[Predictive factors of conservative breast surgery after neoadjuvant chemotherapy for breast cancer]. / Facteurs prédictifs de traitement conservateur après chimiothérapie néo-adjuvante dans le cancer du sein.

OBJECTIVES: The aim of our study was to evaluate the existence of predictive factors of conservative breast surgery after neoadjuvant chemotherapy (NAC) for breast cancer. METHODS: We included all women with invasive breast cancer who received NAC and underwent breast surgery between January 2007 and December 2013 in our institution. Univariable and multivariable analyses were performed to determine the association between clinical and histological factors and conservative breast surgery. RESULTS: During the study period, 229 women were included of whom 73 had breast conservative surgery (32%). At univariable analysis, significant predictive factors were age (OR 0.97 [CI 95% 0.95-0.99], P=0.02), radiological size (OR 0.97 [CI 95% 0.96-0.99], P<0.001), multifocality (OR 0.53 [CI 95% 0.27-1.05], P=0.06), breast inflammation (OR 0.15 [CI 95% 0.07-0.32], P<0.001) and the type of hormone receptors (P=0.12). In multivariable analysis, all these factors but age were significant factors and thus considered as independent predictive factors. CONCLUSION: This work permitted to identify independent predictive factors of breast conservative surgery after NAC for breast cancer that will be included in a risk scoring system that we aim to evaluate prospectively.

[Impact of pathological complete response to neoadjuvant chemotherapy in invasive breast cancer according to molecular subtype]. / Impact de la réponse histologique complète à la chimiothérapie néo-adjuvante pour cancer du sein selon le sous-type moléculaire.

OBJECTIVES: The aim of this study was to evaluate the impact of pathological complete response (pCR) on overall survival (OS) and recurrence-free survival (RFS) according to molecular subtypes in women treated for an invasive breast cancer after neoadjuvant chemotherapy (NAC). METHODS: All women (n=225) managed with a neoadjuvant chemotherapy for an invasive breast cancer in our institution between January 2007 and December 2013 were included. The characteristics of patients with pCR (pCR-1), breast pCR and axillary pCR were compared to those without pCR (pCR-0) according to the molecular subtypes: luminal A (n=62), luminal B (n=77), Her-2 (n=31) and triple negative (n=55). RESULTS: NAC concerned 225 patients of whom 36 (16%) had pCR. Achievement of pCR led to significantly better overall survival in women with Her-2 tumors (35% versus 100%, P=0.035) and also to significantly better locoregional survival in women treated for triple negative tumors (P=0.026). Predictive factors of pCR were a high pathologic grade: OR=2.39, IC 95% (1.19-4.83), P=0.008; Her-2 molecular subtype (P=0.008); positive estrogenic hormonal receptors (P=0.006), a positive Her-2 receptor: OR=2.58, IC 95% (1.20-5.54), P=0.01. CONCLUSION: Achievement of pCR is an intermediate marker of survival in women managed with NAC for breast cancer.

[Outcomes of patients with breast cancer in function of their body mass index]. / Indice de masse corporelle et facteurs pronostiques du cancer du sein.

OBJECTIVE: The aim of this study was to evaluate outcomes of patients with breast cancer in function of the body mass index (BMI). METHODS: The study cohort consisted of consecutive women undergoing surgery for breast cancer in our institution between January 2009, and September 2013. Individual records of all patients were reviewed and analyzed. Patient BMI was categorized as underweight, normal, overweight and obese. RESULTS: A total of 1599 patients were evaluated. Patients were followed for one to 265months with a mean of 36.4months. The number of patients in each of the BMI categories was 66, 779, 463 and 291 for underweight, normal, overweight and obese women respectively. Women with higher BMI were more frequently affected by hypertension (18, 21, 35 and 47% respectively, P<0.0001) and diabetes (3, 2, 7 and 7% respectively, P<0.0001). Obese women had more frequently an inflammatory presentation (P=0.006), larger tumour size (P=0.038) and axillary lymph node involvement (P=0.03) with much more positive lymph nodes (P=0.02). Patients had the same protocols of treatment (surgery and adjuvant treatment). There was no statistically significant difference in overall 5-years survival between groups (P=0.30). CONCLUSIONS: Our study demonstrate a more aggressive clinical and histological presentation for obese women with breast cancer.

Embolization biomaterial reinforced with nanotechnology for an in-situ release of anti-angiogenic agent in the treatment of hyper-vascularized tumors and arteriovenous malformations.

A polymer based material was developed to act as an embolic agent and drug reservoir for the treatment of arteriovenous malformations (AVM) and hyper vascularized solid tumors. The aim was to combine the blocking of blood supply to the target region and the inhibition of the embolization-stimulated angiogenesis. The material is composed of an ethanolic solution of a linear acrylate based copolymer and acrylate calibrated microparticles containing nanospheres loaded with sunitinib, an anti-angiogenic agent. The precipitation of the linear copolymer in aqueous environment after injection through microcatheter results in the formation of an in-situ embolization gel whereas the microparticles serve to increase the cohesive properties of the embolization agent and to form a reservoir from which the sunitinib-loaded nanospheres are released post-embolization. The swollen state of the microparticles in contact with aqueous medium results in the release of the nanospheres out of microparticles macromolecular structure. After the synthesis, the formulation and the characterization of the different components of the material, anti-angiogenic activity was evaluated in vitro using endothelial cells and in vivo using corneal neovascularization model in rabbit. The efficiency of the arterial embolization was tested in vivo in a sheep model. Results proved the feasibility of this new system for vascular embolization in association with an in situ delivery of anti-angiogenic drug. This combination is a promising strategy for the management of arteriovenous malformations and solid tumors.

