Buttiauxella massiliensis sp. nov., Isolated from a Human Bone Infection.

Strain Marseille-P9829 was isolated from a bone sample collected from an open right fibula fracture from a 46-years old patient. Strain Marseille-P9829 (= CSUR P9829 = DSM 110695) was a Gram-negative, non-spore-forming and non-motile bacterium. This strain had a positive catalase activity but was oxidase-negative. The major fatty acids methyl esters were hexadecanoic acid (45.6%) and 9-hexadecenoic acid (28.4%). Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry analysis suggested that this strain belongs to the species Buttiauxella gaviniae. Since there were few reports of clinical infections with this species in humans, whole genome sequencing was performed and a polyphasic taxono-genomic approach was followed in order to verify the classification of strain Marseille-P9829. The 16S rRNA gene sequence BLAST against the NCBI database yielded the highest similarity of 99.8% with Buttiauxella agrestis, suggesting that strain Marseille-P9829 belongs to this species. However, genomic comparison by digital DNA-DNA hybridization showed that values between strain Marseille-P9829 and other validly published Buttiauxella species were all lower than 70%. Furthermore, all average nucleotide identities were lower than 95-96%. Therefore, these results confirmed that strain Marseille-P9829 belonged to a new Buttiauxella species for which we propose the name Buttiauxella massiliensis sp. nov., with strain Marseille-P9829 as type strain.

Konateibacter massiliensis gen. nov. sp. nov. and Paenibacillus faecalis sp. nov., Two New Species Isolated from the Stool Samples of Infants Suffering from Marasmus.

Two bacterial strains were isolated and identified using microbial culturomics and characterised according to the taxono-genomics strategy. The strictly anaerobic strain, Marseille-P3773T, forms smooth and translucent colonies consisting of Gram-stain negative, non-motile and non-spore-forming rod-shaped cells. Strain Marseille-P3787T consists of Gram-stain positive, motile and spore-forming cells resulting in grey and translucent colonies. The phylogenetic analysis of the 16S rRNA gene of strains Marseille-P3773T and Marseille-P3787T revealed a 96.9% similarity level with Lachnotalea glycerini strain DLD10 and 97% identity with Paenibacillus uliginis strain N3/975, respectively. The genome of strain Marseille-P3773 is 4,260,534 bp long with a 40.3 mol% G + C content and includes 3879 predicted genes of which 3769 are protein-coding genes, 76 RNAs and 34 are pseudo-genes. Strain Marseille-P3787 had a genome size of 4,833,032 bp with a 47.9 mol% G + C and has 4481 predicted genes of which 4265 are protein-coding genes, 101 RNAs and 115 are pseudo-genes. According to the data collected on these strains and, more specifically to the genomic comparison, we suggest the creation of a new genus and species, Konateibacter massiliensis gen. nov., sp. nov. with strain Marseille-P3773T (=CSURP3773 and CCUG71331) as its type strain within the Lachnospiraceae family, as well as a new species, Paenibacillus faecalis sp. nov. with strain Marseille-P3787T (=CSURP3787 and CCUG71650) as its type strain within the Paenibacillus genus.

Pediatric cannabis poisonings in France: more and more frequent and severe.

