U-S was prescribed for all patients and performed in 100/118. Sex ratio (F/M) was 2/98, and mean age 24.4 years. Patients were from West Africa (73% from Mali) and consulted 8 months (median) after their arrival. Among the 95 patients with interpretable findings, 32 (33.7%) had abnormalities related to UGS, considered as major in 6 cases (6.3%), and mostly localized at the bladder (31/32) without detection of cancer. No sociodemographic, clinical, or biological factors were found to be associated with U-S abnormalities. All 100 patients were treated by praziquantel (PZQ). Among those with abnormalities, 20/32 received two to four doses at various time intervals. Post-cure imaging control performed in 19/32 showed persistent abnormalities in 6 patients, on average 5 months after the last PZQ uptake. CONCLUSION: Urinary tract abnormalities associated with UGS were common and predominated at the bladder. U-S should be prescribed to any patient with positive urine microscopy. Schedules for PZQ uptake and U-S monitoring for patients with complications remain to be determined.

Clostridium difficile carriage in healthy infants in the community: a potential reservoir for pathogenic strains.

BACKGROUND: Clostridium difficile has long been considered to be a nosocomial pathogen but has emerged in the community in recent years. During infancy, asymptomatic C. difficile colonization is common. However, knowledge of colonization determinants and strain characteristics is limited. We studied the dynamics of C. difficile colonization in healthy infants from the community. Determinants of colonization and strain genotypes were also determined in a cohort of infants attending day nurseries. METHODS: A 1-year follow-up study involving 10 healthy infants was performed to determine the incidence and kinetics of intestinal C. difficile colonization. In addition, a 1-point study involving 85 healthy infants (age, 0-3 years) from 2 day nurseries was performed. C. difficile isolates were typed by polymerase chain reaction-ribotyping and analyzed for the presence of toxin genes. RESULTS: During the follow-up study, all infants acquired C. difficile and were colonized for several months. An early (neonatal) and a late (4-6 months of age) acquisition period were identified. In day nurseries, 38 infants (45%) carried C. difficile, with 11 (13%) carrying a toxigenic isolate. Age and several environmental factors were associated with the C. difficile carrier state. Strains causing disease in adults were identified in infants. Interestingly, no infant carried the common epidemic 027 or 078 strains. CONCLUSIONS: This study provides information on the dynamics of colonization in infants in the community and on the genotype of involved strains. C. difficile colonization appears mainly as an age-dependent process. Pathogenic strains circulate in asymptomatic infants from the community, who represent a potential reservoir of pathogenic strains.

The effect of in vitro growth conditions on the resistance of Acanthamoeba cysts.

Despite increasing concerns of direct pathogenicity and/or their role as hosts for other microorganisms there are currently no standard methods for the inactivation of amoebae that belong to the genus Acanthamoeba. Methods used to grow amoebae and produce cysts for these tests may be important as they can dramatically modify cyst susceptibility. We compared resistance of cysts produced from trophozoites grown in peptone-yeast extract-glucose broth or by feeding on HEp-2 cells and then encysted in Neff's medium. We observed that trophozoites grown using HEp-2 cells as a nutrient source produce cysts that are significantly more resistant to SDS and to most biocides tested, including heat. Increased resistance is likely due to a higher proportion of mature cysts presenting thicker cell walls as demonstrated using transmission electron microscopy. This was confirmed by calcofluor white staining demonstrating higher cellulose content in cysts produced from trophozoites grown using HEp-2 cells as a feeding source. These results demonstrate that not only methods used to produce cysts from trophozoites are critical, but that methods used to grow trophozoites before encystment should also be chosen carefully. This should be taken into account for the development of protocols to evaluate biocides and antimicrobials against amoebal cysts.

Osteogenic Effect of Fisetin Doping in Bioactive Glass/Poly(caprolactone) Hybrid Scaffolds.

Treating large bone defects or fragile patients may require enhancing the bone regeneration rate to overcome a weak contribution from the body. This work investigates the osteogenic potential of nutrient fisetin, a flavonoid found in fruits and vegetables, as a doping agent inside the structure of a SiO2-CaO bioactive glass-poly(caprolactone) (BG-PCL) hybrid scaffold. Embedded in the full mass of the BG-PCL hybrid during one-pot synthesis, we demonstrate fisetin to be delivered sustainably; the release follows a first-order kinetics with active fisetin concentration being delivered for more than 1 month (36 days). The biological effect of BG-PCL-fisetin-doped scaffolds (BG-PCL-Fis) has been highlighted by in vitro and in vivo studies. A positive impact is demonstrated on the adhesion and the differentiation of rat primary osteoblasts, without an adverse cytotoxic effect. Implantation in critical-size mouse calvaria defects shows bone remodeling characteristics and remarkable enhancement of bone regeneration for fisetin-doped scaffolds, with the regenerated bone volume being twofold that of nondoped scaffolds and fourfold that of a commercial trabecular bovine bone substitute. Such highly bioactive materials could stand as competitive alternative strategies involving biomaterials loaded with growth factors, the use of the latter being the subject of growing concerns.

