Dissection of the anti-Candida albicans mannan immune response using synthetic oligomannosides reveals unique properties of ß-1,2 mannotriose protective epitopes.

Candida albicans mannan consists of a large repertoire of oligomannosides with different types of mannose linkages and chain lengths, which act as individual epitopes with more or less overlapping antibody specificities. Although anti-C. albicans mannan antibody levels are monitored for diagnostic purposes nothing is known about the qualitative distribution of these antibodies in terms of epitope specificity. We addressed this question using a bank of previously synthesized biotin sulfone tagged oligomannosides (BSTOs) of α and ß anomery complemented with a synthetic ß-mannotriose described as a protective epitope. The reactivity of these BSTOs was analyzed with IgM isotype monoclonal antibodies (MAbs) of known specificity, polyclonal sera from patients colonized or infected with C. albicans, and mannose binding lectin (MBL). Surface plasmon resonance (SPR) and multiple analyte profiling (MAP) were used. Both methods confirmed the usual reactivity of MAbs against either α or ß linkages, excepted for MAb B6.1 (protective epitope) reacting with ß-Man whereas the corresponding BSTO reacted with anti-α-Man. These results were confirmed in western blots with native C. albicans antigens. Using patients' sera in MAP, a significant correlation was observed between the detection of anti-mannan antibodies recognizing ß- and α-Man epitopes and detection of antibodies against ß-linked mannotriose suggesting that this epitope also reacts with human polyclonal antibodies of both specificities. By contrast, the reactivity of human sera with other α- and ß-linked BSTOs clearly differed according to their colonized or infected status. In these cases, the establishment of an α/ß ratio was extremely discriminant. Finally SPR with MBL, an important lectin of innate immunity to C. albicans, classically known to interact with α-mannose, also interacted in an unexpected way with the protective epitope. These cumulative data suggest that structure/activity investigations of the finely tuned C. albicans anti-mannose immune response are worthwhile to increase our basic knowledge and for translation in medicine.

Retrospective study of COVID-19 seroprevalence among tissue donors at the onset of the outbreak before implementation of strict lockdown measures in France.

Background The COVID-19 pandemic has altered organ and tissue donations as well as transplantation practices. SARS-CoV-2 serological tests could help in the selection of donors. We assessed COVID-19 seroprevalence in a population of tissue donors, at the onset of the outbreak in France, before systematic screening of donors for SARS-CoV-2 RNA. Methods 235 tissue donors at the Lille Tissue Bank between November 1, 2019 and March 16, 2020 were included. Archived serum samples were tested for SARS-CoV-2 antibodies using two FDA-approved kits. Results Most donors were at higher risks for severe COVID-19 illness including age over 65 years (142/235) and/or presence of co-morbidities (141/235). According to the COVID-19 risk assessment of transmission, 183 out of 235 tissue donors presented with a low risk level and 52 donors with an intermediate risk level of donor derived infection. Four out of the 235 (1.7%) tested specimens were positive for anti-SARS-CoV-2 antibodies: 2 donors with anti-N protein IgG and 2 other donors with anti-S protein total Ig. None of them had both type of antibodies. Conclusion Regarding the seroprevalence among tissue donors, we concluded that the transmission probability to recipient via tissue products was very low at the beginning of the outbreak.

How to improve donor skin availability: Pragmatic procedures to minimize the discard rate of cryopreserved allografts in skin banking.