CONTEXT: Accidental pediatric cannabis poisonings are an incidental effect of cannabis use. The average THC content of cannabis resin and the number of consumers are rising sharply in the USA and in most European countries. The objective is to study the evolution of prevalence and severity of pediatric exposures to cannabis in France. METHOD: This is a retrospective observational study of cases detected by French poison centers between January 1st 2010 and December 31st 2017 of cannabis exposure by ingestion in children aged ten or younger. The clinical severity was assessed using the Poisoning Severity Score (PSS). The criteria used for assessing the overall severity were as follows: PSS ≥ 2, admission to pediatric intensive care, coma and respiratory depression (univariate and multivariate logistic regression). RESULTS: A total of 965 cases of poisoning were covered. The annual average number of cases was 93 between 2010 and 2014 and 167 between 2015 and 2017. The median age was 15 months (range, 6 months-10 years) and the sex ratio was 1:1. The form of cannabis ingested was mainly resin (75%). During the period covered by the study, 26.1% of children (n = 252) presented with a PSS ≥ 2, 4.5% (n = 43) coma, 4.6% (n = 44) with respiratory depression and 11.7% (n = 113) were admitted into pediatric intensive care (out of 819 hospitalizations). No fatal cases were reported. In comparison to the 2010-2014 period, the length of hospital stays was significantly higher (p < 0.0001) and the comas were significantly deeper (lower score on the Glasgow coma scale, p < 0.005) in 2015-2017. Following adjustments made for the sex, age and weight of the children, the data show that the severity of the poisonings was significantly greater in 2015-2017 in terms of PSS score, the number of comas and monitoring in intensive care (p < 0.001). CONCLUSION: The data indicates a significant increase in the number of cases of pediatric exposure to cannabis and a rise in the seriousness of poisonings between 2010 and 2017.

Defective Granuloma Formation in Elderly Infected Patients.

Granulomas are compact structures formed in tissues by the immune system in response to aggressions. The in vitro formation of granulomas using circulating mononuclear cells is an innovative method to easily assess the immune response of patients. Monitoring the efficiency of mononuclear cells from patients to form granulomas in vitro would help improve their therapeutic management. Circulating mononuclear cells from 23 elderly patients with sepsis and 24 elderly controls patients were incubated with Sepharose beads coated with either BCG or Coxiella burnetii extracts. The formation of granulomas was measured over 9 days. Most healthy elderly patients (92%) were able to form granulomas in response to BCG and Coxiella burnetii extracts compared to only 48% of infected elderly patients. Undernutrition was significantly associated with impaired granuloma formation in healthy and infected patients. Granulomas typically comprise epithelioid cells and multinucleated giant cells, however, these cells were not detected in samples obtained from patients unable to form granulomas. We also found that the impairment of granuloma formation was associated with reduced production of tumor necrosis factor without overproduction of interleukin-10. Finally, all genes specifically modulated in granulomatous cells were down-modulated in patients with defective granuloma formation. TNFSF10 was the only M1 gene markedly upregulated in patients who did not form granulomas. Our study suggest that defective granuloma formation may be a measurement of altered activation of immune cells which can predispose to nosocomial infections in elderly patients.

Full-Term Human Placental Macrophages Eliminate Coxiella burnetii Through an IFN-γ Autocrine Loop.

The intracellular bacterium Coxiella burnetii is responsible for Q fever, an infectious disease that increases the risk of abortion, preterm labor, and stillbirth in pregnant women. It has been shown that C. burnetii replicates in BeWo trophoblast cell line and inhibits the activation and maturation of decidual dendritic cells. Although tissue macrophages are known to be targeted by C. burnetii, no studies have investigated the interplay between placental macrophages and C. burnetii. Here, CD14+ macrophages from 46 full-term placentas were isolated by positive selection. They consisted of a mixed population of maternal and fetal origin as shown by genotype analysis. We showed that C. burnetii organisms infected placental macrophages after 4 h. When these infected macrophages were incubated for an additional 9-day culture, they completely eliminated organisms as shown by quantitative PCR. The ability of placental macrophages to form multinucleated giant cells was not affected by C. burnetii infection. The transcriptional immune response of placental macrophages to C. burnetii was investigated using quantitative real-time RT-PCR on 8 inflammatory and 10 immunoregulatory genes. C. burnetii clearly induced an inflammatory profile. Interestingly, the production by placental macrophages of interferon-γ, a cytokine known to be involved in efficient immune responses, was dramatically increased in response to C. burnetii. In addition, a clear correlation between interferon-γ production and C. burnetii elimination was found, suggesting that macrophages from full-term placentas eliminate C. burnetii under the control of an autocrine production of interferon-γ.

High Concentrations of Serum Soluble E-Cadherin in Patients With Q Fever.

