New insight on spatial localization and microstructures of calcite-aragonite interfaces in adult shell of Haliotis tuberculata: Investigations of wild and farmed abalones by FTIR and Raman mapping.

In the present study, we investigated the shell microstructures of the gastropod European abalone Haliotis tuberculata in order to clarify the complex spatial distribution of the different mineral phases. Our studies were carried out with a standardized methodology on thirty adult European abalone H. tuberculata (5-6 cm long) composed of 15 wild individuals and 15 individuals taken from the France Haliotis hatchery. The macroscopic (binocular) and microscopic observations coupled with Fourier Transform Infrared Spectroscopy (FTIR) and Raman vibrational analysis allowed to unambiguously detect, identify and localize calcite and aragonite. For the first time it has been shown that calcite is present in 100% of farmed and wild adult shell. The microstructural details of the calcite-aragonite interfaces were revealed by using both confocal micro-Raman mapping and Scanning Electron Microscopy (SEM) observations. Calcite zones are systematically found in the spherulitic layer without direct contact with the nacreous layer. The calcite area - nacreous layer interface is made of a thin spherulitic layer with variable thickness from a few micrometers to several millimeters. In order to contribute to a better understanding of the biomineralization process, a model explaining the hierarchical arrangement of the different phases of calcium carbonate is presented and discussed. Finally, it has been shown that these calcitic zones can be connected to each other within the shells and that their spatial distributions correspond to streaks perpendicular to the direction of length growth.

Acid-base balance in the hæmolymph of European abalone (Haliotis tuberculata) exposed to CO2-induced ocean acidification.

Ocean acidification (OA) and the associated changes in seawater carbonate chemistry pose a threat to calcifying organisms. This is particularly serious for shelled molluscs, in which shell growth and microstructure has been shown to be highly sensitive to OA. To improve our understanding of the responses of abalone to OA, this study investigated the effects of CO2-induced ocean acidification on extra-cellular acid-base parameters in the European abalone Haliotis tuberculata. Three-year-old adult abalone were exposed for 15 days to three different pH levels (7.9, 7.7, 7.4) representing current and predicted near-future conditions. Hæmolymph pH and total alkalinity were measured at different time points during exposure and used to calculate the carbonate parameters of the extracellular fluid. Total protein content was also measured to determine whether seawater acidification influences the composition and buffer capacity of hæmolymph. Extracellular pH was maintained at seawater pH 7.7 indicating that abalones are able to buffer moderate acidification (-0.2 pH units). This was not due to an accumulation of HCO3- ions but rather to a high hæmolymph protein concentration. By contrast, hæmolymph pH was significantly decreased after 5 days of exposure to pH 7.4, indicating that abalone do not compensate for higher decreases in seawater pH. Total alkalinity and dissolved inorganic carbon were also significantly decreased after 15 days of low pH exposure. It is concluded that changes in the acid-base balance of the hæmolymph might be involved in deleterious effects recorded in adult H. tuberculata facing severe OA stress. This would impact both the ecology and aquaculture of this commercially important species.

Structural description of surfaces and interfaces in biominerals by DNP SENS.

Biological mineralized tissues are hybrid materials with complex hierarchical architecture composed of biominerals often embedded in an organic matrix. The atomic-scale comprehension of surfaces and organo-mineral interfaces of these biominerals is of paramount importance to understand the ultrastructure, the formation mechanisms as well as the biological functions of the related biomineralized tissue. In this communication we demonstrate the capability of DNP SENS to reveal the fine atomic structure of biominerals, and more specifically their surfaces and interfaces. For this purpose, we studied two key examples belonging to the most significant biominerals family in nature: apatite in bone and aragonite in nacreous shell. As a result, we demonstrate that DNP SENS is a powerful approach for the study of intact biomineralized tissues. Signal enhancement factors are found to be up to 40 and 100, for the organic and the inorganic fractions, respectively, as soon as impregnation time with the radical solution is long enough (between 12 and 24 h) to allow an efficient radical penetration into the calcified tissues. Moreover, ions located at the biomineral surface are readily detected and identified through 31P or 13C HETCOR DNP SENS experiments. Noticeably, we show that protonated anions are preponderant at the biomineral surfaces in the form of HPO42- for bone apatite and HCO32- for nacreous aragonite. Finally, we demonstrate that organo-mineral interactions can be probed at the atomic level with high sensitivity. In particular, reliable 13C-{31P} REDOR experiments are achieved in a few hours, leading to the determination of distances, molar proportion and binding mode of citrate bonded to bone mineral in native compact bone. According to our results, only 80% of the total amount of citrate in bone is directly interacting with bone apatite through two out of three carboxylic groups.

Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with Positive Contrast in Magnetic Resonance Imaging for Specific Biomedical Applications.

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH) are well-known for their anticoagulant properties. There is also currently a growing interest in using LMWH in targeted cancer therapy. In particular, several types inhibit heparanase, a key enzyme overexpressed in the tumor microenvironment that promotes angiogenesis progression and metastasis spreading. Here, we propose iron oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging. As a proof of concept, magnetic resonance angiography (MRA) was conducted in mice up to 3 h after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enable innovative theranostic applications if combined with the various bioactivities of the heparins. Indeed, we showed, using advanced in vitro tests, how HEP-IONP anticoagulant or anti-heparanase activities were maintained depending on the heparin species used for the coating. Overall, the study allowed presenting an IONP coated with a commercial LMWH (Lovenox) suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.

Four-year clinical update and treatment switching-adjusted outcomes with first-line nivolumab plus ipilimumab with chemotherapy for metastatic non-small cell lung cancer in the CheckMate 9LA randomized trial.

BACKGROUND: In CheckMate 9LA, nivolumab plus ipilimumab with chemotherapy prolonged overall survival (OS) versus chemotherapy regardless of tumor PD-L1 expression or histology. We report updated efficacy and safety in all randomized patients with a minimum 4-year follow-up and an exploratory treatment-switching adjustment analysis in all treated patients who received chemotherapy and subsequent immunotherapy. METHODS: Adults with stage IV/recurrent non-small cell lung cancer (NSCLC), no sensitizing EGFR/ALK alterations, and ECOG performance status ≤1 were randomized 1:1 to nivolumab 360 mg every 3 weeks plus ipilimumab 1 mg/kg every 6 weeks with chemotherapy (two cycles) or chemotherapy (four cycles, with optional maintenance pemetrexed for the nonsquamous population). Assessments included OS, progression-free survival, and objective response rate. Exploratory analyses included efficacy by tumor PD-L1 expression and histology and in patients who discontinued nivolumab plus ipilimumab with chemotherapy due to treatment-related adverse events (TRAEs), and a treatment-switching adjustment analysis using inverse probability of censoring weighting. RESULTS: With a 47.9-month minimum follow-up for OS, nivolumab plus ipilimumab with chemotherapy continued to prolong OS over chemotherapy in all randomized patients (HR 0.74, 95% CI 0.63 to 0.87; 4-year OS rate: 21% versus 16%), regardless of tumor PD-L1 expression (HR (95% CI): PD-L1<1%, 0.66 (0.50 to 0.86) and ≥1%, 0.74 (0.60 to 0.92)) or histology (squamous, 0.64 (0.48 to 0.84) and non-squamous, 0.80 (0.66 to 0.97)). In patients who discontinued all components of nivolumab plus ipilimumab with chemotherapy due to TRAEs (n=61), the 4-year OS rate was 41%. With treatment-switching adjustment for the 36% of patients receiving subsequent immunotherapy in the chemotherapy arm, the estimated HR of nivolumab plus ipilimumab with chemotherapy versus chemotherapy was 0.66 (95% CI 0.55 to 0.80). No new safety signals were observed. CONCLUSIONS: In this 4-year update, patients treated with nivolumab plus ipilimumab with chemotherapy continued to have long-term, durable efficacy benefit over chemotherapy regardless of tumor PD-L1 expression and/or histology. A greater estimated relative OS benefit was observed after adjustment for subsequent immunotherapy use in the chemotherapy arm. These results further support nivolumab plus ipilimumab with chemotherapy as a first-line treatment for patients with metastatic/recurrent NSCLC, including those with tumor PD-L1<1% or squamous histology, populations with high unmet needs.