BACKGROUND: Microbial contamination of human skin allografts is a frequent cause of allograft discard. Our purpose was to evaluate the discard rate of skin bank contaminated allografts and specific procedures used to reduce allograft contamination without affecting safety. METHODS: We conducted at the Lille Tissue Bank a retrospective study of all deceased donors (n = 104) harvested from January 2018 to December 2018. Skin procurement was split into 3 zones: the back of the body and the two legs that were processed separately. It represented 433 cryopreserved skin allograft pouches of approximatively 500 cm² each. Donors were almost equally split between brain-dead (53%, 55/104) and cadaveric (47%, 49/104) donors. RESULTS: Out of all donors, 42 (40.5%) had at least one sampling zone with a positive microbiological test resulting in 106 (24%) contaminated skin pouches. The contamination rate did not vary according to the harvested zone or type of donor. Traumatic deaths showed significantly less contamination rates than other death types (p < 0.05). Contamination rate decreased with time spent in the antibiotic solution. The risk of having contaminated allografts was five-fold higher when the skin spent less than 96 h in the antibiotic cocktail (p < 0.05). According to our validation protocol, most donors (32/42, 76%) had skin allografts contaminated with bacteria (mainly Staphylococcus spp) compatible with clinical use. No recipient infection was recorded as a result of skin graft contaminated with saprophytic or non-pathogenic germs. By harvesting 3 separate zones per donor, the total surface area for clinical use increased by 53% for contaminated donors. Overall, the proportion of contamination-related discarded allografts was 3.2% (14/433 of pouches). CONCLUSION: Few simple pragmatic measures (including skin incubation in the antibiotic bath for at least 96 h at 4 °C, splitting the skin harvesting areas to minimize the risk of cross-infection and clinical use of allografts contaminated with saprophytic and non-pathogenic germs) can reduce the discard rate of contaminated allografts without affecting clinical safety.

Rationale for the design of 3D-printable bioresorbable tissue-engineering chambers to promote the growth of adipose tissue.

Tissue engineering chambers (TECs) bring great hope in regenerative medicine as they allow the growth of adipose tissue for soft tissue reconstruction. To date, a wide range of TEC prototypes are available with different conceptions and volumes. Here, we addressed the influence of TEC design on fat flap growth in vivo as well as the possibility of using bioresorbable polymers for optimum TEC conception. In rats, adipose tissue growth is quicker under perforated TEC printed in polylactic acid than non-perforated ones (growth difference 3 to 5 times greater within 90 days). Histological analysis reveals the presence of viable adipocytes under a moderate (less than 15% of the flap volume) fibrous capsule infiltrated with CD68+ inflammatory cells. CD31-positive vascular cells are more abundant at the peripheral zone than in the central part of the fat flap. Cells in the TEC exhibit a specific metabolic profile of functional adipocytes identified by 1H-NMR. Regardless of the percentage of TEC porosity, the presence of a flat base allowed the growth of a larger fat volume (p < 0.05) as evidenced by MRI images. In pigs, bioresorbable TEC in poly[1,4-dioxane-2,5-dione] (polyglycolic acid) PURASORB PGS allows fat flap growth up to 75 000 mm3 at day 90, (corresponding to more than a 140% volume increase) while at the same time the TEC is largely resorbed. No systemic inflammatory response was observed. Histologically, the expansion of adipose tissue resulted mainly from an increase in the number of adipocytes rather than cell hypertrophy. Adipose tissue is surrounded by perfused blood vessels and encased in a thin fibrous connective tissue containing patches of CD163+ inflammatory cells. Our large preclinical evaluation defined the appropriate design for 3D-printable bioresorbable TECs and thus opens perspectives for further clinical applications.

Benefits of cryopreserved human amniotic membranes in association with conventional treatments in the management of full-thickness burns.

The use of split-thickness skin autografts (STSA) with dermal substitutes is the gold standard treatment for third-degree burn patients. In this article, we tested whether cryopreserved amniotic membranes could be beneficial to the current treatments for full-thickness burns. Swines were subjected to standardised full-thickness burn injuries, and then were randomly assigned to treatments: (a) STSA alone; (b) STSA associated with the dermal substitute, Matriderm; (c) STSA plus human amniotic membrane (HAM); and (d) STSA associated with Matriderm plus HAM. Clinical and histological assessments were performed over time. We also reported the clinical use of HAM in one patient. The addition of HAM to classic treatments reduced scar contraction. In the presence of HAM, skin wound healing displayed high elasticity and histological examination showed a dense network of long elastic fibres. The presence of HAM increased dermal neovascularization, but no effect was observed on the recruitment of inflammatory cells to the wound. Moreover, the use of HAM with classical treatments in one human patient revealed a clear benefit in terms of elasticity. These results give initial evidence to consider the clinical application of HAM to avoid post-burn contractures and therefore facilitate functional recovery after deep burn injury.