Cadherins switching is a hallmark of neoplasic processes. The E-cadherin (E-cad) subtype is one of the surface molecules regulating cell-to-cell adhesion. After its cleavage by sheddases, a soluble fragment (sE-cad) is released that has been identified as a pro-carcinogenic inflammatory signal in several bacteria-induced cancers. Recently we reported that Q fever, a disease due to Coxiella burnetii infection, can be complicated by occurrence of non-Hodgkin lymphoma (NHL). Therefore, we studied E-cad switching in Q fever. The sE-cad levels were found increased in the sera of acute and persistent Q fever patients, whereas they remained at the baseline in controls groups of healthy donors, people cured of Q fever, patients suffering from unrelated inflammatory diseases, and past Q fever patients who developed NHL. These results indicate that sE-cad can be considered as a new biomarker of C. burnetii infection rather than a marker of NHL-associated to Q fever. We wondered if changes in sE-cad reflected variations in the CDH1 gene transcription. The expression of E-cad mRNA and its intracellular ligand ß-catenin was down-regulated in peripheral blood mononuclear cells (PBMCs) of patients with either acute or persistent forms of Q fever. Indeed, a lower cell-surface expression of E-cad was measured in a minority (<5%) subpopulation of HLADR+/CD16+ monocytes from patients with acute Q fever. However, a very strong increase in E-cad expression was observed on more than 30% of the HLADR+/CD16+ monocytes of persistent Q fever patients, a cell subpopulation known to be a target for C. burnetii in humans. An experimental in vitro infection of healthy donors' PBMCs with C. burnetii, was performed to directly evaluate the link between C. burnetii interaction with PBMCs and their E-cad expression. A significant increase in the percentage of HLADR+/CD16+ monocytes expressing E-cad was measured after PBMCs had been incubated for 8 h with C. burnetii Nine Mile strain. Altogether, these data demonstrate that C. burnetii severely impairs the E-cad expression in circulating cells of Q fever patients.

Progenitor mast cells and tryptase in Q fever.

Q fever is an infectious disease due to Coxiella burnetii. Following a primary-infection, C. burnetii may persist in some patients, leading to endocarditis and vascular infections. Mast cells (MCs), known for their role in allergic diseases, innate immunity and cardiac function, are produced by bone marrow, circulate as progenitors in the bloodstream and reach tissues for their maturation and activation. The latter may be estimated by measuring serum tryptase levels. We wondered if MC progenitors and tryptase were affected in Q fever. We showed a decrease in MC progenitor count in Q fever patients whereas serum tryptase levels were increased. Taken together, our results show alterations of MC numbers and activity in Q fever patients, suggesting that MC are involved in Q fever pathophysiology.

A Fast and Reliable Method to Isolate Human Placental Macrophages.

Macrophages are specialized cells involved in recognition, uptake, and destruction of microorganisms. Human placental macrophages are poorly investigated because of the lack of a convenient protocol for their isolation. Here, we present a straightforward and reliable method to isolate macrophages from full-term human placentas. After enzymatic digestion of placental tissue and centrifugation of the cell suspension on a Ficoll cushion, placental macrophages are selected using magnetic beads coated with anti-CD14 antibodies. Isolated cells are characterized by flow cytometry. Ninety eight percent of isolated CD14+ placental macrophages also express the macrophage marker CD68. Thus, this efficient and reliable method yields placental macrophages at high purity and sufficient quantity for functional studies. © 2019 by John Wiley & Sons, Inc.

Evaluation of Cellular Responses for the Diagnosis of Allergic Bronchopulmonary Mycosis: A Preliminary Study in Cystic Fibrosis Patients.