Factors and Mechanisms Involved in Acquired Developmental Defects of Enamel: A Scoping Review.

Background: Developmental Defects of Enamel (DDE) is a pathology of the teeth that can greatly alter the quality of life of patients (hypersensitivity, esthetic issues, loss of function, etc.). The acquired DDE may occur as a result of a wide range of acquired etiological factors and his prevalence of this pathology may reach up to 89.9%. The main objective of this research was to identify and analyze, in current literature, the factors related to acquired DDE, in order to propose a general theory about the mechanisms involved. Methods: The search of the primary literature was conducted until [December 31, 2021]. Our search strategy uses the Pubmed/MEDLINE database and was structured around 3 terms ["Development," "Defect," and "Enamel"]. To be included, references had to be primary studies, written in English. Exclusion criteria were reviews, in vitro, animal, genetic or archeology studies, and studies focused on clinical management of DDE. One hundred and twenty three articles were included in this scoping review: 4 Randomized clinical trials, 1 letter, 5 cases reports, 2 fundamentals studies, and 111 observational studies (33 Cross-sectional studies, 68 Cohort study and 10 Case-control study). The quality of evidence was assessed using the PEDro scale for clinical trials, the Newcastle-Ottawa scale for observational studies, and a published tool to assess the quality of case reports and case series. Results: A scoping review of the literature identified 114 factors potentially involved in acquired DDE. The most frequently encountered pathologies are those causing a disorder of calcium homeostasis or a perturbation of the ARNT pathway in mother or child. The link between the ARNT pathway and metabolism deficiency in uncertain and needs to be defined. Also, the implication of this mechanism in tissue impairment is still unclear and needs to be explored. Conclusions: By identifying and grouping the risk factors cited in the literature, this taxonomy and the hypotheses related to the mechanism allow health practitioners to adopt behaviors that limit the risk of developing aDDE and to set up a prevention of dental pathology. In addition, by reviewing the current literature, this work provides guidance for basic research, clinical studies, and literature searches.

Localization of the Clostridium difficile cysteine protease Cwp84 and insights into its maturation process.

Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp84(30-803) is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84(92-518) (47 kDa). In contrast, a fragment including the propeptide (Cwp84(30-518)) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.

Clostridium difficile colonization in early infancy is accompanied by changes in intestinal microbiota composition.

Clostridium difficile is a major enteric pathogen responsible for antibiotic-associated diarrhea. Host susceptibility to C. difficile infections results partly from inability of the intestinal microbiota to resist C. difficile colonization. During early infancy, asymptomatic colonization by C. difficile is common and the intestinal microbiota shows low complexity. Thus, we investigated the potential relationship between the microbiota composition and the implantation of C. difficile in infant gut. Fecal samples from 53 infants, ages 0 to 13 months, 27 negative and 26 positive for C. difficile, were studied. Dominant microbiota profiles were assessed by PCR-temporal temperature gradient gel electrophoresis (TTGE). Bacterial signatures of the intestinal microbiota associated with colonization by C. difficile were deciphered using principal component analysis (PCA). Resulting bands of interest in TTGE profiles were excised, sequenced, and analyzed by nucleotide BLAST (NCBI). While global biodiversity was not affected, interclass PCA on instrumental variables highlighted significant differences in dominant bacterial species between C. difficile-colonized and noncolonized infants (P = 0.017). Four bands were specifically associated with the presence or absence of C. difficile: 16S rRNA gene sequences related to Ruminococcus gnavus and Klebsiella pneumoniae for colonized infants and to Bifidobacterium longum for noncolonized infants. We demonstrated that the presence of C. difficile in the intestinal microbiota of infants was associated with changes in this ecosystem's composition. These results suggest that the composition of the gut microbiota might be crucial in the colonization process, although the chronology of events remains to be determined.

Heptavalent pneumococcal conjugate vaccine (PCV7): French survey of serious adverse reactions.