Chiral separation of new sulfonamide derivatives and evaluation of their enantioselective affinity for human carbonic anhydrase II by microscale thermophoresis and surface plasmon resonance.

The aim of this study was to develop a method combining chiral separation and biophysical techniques to evaluate the enantioselective affinity of original sulfonamide derivatives towards their therapeutic target, the human carbonic anhydrase II (hACII). The first step consisted in the preparation of the enantiomers by chromatographic separation. The performances of HPLC and Supercritical Fluid Chromatography (SFC) were studied at the analytical scale by optimization of various experimental conditions using adsorbed polysaccharide chiral stationary phases (amylose AD-H and cellulose OD-H). Since SFC allowed obtaining higher enantioresolutions per time unit, it was selected for the semi-preparative scale and successfully used to isolate each enantiomer with a satisfactory enantiomeric purity (>98%). Secondly, microscale thermophoresis (MST) method and surface plasmon resonance (SPR) used as reference method were developed to measure potential enantioselective affinities of these enantiomers towards the hACII. The optimizations of both methods were performed using a reference compound, i.e. acetazolamide, which affinity for hCAII has previously been demonstrated. For all compounds, KD values obtained using MST and SPR were in good agreement, leading to similar affinity scales despite both approaches totally differ (labeling for MST versus immobilization of the protein for SPR). The equilibrium dissociation constants of our original compounds for the hCAII were in the range 100-1000nM and an enantioselectivity was observed using the MST and SPR methods for the diarylpyrazole 2. Finally, by comparing the MST and SPR techniques, MST appears especially adapted for further screening of a series of sulfonamide derivatives due to the lower time required to estimate a binding constant while consuming as little hCAII as SPR.

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

Unconventional surface plasmon resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands.

EthR is a mycobacterial repressor that limits the bioactivation of ethionamide, a commonly used anti-tuberculosis second-line drug. Several efforts have been deployed to identify EthR inhibitors abolishing the DNA-binding activity of the repressor. This led to the demonstration that stimulating the bioactivation of Eth through EthR inhibition could be an alternative way to fight Mycobacterium tuberculosis. We propose a new surface plasmon resonance (SPR) methodology to study the affinity between inhibitors and EthR. Interestingly, the binding between inhibitors and immobilized EthR produced a dose-dependent negative SPR signal. We demonstrate that this signal reveals the affinity of small molecules for the repressor. The affinity constants (K(D)) correlate with their capacity to inhibit the binding of EthR to DNA. We hypothesize that conformational changes in EthR during ligand interaction could be responsible for this SPR signal. Practically, this unconventional result opens perspectives onto the development of an SPR assay that would at the same time reveal structural changes in the target upon binding with an inhibitor and the binding constant of this interaction.

Label-free characterization of carbonic anhydrase-novel inhibitor interactions using surface plasmon resonance, isothermal titration calorimetry and fluorescence-based thermal shift assays.

This work describes the development of biophysical unbiased methods to study the interactions between new designed compounds and carbonic anhydrase II (CAII) enzyme. These methods have to permit both a screening of a series of sulfonamide derivatives and the identification of a lead compound after a thorough study of the most promising molecules. Interactions data were collected using surface plasmon resonance (SPR) and thermal shift assay (TSA). In the first step, experiments were performed with bovine CAII isoform and were extended to human CAII. Isothermal titration calorimetry (ITC) experiments were also conducted to obtain thermodynamics parameters necessary for the processing of the TSA data. Results obtained with this reference methodology demonstrate the effectiveness of SPR and TSA. KD values obtained from SPR data were in perfect accordance with ITC. For TSA, despite the fact that the absolute values of KD were quite different, the same affinity scale was obtained for all compounds. The binding affinities of the analytes studied vary by more than 50 orders of magnitude; for example, the KD value determined by SPR were 6 ± 4 and 299 ± 25 nM for compounds 1 and 3, respectively. This paper discusses some of the theoretical and experimental aspects of the affinity-based methods and evaluates the protein consumption to develop methods for the screening of further new compounds. The double interest of SPR, that is, for screening and for the quick thorough study of the interactions parameters (ka , kd , and KD ), leads us to choose this methodology for the study of new potential inhibitors.