[Bakri balloon tamponade for severe post-partum haemorrhage: efficiency and fertility outcomes]. / Évaluation du ballon de Bakri dans les hémorragies graves du post-partum et fertilité ultérieure.

AIM: To evaluate efficiency of Bakri balloon tamponade (BB) to stop severe post-partum haemorrhage (PPH) and fertility outcomes. METHODS: Retrospective study including all patients who underwent Bakri balloon tamponade for severe PPH between January 2009 and December 2013. The objectives were to stop PPH by BB and to evaluate the fertility after Bakri balloon tamponade. RESULTS: Sixty-one women had a Bakri balloon inserted in utero for severe PPH. The PPH was stopped in 55 patients out of 61 (88%). The reasons of severe PPH were uterine atony in 44 cases (72%), placental retention in 10 cases, placenta praevia in 3 cases, and cervical or vaginal tears in 4 cases. In one third of cases, the pregnancy was complicated by diabetes, placenta praevia, hypertensive troubles. A cesarean section or an instrumental delivery was performed for one third of patients. The mean duration of the Bakri balloon insertion was of 7 hours [5-9] and the mean filling of the balloon was of 350 ml [205-450]. The mean blood loss was of 1600 [1200-2250]. Sixty-three percent of patients (n=38) received red blood cells transfusion. The BB was efficient after a vaginal delivery or after a caesarean section and in all cases of placenta praevia. In 6 cases, the BB was inefficient and uterine embolisation or a surgical procedure was performed to stop PPH. Nine women underwent a new pregnancy after the insertion of Bakri balloon for severe PPH and 3 delivered healthy newborns. CONCLUSION: Bakri balloon tamponade is a minimally invasive intrauterine device efficient to stop severe post-partum haemorrhage. New pregnancies and deliveries are possible after tamponade by Bakri balloon.

Biomineralization of Schlumbergerella floresiana, a significant carbonate-producing benthic foraminifer.

Most foraminifera that produce a shell are efficient biomineralizers. We analyzed the calcitic shell of the large tropical benthic foraminifer Schlumbergerella floresiana. We found a suite of macromolecules containing many charged and polar amino acids and glycine that are also abundant in biomineralization proteins of other phyla. As neither genomic nor transcriptomic data are available for foraminiferal biomineralization yet, de novo-generated sequences, obtained from organic matrices submitted to ms blast database search, led to the characterization of 156 peptides. Very few homologous proteins were matched in the proteomic database, implying that the peptides are derived from unknown proteins present in the foraminiferal organic matrices. The amino acid distribution of these peptides was queried against the uniprot database and the mollusk uniprot database for comparison. The mollusks compose a well-studied phylum that yield a large variety of biomineralization proteins. These results showed that proteins extracted from S. floresiana shells contained sequences enriched with glycine, alanine, and proline, making a set of residues that provided a signature unique to foraminifera. Three of the de novo peptides exhibited sequence similarities to peptides found in proteins such as pre-collagen-P and a group of P-type ATPases including a calcium-transporting ATPase. Surprisingly, the peptide that was most similar to the collagen-like protein was a glycine-rich peptide reported from the test and spine proteome of sea urchin. The molecules, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses, included acid-soluble N-glycoproteins with its sugar moieties represented by high-mannose-type glycans and carbohydrates. Describing the nature of the proteins, and associated molecules in the skeletal structure of living foraminifera, can elucidate the biomineralization mechanisms of these major carbonate producers in marine ecosystems. As fossil foraminifera provide important paleoenvironmental and paleoclimatic information, a better understanding of biomineralization in these organisms will have far-reaching impacts.

The in vitro osteoclastic degradation of nacre.

Osteoclast activity was studied on nacre, the mother of pearl (MOP) in order to assess the plasticity of bone resorbing cells and their capacity to adapt to a biomineralized material with a different organic and mineral composition from that of its natural substrate, bone. Pure MOP, a natural biomineralized CaCO(3) material, was obtained from Pinctada oyster shell. When implanted in the living system, nacre has proven to be a sustainable bone grafting material although a limited surface degradation process. Osteoclast stem cells and mature osteoclasts were cultured on MOP substrate and osteoclast precursor cells were shown to differentiate into osteoclasts capable of resorbing nacre substrate. However, analysis of the organization of the cytoskeleton showed that both a sealing zone and a podosome structure were observed on the nacre substrate. Moreover, MOP resorption efficiency was consistently found to be lower than that of bone and appeared to be a limited process.

Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera.

Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.