PURPOSE: Previous study did not reveal any particular heptavalent pneumococcal conjugate vaccine (PCV7) related risk. However, french drugs agency (Afssaps) requested the continuation of its surveillance. METHODS: All serious PCV7-related adverse drug reactions spontaneously reported between October 1, 2004 and December 31, 2007 to the French pharmacovigilance centers or to Wyeth Pharmaceutical France were included. Vaccine failure was defined as an invasive pneumococcal infection due to vaccine serotype which occurs at least 15 days after the third dose of vaccine. Incidence rates were estimated according to the doses number except for vaccine failure estimated according to the vaccinated children number. RESULTS: During the 39-month follow-up period, 154 serious adverse drug reactions were spontaneously reported: convulsions (17%), fever (13%), hypotonia (10%), sudden death (7%) and thrombopenic purpura (6%). Evolution was recovery in 72% of cases. PCV7 was the only suspect medication in 28% of cases. The median age was 4 months (range 1-108), and the children's sex was male in 53%. The adverse drug reaction recurred after a subsequent injection in six cases. Among the 24 pneumococcal infections PCV7 failure was certain in 4 cases. The incidences of serious adverse drug reactions did not differ from our previous survey, except the incidence of thrombopenic purpura and of PCV7 failure which seems to be increasing. CONCLUSIONS: This new study confirms the risk of vascular purpura, raises the thrombopenic purpura issue, and the emergence of PCV7 failures which will need a strict monitoring of the future 13 valences vaccine.

Acellular dense collagen-S53P4 bioactive glass hybrid gel scaffolds form more bone than stem cell delivered constructs.

Dense collagen (DC) gels facilitate the osteoblastic differentiation of seeded dental pulp stem cells (DPSCs) and undergo rapid acellular mineralization when incorporated with bioactive glass particles, both in vitro and subcutaneously in vivo. However, the potential of DC-bioactive glass hybrid gels in delivering DPSCs for bone regeneration in an osseous site has not been investigated. In this study, the efficacies of both acellular and DPSC-seeded DC-S53P4 bioactive glass [(53)SiO2-(23)Na2O-(20)CaO-(4)P2O5, wt%] hybrid gels were investigated in a critical-sized murine calvarial defect. The incorporation of S53P4, an osteostimulative bioactive glass, into DC gels led to its accelerated acellular mineralization in simulated body fluid (SBF), in vitro, where hydroxycarbonated apatite was detected within 1 day. By day 7 in SBF, micro-mechanical analysis demonstrated an 8-fold increase in the compressive modulus of the mineralized gels. The in-situ effect of the bioactive glass on human-DPSCs within DC-S53P4 was evident, by their osteogenic differentiation in the absence of osteogenic supplements. The production of alkaline phosphatase and collagen type I was further increased when cultured in osteogenic media. This osteostimulative effect of DC-S53P4 constructs was confirmed in vivo, where after 8 weeks implantation, both acellular scaffolds and DPSC-seeded DC-S53P4 constructs formed mineralized and vascularized bone matrices with osteoblastic and osteoclastic cell activity. Surprisingly, however, in vivo micro-CT analysis confirmed that the acellular scaffolds generated larger volumes of bone, already visible at week 3 and exhibiting superior trabecular architecture. The results of this study suggest that DC-S53P4 scaffolds negate the need for stem cell delivery for effective bone tissue regeneration and may expedite their path towards clinical applications.

Resistance of Acanthamoeba cysts to disinfection treatments used in health care settings.

Free-living amoebae that belong to the genus Acanthamoeba are widespread in the environment, including water. They are responsible for human infections and can host pathogenic microorganisms. Under unfavorable conditions, they form cysts with high levels of resistance to disinfection methods, thus potentially representing a threat to public health. In the present study we evaluated the efficacies of various biocides against trophozoites and cysts of several Acanthamoeba strains. We demonstrated that disinfectant efficacy varied depending on the strains tested, with environmental strains demonstrating greater resistance than collection strains. Trophozoites were inactivated by all treatments except those using glutaraldehyde as an active compound: for these treatments, we observed resistance even after 30 min exposure. Cysts resisted many treatments, including certain conditions with glutaraldehyde and other biocides. Moist heat at 55 degrees C was not efficient against cysts, whereas exposure at 65 degrees C was. Several chemical formulations containing peracetic acid, hydrogen peroxide, or ortho-phthalaldehyde presented greater efficacy than glutaraldehyde, as did ethanol and sodium hypochlorite; however, some of these treatments required relatively long incubation times to achieve cyst inactivation. Amoebal cysts can be highly resistant to some high-level disinfectants, which has implications for clinical practice. These results highlight the need to consider the effective disinfection of protozoa in their vegetative and resistant forms due to their intrinsic resistance. This is important not only to prevent the transmission of protozoa themselves but also due to the risks associated with a range of microbial pathogens that are found to be associated intracellularly with these microorganisms.