Characterization of the recognition of Candida species by mannose-binding lectin using surface plasmon resonance.

The interaction of mannose-binding lectins (MBLs) with Candida albicans has been analyzed previously by microscopy and flow cytometry. We recently demonstrated that serum MBL levels vary during infection with Candida spp. and that serum MBLs are capable of interacting with yeast cell wall components. The aim of this study was to use, for the first time, surface plasmon resonance (SPR) technology to characterize the interaction between living label-free yeasts and non-mutated MBL purified from human serum. Our preliminary results demonstrate the robustness of this tool, which revealed specific and differential reactivities between the principal Candida spp. of medical interest. This model offers new perspectives as a tool for the characterization of yeast strains carrying mutations in gene coding for the mannosylation of fungal cell wall glycans and will enable better characterization of the interactions between C-lectins and glycan motifs expressed on the surface of yeasts.

Comparison of surface plasmon resonance transferrin quantification with a common immunoturbidimetric method.

OBJECTIVES: The variation in the results from different clinical laboratories testing the same patient sample using classical methods for serum transferrin determination can affect interpretation. We developed a surface plasmon resonance (SPR) method for serum transferrin assay that may aid standardization. DESIGN AND METHODS: Quantification of transferrin was performed by direct analysis on a Biacore system (GE Healthcare). We assessed analytical performance of this new assay and compared it to a common immunoturbidimetric method (Beckman Coulter). RESULTS: Intra-run coefficients of variation (CV) were up to 1.10% and inter-day CVs were up to 2.10%. The comparison of this new SPR assay (Biacore) with a classical immunoturbidimetric method (Beckman Coulter) showed a good correlation (r=0.959), and the Bland and Altman plot with no global significant difference. CONCLUSIONS: According excellent achievement of SPR transferrin determination, we suggest its use as an independent method for comparison different immunoassays. This could be an important tool to help clinical laboratories to deliver standardized values, essential for the decision to investigate genetic hereditary hemochromatosis.

Resistance of the cell wall to degradation is a critical parameter for isolation of high quality RNA from natural isolates of Bacillus subtilis.

Natural isolates of Bacillus subtilis are known for their ability to produce a large panel of bioactive compounds. Unfortunately, their recalcitrance to conventional molecular techniques limits their transcript studies. In this work, difficulties to isolate RNA attributed to the cell wall were overcome, finally authorising powerful RT-PCR's.

Biotin sulfone as a new tool for synthetic oligosaccharide immobilization: application to multiple analysis profiling and surface plasmonic analysis of anti-Candida albicans antibody reactivity against alpha and beta (1-->2) oligomannosides.

As a part of our glycoantigen synthetic program for diagnosis and basic analysis of yeast-related pathogenic mechanisms, a library of 1-->2 oligomannosides suitable for immunoanalysis was prepared. The use of biotin sulfone, an oxidized form of biotin, offers a convenient solution for both oligosaccharide synthesis and immobilization on microspheres and surface plasmon resonance sensors. The application of this new strategy for the analysis of anti- Candida albicans antibody response through multiple-analyte profiling technology (Luminex) and with surface plasmonic analysis using biotin tagged synthetic oligosaccharides on avidin coated surfaces was validated using monoclonal antibodies.

Phenotype and HFE genotype in a population with abnormal iron markers recruited from an Endocrinology Department.

