PLoS ONE

Lyme disease is a multisystemic disorder caused by B. burgdorferi sl. The molecular basis for specific organ involvement is poorly understood. The skin plays a central role in the development of Lyme disease as the entry site of B. burgdorferi in which specific clones are selected before dissemination. We compared the skin inflammatory response (antimicrobial peptides, cytokines and chemokines) elicited by spirochete populations recovered from patients presenting different clinical manifestations. Remarkably, these spirochete populations induced different inflammatory profiles in the skin of C3H/HeN mice. As spirochete population transmitted into the host skin is heterogeneous, we isolated one bacterial clone from a population recovered from a patient with neuroborreliosis and compared its virulence to the parental population. This clone elicited a strong cutaneous inflammatory response characterized by MCP-1, IL-6 and antimicrobial peptides induction. Mass spectrometry of this clone revealed 110 overexpressed proteins when compared with the parental population. We further focused on the expression of nine bacterial surface proteins. bb0347 coding for a protein that interacts with host fibronectin, allowing bacterial adhesion to vascular endothelium and extracellular matrix, was found to be induced in host skin with another gene bb0213 coding for a hypothetical protein. These findings demonstrate the heterogeneity of the B. burgdorferi ss population and the complexity of the interaction involved early in the skin

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

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Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

International audienceGeophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Inflammatory profiles of <i>B</i>. <i>burgdorferi</i> ss, parental strain and its clone 297/4.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least, three mice were analyzed for each time point.</p

Proteomic analyses of <i>B</i>. <i>burgdorferi</i> ss, 297 parental strain and its clone 297/4.

<p>(A) The Venn diagram shows the protein overlap and the proteins specifically identified in the parental strain and the clone 297/4. (B) Graphic representation of the breakdown of the proteins specifically identified in the clone. Categorization was based upon JCVI annotation. The percentages represent the fraction of that category within the proteins. (C) Gene location of the specific proteins identified in the clone 297/4.</p

Bacterial loads in the skin.

<p>The spirochetal burden in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>. Values represent relative expression + SD of three independent experiments. Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). (d: days).</p

Innovative Native MS Methodologies for Antibody Drug Conjugate Characterization: High Resolution Native MS and IM-MS for Average DAR and DAR Distribution Assessment

Antibody drug conjugates (ADCs) are macromolecules composed of cytotoxic drugs covalently attached via a conditionally stable linker to monoclonal antibodies (mAbs). ADCs are among the most promising next generation of empowered mAbs foreseen to treat cancers. Compared to naked mAbs, ADCs have an increased level of complexity as the heterogeneity of conjugation cumulates with the inherent microvariability of the biomolecule. An increasing need underlying ADC’s development and optimization is to improve its analytical and bioanalytical characterization by assessing three main ADC quality attributes: drug distribution, amount of naked antibody, and average drug to antibody ratio (DAR). Here, the analytical potential of native mass spectrometry (MS) and native ion mobility MS (IM-MS) is compared to hydrophobic interaction chromatography (HIC), the reference method for quality control of interchain cysteinyl-linked ADCs. Brentuximab vedotin, first in class and gold standard, was chosen for a proof of principle. High resolution native MS provided accurate mass measurement (<30 ppm) of intact ADCs together with average DAR and drug distribution, confirming the unique ability of native MS for simultaneous detection of mixtures of covalent and noncovalent products. Native IM-MS was next used for the first time to characterize an ADC. IM-MS evidenced ADC multiple drug loading, collisional cross sections measurement of each payload species attesting slight conformational changes. A semiquantitative interpretation of IM-MS data was developed to directly extrapolate average DAR and DAR distribution. Additionally, HIC fractions were collected and analyzed by native MS and IM-MS, assessing the interpretation of each HIC peak. Altogether, our results illustrate how native MS and IM-MS can rapidly assess ADC structural heterogeneity and how easily these methods can be implemented into MS workflows for in-depth ADC analytical characterization

Bacterial loads in the skin at the inoculation site was measured by quantitative PCR for the <i>B</i>. <i>burgdorferi flaB</i> gene and normalized to copies of mouse <i>gapdh</i>.

<p>Values between strains were not statistically different (two-way ANOVA test using the Sidak-Bonferroni method). # values close to the detection limit.</p

Inflammatory profiles of the different strains of <i>B</i>. <i>burgdorferi</i> ss.

<p>Levels of transcripts were measured by RT-qPCR from the skin at the inoculation site. The values were calculated using the 2-delta delta Ct method after normalization with <i>gapdh</i>. (h: hours, d: days). Two-way ANOVA was used to analyze the data. At least three mice were analyzed for each time point.</p