Claire Carrion

Dynamic Assessment of the Floc Morphology, Bacterial Diversity, and Integron Content of an Activated Sludge Reactor Processing Hospital Effluent

The treatment of hospital effluents (HE) is a major concern, as they are suspected of disseminating drugs and antibiotic resistance determinants in the environment. In order to assess HE influence on wastewater treatment plant biomass, lab-scale conventional activated sludge systems (CAS) were continuously fed with real HE or urban effluent as a control. To gain insights into the main hurdles linked to HE treatment, we conducted a multiparameter study using classical physicochemical characterization, phase contrast and confocal laser scaning microscopy, and molecular biology (i.e., pyrosequencing) tools. HE caused erosion of floc structure and the production of extracellular polymeric substances attributed to the development of floc-forming bacteria. Adaptation of the sludge bacterial community to the HE characteristics, thus maintaining the purification performance of the biomass, was observed. Finally, the comparative metagenomic analysis of the CAS showed that HE treatment resulted in an increase of class 1 resistance integrons (RIs) and the introduction of Pseudomonas spp. into the bacterial community. HE treatment did not reduce the CAS process performance; nevertheless it increases the risk of dissemination into the environment of bacterial species and genetic determinants (RIs) involved in antibiotic resistance acquisition.

Self-Restrained B Cells Arise following Membrane IgE Expression

Among immunoglobulins (Igs), IgE can powerfully contribute to antimicrobial immunity and severe allergy despite its low abundance. IgE protein and gene structure resemble other Ig classes, making it unclear what constrains its production to thousand-fold lower levels. Whether class-switched B cell receptors (BCRs) differentially control B cell fate is debated, and study of the membrane (m)IgE class is hampered by its elusive inxa0vivo expression. Here, we demonstrate a self-controlled mIgE+ B cell stage. Primary or transfected mIgE+ cells relocate the BCRs into spontaneously internalized lipid rafts, lose mobility to chemokines, and change morphology. We suggest that combined proapoptotic mechanisms possibly involving Hax1 prevent mIgE+ memory lymphocyte accumulation. By uncoupling inxa0vivo IgE switching from cytokine and antigen stimuli, we show that these features are independent from B cell stimulation and instead result from mIgE expression per se. Consequently, few cells survive IgE class switching, which might ensure minimal long-term IgE memory upon differentiation into plasma cells.

Sense transcription through the S region is essential for immunoglobulin class switch recombination.

Class switch recombination (CSR) occurs between highly repetitive sequences called switch (S) regions and is initiated by activation‐induced cytidine deaminase (AID). CSR is preceded by a bidirectional transcription of S regions but the relative importance of sense and antisense transcription for CSR in vivo is unknown. We generated three mouse lines in which we attempted a premature termination of transcriptional elongation by inserting bidirectional transcription terminators upstream of Sμ, upstream of Sγ3 or downstream of Sγ3 sequences. The data show, at least for Sγ3, that sense transcriptional elongation across S region is absolutely required for CSR whereas its antisense counterpart is largely dispensable, strongly suggesting that sense transcription is sufficient for AID targeting to both DNA strands.

Interallelic class switch recombination can reverse allelic exclusion and allow trans-complementation of an IgH locus switching defect

The predominant path of immunoglobulin class switch recombination follows the paradigm of intra‐chromosomal deletion enabling expression of another heavy chain instead of µ and δ. This was, however, challenged by observations of inter‐allelic class switch recombination in rabbit or mouse IgG3‐ or IgA‐producing B cells. Assuming that the conditions of inter‐chromosomal exchange are likely present at any target S regions in stimulated B cells, we explored trans‐association of VH and C genes in a model allowing all C genes to be checked simultaneously. Heterozygous mutant mice are thus studied, which carry one non‐functional IgH allele inactivated by a non‐translatable mutation of VDJ‐CH transcripts, while the functional allele is deficient for class switching due to a truncated 3′regulatory region. A fair level of switching to all Ig classes is restored in heterozygous mice despite the fact that cis‐recombination is either non productive on one allele or deficient on the other. Molecular evidence at the DNA level of trans‐CSR to IgG3 was demonstrated by cloning and sequencing Sµ‐Sγ3 hybrid junctions. These data demonstrate that inter‐allelic recombination may broadly rescue the production of various class‐switched isotypes and allow complementation between mutations located at both ends of the IgH constant gene cluster.