OBJECTIVE: The aim of this study was to describe HFE genotype in a population of patients with altered iron markers recruited in an Endocrinology Department and to define the possible phenotype-genotype relationships. METHODS: A total of 156 patients with high serum ferritin concentrations (>300 ng/ml) or transferrin saturation (>45%) (I group), and a control group of 106 healthy subjects (C group) underwent HFE genotyping (classical C282Y and H63D mutations). We also examined the main genetic features of subgroups in I according to the presence (D) or the absence (ND) of diabetes. RESULTS: (1) The genotypes were significantly different in the I and C groups (P<0.001), with an increased frequency of major 282Y allele in the I group (35% vs 7.5%), but not of minor 63D allele (17 vs 18.5%). (2) The genotype of D and ND groups also differed (P<0.0001), with a lower frequency of C282 heterozygosity (P<0.0001) in the D group, and a higher prevalence of H63D heterozygosity in the D vs ND groups (P<0.01). (3) The phenotypic comparison of D and ND groups also showed a higher mean body mass index, age, and serum ferritin concentration, as well as an increased proportion of males with increased liver enzymes in the D group. CONCLUSION: This population harboring abnormal iron markers had a different HFE genotype and a higher 282Y allele frequency than the control population. This suggests that blood iron markers could be checked in etiological investigations of metabolic disturbances to identify patients who should undergo genotyping, since approximately 20% were diagnosed with C282Y homozygosity.

Expression of progenitor cell markers during expansion of sorted human pancreatic beta cells.

Functional pancreatic beta cell mass is dynamic and although fully differentiated, beta cells are capable of reentering the cell cycle upon appropriate stimuli. Stimulating regeneration-competent cells in situ is clearly the most desirable way to restore damaged tissue. Regeneration by dedifferentiation and transdifferentiation is a potential source of cells exhibiting a more developmentally immature phenotype and a wide differentiation potential. In this context and to gain a better understanding of the transformation induced in human beta cells during forced in vitro expansion, we focused on identifying differences in gene expression along with phenotypical transformation between proliferating and quiescent human beta cells. FACS-purified beta cells from three different human pancreata were cultured during 3-4 months (8-10 subcultures) on HTB-9 cell matrix with hepatocyte growth factor. Gene expression profiling was performed on cells from each subculture on "in-house" pancreas-specific microarrays consisting of 218 genes and concomitant morphological transformations were studied by immunocytochemistry. Immunocytochemical studies indicated a shift from epithelial to neuroepithelial cell phenotype, including progenitor cell features such as protein gene product 9.5 (PGP 9.5), Reg, vimentin, and neurogenin 3 protein expression. The expression of 49 genes was downregulated, including several markers of endocrine differentiation while 76 were induced by cell expansion including several markers of progenitor cells. Their pattern also argues for the transdifferentiation of beta cells into progenitor cells, demonstrating neuroepithelial features and overexpressing both PBX1, a homeodomain protein that can bind as a heterodimer with PDX1 and could switch the nature of its transcriptional activity, and neurogenin 3, a key factor for the generation of endocrine islet cells. Our study of the machinery that regulates human beta cell expansion and dedifferentiation may help elucidate some of the critical genes that control the formation of adult pancreatic progenitor cells and hence design targets to modify their expression in view of the production of insulin-secreting cells.

Expression of apoptosis regulatory factors during myocardial dysfunction in endotoxemic rats.

OBJECTIVES: To document the time course of apoptosis pathway activation in sepsis and to determine whether Bcl-2 overexpression would improve endotoxin-induced myocardial dysfunction and mortality rate. DESIGN: Randomized, controlled trial. SETTING: Experimental laboratory. SUBJECTS: Male Sprague Dawley rats, wild-type C57BL/6 female mice, C57BL/6 female mice overexpressing Bcl-2. INTERVENTIONS: Hearts were isolated from rats treated with endotoxin (10 mg/kg, intravenously) to perform heart function, immunohistochemistry (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick 3'-end labeling, caspase 3), RNase protection assay, reverse transcriptase polymerase chain reaction, Western blotting (caspase 3), and radiolabeled annexin V studies. Twenty-four hours before endotoxin challenge (10 mg/kg, intravenously), rats were pretreated with saline or endotoxin (0.5 mg/kg, intraperitoneally), with or without parthenolide (1 mg/kg, intraperitoneally). Isolated hearts were used to test myocardial function. Mortality induced by endotoxin (10 mg/kg, intraperitoneally) was tested on wild-type or mice overexpressing Bcl-2. MEASUREMENTS AND MAIN RESULTS: Endotoxin-induced heart dysfunction was maximal at 4 and 8 hrs postinjection, started to improve, and was fully restored at 24 hrs after endotoxin treatment. Endotoxin also induced phosphatidylserine outer leaflet membrane exposure, caspase 3 activation, nuclear apoptosis, and changes in apoptosis gene expression. Bcl-2 overexpression induced by endotoxin pretreatment prevented endotoxin-induced myocardial dysfunction. Mice overexpressing Bcl-2 had dramatic improvement in survival rate compared with wild-type mice. CONCLUSIONS: These observations suggest that both death receptor and caspase-mediated apoptosis processes are activated in this sepsis model. Bcl-2 overexpression before endotoxin challenge prevents myocardial dysfunction in rats and improves survival rate in mice.