Methods for obtaining neutral and acid oligosaccharides from flax pectins.

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, (1)H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.

Soluble proteins of the nacre of the giant oyster Pinctada maxima and of the abalone Haliotis tuberculata: extraction and partial analysis of nacre proteins.

Several proteins from nacre of the oyster Pinctada maxima and the abalone Haliotis tuberculata were extracted and partly characterized. Proteins dispersed in aragonite were solubilized during demineralization with acetic acid whereas proteins adsorbed on conchiolin were extracted with sodium dodecyl sulfate and beta-mercaptoethanol. The matrix of Pinctada maxima nacre is composed of one main protein with an apparent molecular weight of 20 kDa (p20). This protein was found in the acetic acid soluble fraction of nacre, as well as in the Laemmli-solubilized extract of conchiolin. In addition, the p20 solubilized with acetic acid can form oligomers made of 6 monomers linked together by disulfide bridges. The first N-terminal 21 amino acids of p20 were determined and no homology with known proteins was found. In Haliotis tuberculata nacre, 5 main proteins were solubilized during demineralization and 3 glycoproteins were detected. Stains-all and Alcian blue staining revealed polyanionic proteins in the extracts isolated from Pinctada maxima and Haliotis tuberculata nacre.

Uptake of dimannoside clusters and oligomannosides by human dendritic cells.

Knowing that human blood monocyte-derived dendritic cells express cell-surface mannose-specific lectins, we prepared various mannoses containing glycoconjugates with the aim of developing highly specific synthetic carriers of oligonucleotides and genes. Conjugates were prepared from oligosaccharides obtained by hydrazinolysis of Saccharomyces cerevisiae invertase glycopeptides. The reducing saccharides were converted into glycosynthons, i.e., into glyco-amino acids. Fluorescein derivatives were obtained by coupling the free carboxyl group of oligosaccharyl-pyroglutamate to the alpha-amino group of epsilon-fluoresceinyl-thiocarbamyl lysine methyl ester. It has been shown by others that glycosylated linear oligolysines containing up to six alpha-D-mannopyranosylphenylthiocarbamyl units have a high affinity for the human mannose receptor. In order to obtain fully biodegradable clusters and to improve both the specificity and the selectivity, disaccharides transformed into glycosynthons were coupled to pentalysine carriers (Lys5-Ala-Cys-NH2). Glycosylated pentalysyl cysteine conjugates were made fluorescent upon substitution of the cysteine thiol group with fluorescein iodoacetamide. As shown by flow cytofluorimetry, both the dimannoside clusters and yeast oligomannosides were very efficiently taken up by DC, conversely lactoside clusters were not.

Characterization and quantification of chitosan extracted from nacre of the abalone Haliotis tuberculata and the oyster Pinctada maxima.

This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (eta) of extraction was calculated (eta = 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (eta = 0.053%) was isolated rather than chitosan.

Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755.

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.

Evidence for an heterogeneous glycosylation of the Clostridium tyrobutyricum ATCC 25755 flagellin.

Glycosylation analysis of the flagellin from the Gram-positive species Clostridium tyrobutyricum has been supplemented. Amino acid analysis of the glycopeptides obtained after pronase digestion of flagellin indicated that O-glycosylation which was previously demonstrated after nonreductive beta-elimination, probably occurred via the hydroxyl group of serine. Otherwise, beta-elimination partly deglycosylated flagellin. After this treatment carbohydrates were still linked to protein as shown by a digoxigenin-hydrazide labelling. Therefore, in addition to linkages via serine, alkaline resistant linkages exist on the flagellin and some glycans may be linked to the protein core via the amide nitrogen of asparagine or via the hydroxyl group of tyrosine. Furthermore, according to an immunological analysis, glycans attached to flagellin via alkaline sensitive linkages may be different from those attached via alkaline resistant linkages.

Partial analysis of the flagellar antigenic determinant recognized by a monoclonal antibody to Clostridium tyrobutyricum.

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D-glucose and N-acetyl-glucosamine in a molar ration of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D-glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The over-all stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain alpha (1-->4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.

Biochemical and immunological analyses of the flagellin of Clostridium tyrobutyricum ATCC 25755.

The monoclonal antibody 21E7-B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and beta-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.

Monoclonal antibody detection of Clostridium microcolonies directly on membrane used for milk filtration.

The normal procedure for bacterial colony detection requires a nitrocellulose transfer step after membrane filtration and culture to prevent the development of a high background during the immunodetection. In this paper, we describe a modification of the basic protocol that omits the transfer step and reduces the risk of background. Previous observations indicated that interactions between milk components (principally cream) and membrane are responsible for the high non-specific staining observed. Experiments were performed to remove lipid components or to block the membrane binding sites before milk filtration. Samples of milks of different origin (collected at different times of the year) and different membranes were tested. The results obtained showed that removing lipids did not significantly improve the test but, on the contrary, led to an antigen diffusion. Incubation of the membrane in 0.1% (w/v) of Tween 20 in phosphate-buffered saline before milk filtration prevented non-specific binding, and allowed performance of the detection without any noticeable background.