Risk Factors of Venous Thromboembolic Disease in Cancer Patients Treated with Immune Checkpoint Inhibitor.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the management of cancers. The risk factors and pathophysiological mechanisms of venous thromboembolic events (VTEs) of this new therapeutic class are still to be specified. METHODS: The included patients had to have cancer and should be treated with ICI. Data analyzed included demographic data, biological data, and immune-related adverse events (IRAEs). We studied the prevalence of VTEs and the factors associated with VTEs. RESULTS: Of 374 patients on ICI, over a median follow-up period of 15.2 months, the number of VTE was 50 (13.4%). The majority of patients were treated for metastatic melanoma or nonsmall cell lung cancer. There was no difference in prevalence or survival between cancer types. Patients with combined therapy composed of nivolumab and ipilimumab had higher 1-year cumulative VTE occurrence (29.3% [95% confidence interval [CI]: 9.7; 44.6]) than patients with pembrolizumab (14.9%, [95%CI: 2.5; 25.8], p = 0.03) or nivolumab (9.1%, [95% CI: 5.0; 12.9], p < 0.01). The presence of IRAE was associated with a higher risk of VTE occurrence compared with patients without any IRAE (1-year VTE cumulative incidence: 17.42% [95% CI: 9.5; 24.65] vs. 9.46% [95% CI: 5.18; 13.55], p = 0.04). There was a higher risk of VTE in patients treated with the combination of nivolumab and ipilimumab (adjusted subdistribution hazard ratio [SHR]: 3.71 [95% CI: 1.74; 7.90], p < 0.001) and in patients with IRAE (adjusted SHR: 2.14 [95% CI: 1.22; 3.75], p < 0.01). CONCLUSION: The prevalence of VTE was 14.2% under ICIs. IRAE and combine treatment of nivolumab and ipilimumab were associated with VTE. The pathophysiological mechanisms are multiple and complex with a possible link to aberrant activation of the immune system.

First-line nivolumab plus ipilimumab for metastatic non-small cell lung cancer, including patients with ECOG performance status 2 and other special populations: CheckMate 817.

BACKGROUND: CheckMate 817, a phase 3B study, evaluated flat-dose nivolumab plus weight-based ipilimumab in patients with metastatic non-small cell lung cancer (NSCLC). Here, in this research, we report on first-line treatment in patients with Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0-1 (cohort A) and special populations (cohort A1: ECOG PS 2; or ECOG PS 0-1 with untreated brain metastases, renal impairment, hepatic impairment, or controlled HIV infection). METHODS: Cohorts A and A1 received nivolumab 240 mg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary endpoint was the incidence of grade 3-4 and grade 5 immune-mediated adverse events (IMAEs; adverse events (AEs) deemed potentially immune-related, occurring <100 days of last dose, and treated with immune-modulating medication (except endocrine events)) and treatment-related select AEs (treatment-related AEs with potential immunological etiology requiring frequent monitoring/intervention, reported between first dose and 30 days after the last dose) in cohort A; efficacy endpoints were secondary/exploratory. In cohort A1, safety/efficacy assessment was exploratory. RESULTS: The most common grade 3-4 IMAEs were pneumonitis (5.1%), diarrhea/colitis (4.9%), and hepatitis (4.6%) in cohort A (N=391) and diarrhea/colitis (3.5%), hepatitis (3.5%), and rash (3.0%) in cohort A1 (N=198). The most common grade 3-4 treatment-related select AEs were hepatic (5.9%), gastrointestinal (4.9%), and pulmonary (4.6%) events in cohort A and gastrointestinal (4.0%), skin (3.5%), and endocrine (3.0%) events in cohort A1. No grade 5 IMAEs or treatment-related select AEs occurred. Treatment-related deaths occurred in 4 (1.0%) and 3 (1.5%) patients in cohorts A and A1, respectively. Three-year overall survival (OS) rates were 33.7% and 20.5%, respectively. CONCLUSIONS: Flat-dose nivolumab plus weight-based ipilimumab was associated with manageable safety and durable efficacy in cohort A, consistent with data from phase 3 metastatic NSCLC studies. Special populations of cohort A1 including patients with ECOG PS 2 or ECOG PS 0-1 with untreated brain metastases had manageable treatment-related toxicity and clinically meaningful 3-year OS rate. TRIAL REGISTRATION NUMBER: NCT02869789.