Analysis of the in vitro and in vivo effects of photodynamic therapy on prostate cancer by using new photosensitizers, protoporphyrin IX-polyamine derivatives

BACKGROUNDnPhotodynamic therapy, using porphyrins as photosensitizers (PS), has been approved in treatment of several solid tumors. However, commonly used PS induce death but also resistance pathways in cancer cells and an alteration of surrounding normal tissues. Because polyamines (PA) are actively accumulated in cancer cells by the Polyamine Transport System (PTS), they may enable PS to specifically target cancer cells. Here, we investigated whether new protoporphyrin IX-polyamine derivatives were effective PS against prostate cancer and whether PA increased PDT specificity after 630nm irradiation.nnnMETHODSnCHO and CHO-MG cells (differing in their PTS activity) were used to assess efficacy of polyamine vectorization. MTT assays were performed on human prostate non-malignant (RWPE-1) and malignant (PC-3, DU 145 and LNCaP) cell lines to test PS phototoxicity. ROS generation, DNA fragmentation and cell signalling were assessed by ELISA/EIA, western-blots and gel shift assays. Finally, PS effects were studied on tumor growth in nude mice.nnnRESULTSnOur PS were more effective on cancer cells compared to non-malignant cells and more effective than PpIX alone. PpIX-PA generated ROS production involved in induction of apoptotic intrinsic pathways. Different pathways involved in apoptosis resistance were studied: PS inhibited Bcl-2, Akt, and NF-κB but activated p38/COX-2/PGE2 pathways which were not implicated in apoptosis resistance in our model. In vivo experiments showed PpIX-PA efficacy was greater than results obtained with PpIX.nnnCONCLUSIONSnAll together, our results showed that PpIX-PA exerted its maximum effects without activating resistance pathways and appears to be a good candidate for prostate cancer PDT treatment.

The IgH 3′ regulatory region super-enhancer does not control IgA class switch recombination in the B1 lineage

The bone marrow-derived B2 population represents the vast majority of bone marrow, blood, lymph node and splenic B-cells. Mouse B1 B-cells mostly originate during embryonic life in the liver and represent the main B-cell population in the pleural and peritoneal cavities.1–5 B1 and B2 B-cells differ in their origin, antigen specificity, cell surface markers, tissue distribution and capacity for class switch recombination (CSR). Schematically, B1 B-cells appear earlier than B2 B-cells during fetal development and maintain their self-renewal ability throughout their life. Furthermore, they display a CSR biased toward IgA.1–5 IgH cis-regulatory regions, especially transcriptional super-enhancers, are major locus regulators.6 The IgH 3′ regulatory region (3′RR) super-enhancer promotes CSR in B2 B-cells.7,8 As B1 and B2 B-cells originate from different precursors and have clearly different development, function and regulation, we postulated that the 3′RR super-enhancer might differently regulate B1 and B2 B-cell IgA CSR. The 3′RR super-enhancer plays a key regulatory role in B2 B-cell CSR by poising the S acceptor region for efficient recombination toward all isotypes8 except IgD.9,10 Peritoneal cavity B1 B-cells switch preferentially to IgA.11,12 We thus investigated B1 B-cell IgA CSR in 3′RR-deficient mice.7 Our research was approved by our local ethics committee review board (Comité Régional dEthique sur lExpérimentation Animale du Limousin, Limoges, France) and carried out according to the European guidelines for animal experimentation. The 3′RR deletion was performed in a 129 ES cell line (IgH a allotype) and developed in a 129 background (IgH awt/awt). In this study, homozygous 3′RR-deficient mice (IgH aΔ3′RR/aΔ3′RR) were compared with 129 IgH awt/awt mice. Mouse B1 and B2 B-cells are distinguished on the basis of membrane cell surface markers. B1 B-cells are B220lowIgMhighIgDlowCD23−CD11b+/low, whereas B2 B-cells are B220highIgMhighIgDhighCD23+ CD11b−.2,4,13 As shown in Figure 1a, similar (P= 0.5) percentages of IgA+ B1 B-cells were found in the peritoneal cavity of 3′RR-deficient and wild type (wt) mice in response to a pristane-induced local inflammatory reaction. Almost all recruited inflammatory B-cells in response to pristane had a B1 B-cell phenotype. We next investigated their ability to switch in vitro toward IgA (LPS+BAFF+TGFβ stimulation). As shown in Figure 1b, no difference (P= 0.3) was found in B1 B-cell IgA CSR between 3′RR-deficient mice and wt mice. Despite similar in vivo and in vitro IgA CSR, lowered (P= 0.0003) levels of IgA were found in peritoneal ascites of 3′RR-deficient mice compared with wt mice (Figure 1c) and in IgA+ cell culture supernatants (P= 0.002) of 3′RR-deficient B1 B-cells compared with wt B1 B-cells (Figure 1d). q-PCR analysis indicated that the latter result originated from a significant (P= 0.01) decrease in Iμ-Cα transcripts in 3′RR-deficient mice compared with wt mice (Figure 1e). Taken together, these results suggest that in contrast to B2 B-cells, 3′RR deletion did not affect the B1 B-cell CSR toward IgA but only decreased Cα transcription after CSR. The IgA-producing B1 B-cells contribute substantially to mucosal IgA production and thus play a key role in regulating commensal microbiota.4,14 We thus investigated IgA production in the intestinal tract of 3′RR-deficient mice and wt mice. Mouse feces were recovered and tested for the presence of IgA. We found a significant (P= 0.002) and marked (up to 99%) decrease in secreted IgA levels in feces of 3′RR-deficient mice (0.08± 0.03 μg/g, six mice) compared with wt mice (11.34± 0.63 μg/g, six mice). Confirming that this result is related to an IgA secretion deficiency but not to an IgA CSR or a plasma cell maturation defect, immunohistochemistry experiments indicated similar IgA+ CNRS UMR 7276, CRIBL, Université de Limoges, Limoges, France