Background: Allergic bronchopulmonary mycosis (ABPM) is an underestimated allergic disease due to fungi. Most reported cases are caused by Aspergillus fumigatus (Af) and are referred to as allergic bronchopulmonary aspergillosis (ABPA). The main risk factor of ABPA is a history of lung disease, such as cystic fibrosis, asthma, or chronic obstructive pulmonary disease. The main diagnostic criteria for ABPA rely on the evaluation of humoral IgE and IgG responses to Af extracts, although these cannot discriminate Af sensitization and ABPA. Moreover, fungi other than Af have been incriminated. Flow cytometric evaluation of functional responses of basophils and lymphocytes in the context of allergic diseases is gaining momentum. Objectives: We hypothesized that the detection of functional responses through basophil and lymphocyte activation tests might be useful for ABPM diagnosis. We present here the results of a pilot study comparing the performance of these cellular assays vs. usual diagnostic criteria in a cystic fibrosis (CF) cohort. Methods:Ex vivo basophil activation test (BAT) is a diagnostic tool highlighting an immediate hypersensitivity mechanism against an allergen, e.g., through CD63 upregulation as an indirect measure of degranulation. Lymphocyte stimulation test (LST) relies on the upregulation of activation markers, such as CD69, after incubation with allergen(s), to explain delayed hypersensitivity. These assays were performed with Af, Penicillium, and Alternaria extracts in 29 adult CF patients. Results: BAT responses of ABPA patients were higher than those of sensitized or control CF patients. The highest LST result was for a woman who developed ABPA 3 months after the tests, despite the absence of specific IgG and IgE to Af at the time of the initial investigation. Conclusion: We conclude that basophil and lymphocyte activation tests could enhance the diagnosis of allergic mycosis, compared to usual humoral markers. Further studies with larger cohorts and addressing both mold extracts and mold relevant molecules are needed in order to confirm and extend the application of this personalized medicine approach.

Cigarette smoke extract interferes with placenta macrophage functions: A new mechanism to compromise placenta functions?

The success of pregnancy depends on the maternal immune system's ability to promote tolerance and host defense. This equilibrium is compromised in inflammatory and infectious impairment of placenta. Smoking during pregnancy exposes the fetus to severe complications which might result from an alteration in placenta macrophages (pMφ) functions. In this study, we assessed the effect of cigarette smoke extract (CSE) on the functions of third trimester pMφs.CSE inhibited particles uptake and the formation of multinucleated giant cells, a recently reported property of pMφs based on their ability to fuse in vitro. These alterations were associated with a CSE-induced abnormal activation of pMφs, which was characterized by an increased release of TNF, interleukin (IL)-33, and decreased IL-6 and IL-10 release. Furthermore, CSE enhanced the expression of metalloproteinase genes known to be involved in tissue remodeling. This effect of CSE on pMφs was specific because CSE affected circulating monocytes in a different way. Finally, we showed that nicotine affected in part the functional properties of pMφs. Taken together, these results showed that CSE modulated the functional activity of pMφs, which may compromise pregnancy.

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in ß-thalassemia.

The pathophysiology of ineffective erythropoiesis in ß-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of ß-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with ß-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in ß-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in ß-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Evaluation of two automated capillary electrophoresis systems for human serum protein analysis.

OBJECTIVES: We compared automated capillary electrophoresis (CE) systems Capillarys 2 from Sebia and V8 from Helena Biosciences Europe (Elitech) with the semi-automated Hydrasys-Hyrys agarose gel electrophoresis (AGE) from Sebia. DESIGN AND METHODS: We evaluated analytical performances and compared 129 fresh routine sera (group A) to 164 frozen pathologic samples with suspicion or antecedent of monoclonal component (MC) (group B). Immunofixation was then compared with immunotyping provided by both CE systems. RESULTS: Analytical performances from both CE systems have proven suitable results for clinical use, with within-run and between-run coefficients of variation inferior or equal to 5.2% and 7.7%, respectively. A good correlation was found between AGE and CE with r-value ranging from 0.81 to 0.96 for both CE systems. We observed high MC detection sensitivities (>85%) of in electrophoretogram readings for both CE systems. MC identification using CE systems provided suitable concordance with immunofixation, although failing to detect some IgM proteins or free light chains. CONCLUSIONS: Both Capillarys 2 and V8 are reliable automated CE systems for patient care.