Identification of two carbonic anhydrases in the mantle of the European Abalone Haliotis tuberculata (Gastropoda, Haliotidae): phylogenetic implications.

Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks.

Combined effects of ocean warming and acidification on the larval stages of the European abalone Haliotis tuberculata.

This study examined the physiological responses of the larval stages of Haliotis tuberculata, an economically important abalone, to combined temperature (17 °C and 19 °C) and pH (ambient pH and -0.3 units, i.e., +200% increase in seawater acidity) in a full factorial experiment. Tissue organogenesis, shell formation, and shell length significantly declined due to low pH. High temperature significantly increased the proportion of fully shelled larvae at 24 h post-fertilization (hpf), but increased the proportion of unshelled larvae at 72 hpf. Percentage of swimming larvae at 24 hpf, 72 hpf and 96 hpf significantly declined due to high temperature, but not because of low pH. Larval settlement increased under high temperature, but was not affected by low pH. Despite the fact that no interaction between temperature and pH was observed, the results provide additional evidence on the sensitivity of abalone larvae to both low pH and high temperature. This may have negative consequences for the persistence of abalone populations in natural and aquaculture environments in the near future.

Inorganic phosphate in growing calcium carbonate abalone shell suggests a shared mineral ancestral precursor.

The presence of phosphate from different origins (inorganic, bioorganic) is found more and more in calcium carbonate-based biominerals. Phosphate is often described as being responsible for the stabilization of the transient amorphous calcium carbonate phase. In order to specify the composition of the mineral phase deposited at the onset of carbonated shell formation, the present study investigates, down to the nanoscale, the growing shell from the European abalone Haliotis tuberculata, using a combination of solid state nuclear magnetic resonance, scanning transmission electron microscope and spatially-resolved electron energy loss spectroscopy techniques. We show the co-occurrence of inorganic phosphate with calcium and carbonate throughout the early stages of abalone shell formation. One possible hypothesis is that this first-formed mixed mineral phase represents the vestige of a shared ancestral mineral precursor that appeared early during Evolution. In addition, our findings strengthen the idea that the final crystalline phase (calcium carbonate or phosphate) depends strongly on the nature of the mineral-associated proteins in vivo.

Responses of early life stages of European abalone (Haliotis tuberculata) to ocean acidification after parental conditioning: Insights from a transgenerational experiment.

CO2 absorption is leading to ocean acidification (OA), which is a matter of major concern for marine calcifying species. This study investigated the effects of simulated OA on the reproduction of European abalone Haliotis tuberculata and the survival of its offspring. Four-year-old abalone were exposed during reproductive season to two relevant OA scenarios, ambient pH (8.0) and low pH (7.7). After five months of exposure, abalone were induced to spawn. The gametes, larvae and juveniles were then exposed for five months to the same pH conditions as their parents. Several biological parameters involved in adult reproduction as well as in larval, post-larval and juvenile fitness were measured. No effects on gametes, fertilisation or larval oxidative stress response were detected. However, developmental abnormalities and significant decreases in shell length and calcification were observed at veliger stages. The expression profile of a GABA A receptor-like gene appeared to be regulated by pH, depending on larval stage. Larval and post-larval survival was not affected by low pH. However, a lower survival and a reduction of growth were recorded in juveniles at pH 7.7. Our results confirm that OA negatively impacts larval and juvenile fitness and suggest the absence of carry-over effects on abalone offspring. This may compromise the survival of abalone populations in the near future.

Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata.