Deletion of the α immunoglobulin chain membrane-anchoring region reduces but does not abolish IgA secretion.

Class switching and plasma cell differentiation occur at a high level within all mucosa‐associated lymphoid tissues. The different classes of membrane immunoglobulin heavy chains are associated with the Igα/Igβ heterodimer within the B‐cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membranes have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA‐class BCR through a ‘αΔtail’ targeted mutation, deleting the Cα immunoglobulin gene membrane exon. This allowed us to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail+/+ B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA+ B lymphocytes. By contrast, in secretions the secretory IgA was very low, indicating that J‐chain‐positive plasma cells producing secretory IgA overwhelmingly differentiate from previously class‐switched membrane IgA+ memory B cells. In addition, although mucosa‐associated lymphoid tissues are a major site for plasma cell accumulation, αΔtail+/+ mice showed that the gut B‐cell lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the membrane IgA BCR.

The IgH locus 3’ cis -regulatory super-enhancer co-opts AID for allelic transvection

Immunoglobulin heavy chain (IgH) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3′ regulatory region (3′RR) includes several transcriptional cis-enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3′RR super-enhancer supports not only legitimate cis-, but also trans-recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3′RR super-enhancer.

Evaluation of the extracellular polymeric substances by confocal laser scanning microscopy in conventional activated sludge and advanced membrane bioreactors treating hospital wastewater

Confocal laser scanning microscopy (CLSM) combined with fluorescent viability indicators, was used in this study to investigate the impact of hospital wastewaters on floc structure and composition. In this work, three pilot-scale projects, two membrane bioreactors (MBRs) with a submerged or external membrane bioreactor and a conventional activated sludge, were installed and operated for 65 days. They were fed with an influent sampled directly from the hospital drainage system, which contained micropollutant concentrations ranging from ng/L to mg/L. Samples of flocs were observed using CLSM to characterize the extracellular polymeric substances (EPS) stained with concanavalin A-tetra methylrhodamine and fluorescein isothiocyanate solution and combined with a fluorescent viability indicator (Baclight(®) Bacterial Viability Kit, Molecular Probes), allowing visualization of isolated stained cells in the three-dimensional structure of flocs (damaged or not). The results of CLSM of the sludge composition were compared with classical biochemical analysis of EPS made through a thermal extraction method. The results showed a good relation between these analyses and the statistical treatment of microscopic pictures.

Vancomycin sorption on activated sludge Gram+ bacteria rather than on EPS; 3D Confocal Laser Scanning Microscopy time-lapse imaging

Antibiotics-bacteria interactions depend on antibiotic concentration at the scale of bacteria. This study investigates how vancomycin penetrates into activated sludge flocs and can be sorbed on the bacteria and extracellular polymeric substances (EPS). The 3D structure of flocs was imaged using EPS autofluorescence. The green fluorescent BODIPY® FL vancomycin was introduced in a microscopic chamber containing activated sludge and penetration of vancomycin into the flocs by diffusion was observed using time-lapse microscopy. The penetration depended on the floc structure, as long and large pores could go through the whole flocs making preferential path. The antibiotic concentration into the flocs was also found to depend on the sorption rate. BODIPY® FL vancomycin was found to bind preferentially into Gram+ bacteria than on EPS. The vancomycin adsorption constant on bacteria according to the linear adsorption model, Kdbacteria was estimated to be 5 times higher (SD 2.6) than the adsorption constant on EPS KdEPS. These results suggest that antibiotic removal by sorption into wastewater treatment plants could change according to the amount of bacteria in the sludge. Moreover, antibiotic concentration at the scale of bacteria could be significantly higher than the concentration in the bulk solution and this should be taken into account when studying antibiotic activity or biodegradation.