Selective alteration of gene expression in response to natural and synthetic retinoids.

BACKGROUND: Retinoids are very potent inducers of cellular differentiation and apoptosis, and are efficient anti-tumoral agents. Synthetic retinoids are designed to restrict their toxicity and side effects, mostly by increasing their selectivity toward each isotype of retinoic acids receptors (RARalpha,beta, gamma and RXRalpha, beta, gamma). We however previously showed that retinoids displayed very different abilities to activate retinoid-inducible reporter genes, and that these differential properties were correlated to the ability of a given ligand to promote SRC-1 recruitment by DNA-bound RXR:RAR heterodimers. This suggested that gene-selective modulation could be achieved by structurally distinct retinoids. RESULTS: Using the differential display mRNA technique, we identified several genes on the basis of their differential induction by natural or synthetic retinoids in human cervix adenocarcinoma cells. Furthermore, this differential ability to regulate promoter activities was also observed in murine P19 cells for the RARbeta2 and CRABPII gene, showing conclusively that retinoid structure has a dramatic impact on the regulation of endogenous genes. CONCLUSIONS: Our findings therefore show that some degree of selective induction or repression of gene expression may be achieved when using appropriately designed ligands for retinoic acid receptors, extending the concept of selective modulators from estrogen and peroxisome proliferator activated receptors to the class of retinoid receptors.

AGEs bind to mesothelial cells via RAGE and stimulate VCAM-1 expression.

BACKGROUND: Excess advanced glycation end-products (AGEs) are formed during renal failure, and AGE formation also may be connected with the high glucose concentration of peritoneal dialysis (PD) fluids. To determine the effect of human peritoneal mesothelial cell (HPMC) exposure to glycated proteins, we studied the HPMC receptor of AGE expression (RAGE), and analyzed the results of AGE-RAGE interaction on adhesion molecule expression and leukocyte binding. METHODS: RAGE was detected by FACS analysis, and RAGE mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Vascular and intercellular cell adhesion molecule (VCAM-1 and ICAM-1) expression was measured by a known radiometric technique under basal conditions, after the addition of an AGE-specific compound, Nepsilon-carboxylmethyllysine (CML-albumin). Leukocyte adhesion on HPMC was analyzed by videomicroscopy after HPMC stimulation. RESULTS: RAGE protein was detected on HPMC, and RAGE mRNA was evidenced by RT-PCR. VCAM-1 expression was stimulated by CML-albumin (P < 0.01), while ICAM-1 was unchanged. By blocking the AGE-RAGE interaction, anti-RAGE antibodies or recombinant RAGE inhibited the increase in VCAM-1 expression. CML-albumin stimulation potentiated leukocyte adhesion to HPMC (P < 0.001). This effect was prevented by the incubation of leukocytes with recombinant VCAM-1 (P < 0.001). CONCLUSIONS: AGE binding to RAGE stimulated mesothelial cell activity, and resulted in the overexpression of VCAM-1, a structure for leukocyte adhesion. The AGE-RAGE interaction resulted in HPMC activation, which may promote local inflammation, and thus is implicated in the peritoneal injury found in long-term PD patients.