An integrated study of shell formation was initiated covering the entire life cycle of the marine gastropod Haliotis tuberculata. Shell microstructure, chemistry and mineralogy were investigated by polarized microscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDX) and infra-red (IR) spectroscopy. SEM images of trochophore and veliger larvae showed the different stages of shell growth from the initial shell field to the late calcified protoconch. Cross-sections revealed the microstructural arrangement of biominerals, showing the progressive mineralization of the organic protoconch prior to metamorphosis. To gain more information on mineralogical composition, EDX analyses and IR spectroscopy were performed along the development stages. The results demonstrated that early protoconch was mostly composed of amorphous calcium carbonate, while veliger stages showed a gradually crystallization under the form of aragonite. Post-metamorphic shell contained two distinct parts, the original protoconch supporting the new juvenile shell characterized by a marked sculptural pattern. The shells from post-larval and juvenile abalones were essentially made of aragonite.

Comparative effects of a non-steroidal ecdysone agonist RH-5992 and 20-hydroxyecdysone in a lepidopteran cell line (IAL-PID2).

The non-steroidal ecdysone agonist, RH-5992, exhibits ecdysteroid activities in vivo as well as in vitro more effectively than 20-hydroxyecdysone (20E). Using the IAL-PID2 cells derived from imaginal wing discs of last larval instar of Plodia interpunctella, we investigated the action of RH-5992 in the control of cell growth. Its effects on the proliferative activity of IAL-PID2 cells, the induction level in G2/M arrest and on the expression rate of Plodia B cyclin (PcycB), ecdysone B1-isoform (PIEcR-B1) and Ultraspiracle-2 isoform (PIUSP-2) were examined. From these cellular and molecular assays, our results brought evidence that RH-5992, like 20E, induced an inhibition on cell proliferation by blocking IAL-PID2 cells in G2/M phase. Moreover, this G2/M arrest was preceded by a decrease in the expression level of PcycB and a high induction of PIEcR-B1, PIUSP-2 mRNAs. Dose-response experiments revealed that RH-5992 was even more potent than 20E. On these parameters, we therefore suggest that the differential observed in the expression level of USP and EcR by RH-5992 and 20E could contribute to the difference observed for the biological potency of these two compounds.

Anti-proliferative effect of 20-hydroxyecdysone in a lepidopteran cell line.

Ecdysteroids are steroid hormones involved in the epidermal growth of arthropods, controlling cell proliferation and further differentiation of target cells. The epidermal cell line IAL-PID2, established from imaginal discs of the Indian meal moth Plodia interpunctella kept its sensitivity to ecdysteroids in vitro, cells being able to respond to them by cytological and biochemical changes. When added to the culture medium, 20-hydroxyecdysone (20E) stopped cell proliferation and induced formation of epithelial-like aggregates. In order to better understand the cellular sequence of ecdysteroids signalling in epidermal cells we used the IAL-PID2 cell line for in vitro investigations of cytological events induced by the moulting hormone. After a 40 h serum deprivation, formazan assay (XTT) was routinely used to evaluate anti-proliferative effects of 20E during cell cycle. We established a more precise timing of the period of cell sensitivity to the hormone during the cell cycle, by the use of the mitotic index and the BrdU incorporation test. These in vitro assays were performed in parallel with the description of some hormone dependant cytological events, using immunofluorescent labelling with anti-beta tubulin/FITC antibodies and DNA staining.

The 20-hydroxyecdysone-induced cellular arrest in G2 phase is preceded by an inhibition of cyclin expression.

We have studied the effect of 20-hydroxyecdysone (20E) on cellular proliferation in IAL-PID2 cell line established from imaginal wing discs of Plodia interpunctella. Flow cytometry analysis demonstrated that 20E induced an arrest of cells in G2 phase. To determine whether this arrest was due to an effect of 20E on cyclin expression, we cloned two cDNA fragments, named PcycA and PcycB, encoding, respectively, Plodia cyclins A and B. Using PcycA and PcycB probes, we have demonstrated that 20E induced a sharp decrease in the levels of cyclin A and B expression. Studies of induction pattern of Plodia HR3 transcription factor by 20E revealed that its induction preceded the decrease of cyclins transcripts. An exposure of cells to 20E in the presence of juvenile hormone (JH) led to a change in the kinetic of PHR3 induction and prevented both the decline of cyclin A and B expression and the G2 arrest. This effect of JH provides an additional argument for the existence of a correlation between cyclin transcripts level and G2 arrest. For the first time in insects, these findings bring evidence that ecdysteroids regulate cellular proliferation by acting on cell cycle regulators as cyclins.

Comparative effects of angiotensin IV and two hemorphins on angiotensin-converting enzyme activity.

The role of angiotensin IV (AngIV) in the regulation of angiotensin-converting enzyme (ACE) was studied in vitro. This study demonstrates that this active fragment appeared as a novel endogenous ACE inhibitor. Inhibitory kinetic studies revealed that AngIV acts as a purely competitive inhibitor with a K(i) value of 35 microM. AngIV was found to be quite resistant to ACE hydrolysis opposite to hemorphins which are both ACE inhibitors and substrates. In order to confirm a putative role of AngIV and hemorphins in the Renin-Angiotensin system (RAS) regulation, we studied their influence on AngI conversion. We noticed that 16.7 microM of both peptides decreased more than 50% of AngI conversion to AngII in vitro. The capacity of hemorphins, particularly LVVH-7, and AngIV to inhibit ACE activity here suggests a synergistic relation between these two peptides and the regulation of RAS.

Expression of biomineralisation genes in tissues and cultured cells of the abalone Haliotis tuberculata.

Mollusc shell biomineralisation involves a variety of organic macromolecules (matrix proteins and enzymes) that control calcium carbonate (CaCO3) deposition, growth of crystals, the selection of polymorph, and the microstructure of the shell. Since the mantle and the hemocytes play an important role in the control of shell formation, primary cell cultures have been developed to study the expression of three biomineralisation genes recently identified in the abalone Haliotis tuberculata: a matrix protein, Lustrin A, and two carbonic anhydrase enzymes. Mantle cells and hemocytes were successfully maintained in primary cultures and were evaluated for their viability and proliferation over time using a semi-automated assay (XTT). PCR and densitometric analysis were used to semi-quantify the gene expression and compare the level of expression in native tissues and cultured cells. The results demonstrated that the three genes of interest were being expressed in abalone tissues, with expression highest in the mantle and much lower in the hemocytes and the gills. Biomineralisation genes were also expressed significantly in mantle cells, confirming that primary cultures of target tissues are suitable models for in vitro investigation of matrix protein secretion.

Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures.

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell-V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.

In vitro effects of triclosan and methyl-triclosan on the marine gastropod Haliotis tuberculata.

Triclosan (2,4,4'-trichloro-2'-hydroxy-diphenyl ether; TCS) is an antibacterial agent incorporated in a wide variety of household and personal care products. Because of its partial elimination in sewage treatment plants, TCS is commonly detected in natural waters and sediments. Moreover, due to its high hydrophobicity, TCS accumulates in fatty tissues in various aquatic organisms. TCS can be converted into methyl-triclosan (2,4,4'-trichloro-2'-methoxydiphenyl ether; MTCS) after biological methylation. In this study, the acute cytotoxicity of TCS and MTCS in short-term in vitro experiments was assessed on cell cultures from the European abalone Haliotis tuberculata. The results showed that morphology and density of hemocyte are affected from a concentration of 8 µM TCS. Using the XTT reduction assay, TCS has been demonstrated to decrease hemocyte metabolism activity in a dose- and time-dependent exposure. The IC(50) was evaluated at 6 µM for both hemocyte and gill cells after a 24 h-incubation with TCS. A significant cytotoxicity of MTCS was also observed from 4 µM in 24 h-old hemocyte culture. Our results reveal a toxic effect of TCS and MTCS on immune (hemocytes) and/or respiratory cells (gill cells) of the abalone, species living in coastal waters areas and exposed to anthropogenic pollution.