Towards the development of a bioengineered uterus: comparison of different protocols for rat uterus decellularization.

Uterus transplantation (UTx) may be the only possible curative treatment for absolute uterine factor infertility, which affects 1 in every 500 females of fertile age. We recently presented the 6-month results from the first clinical UTx trial, describing nine live-donor procedures. This routine involves complicated surgery and requires potentially harmful immune suppression to prevent rejection. However, tissue engineering applications using biomaterials and stem cells may replace the need for a live donor, and could prevent the required immunosuppressive treatment. To investigate the basic aspects of this, we developed a novel whole-uterus scaffold design for uterus tissue engineering experiments in the rat. Decellularization was achieved by perfusion of detergents and ionic solutions. The remaining matrix and its biochemical and mechanical properties were quantitatively compared from using three different protocols. The constructs were further compared with native uterus tissue composition. Perfusion with Triton X-100/dimethyl sulfoxide/H2O led to a compact, weaker scaffold that showed evidence of a compromised matrix organization. Sodium deoxycholate/dH2O perfusion gave rise to a porous scaffold that structurally and mechanically resembled native uterus better. An innovative combination of two proteomic analyses revealed higher fibronectin and versican content in these porous scaffolds, which may explain the improved scaffold organization. Together with other important protocol-dependent differences, our results can contribute to the development of improved decellularization protocols for assorted organs. Furthermore, our study shows the first available data on decellularized whole uterus, and creates new opportunities for numerous in vitro and in vivo whole-uterus tissue engineering applications.

Abnormal expression of 17beta-hydroxysteroid dehydrogenases in breast cancer predicts late recurrence.

The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) enzymes are involved in the interconversion of biologically active and inactive sex steroids and are considered to play a critical role in the in situ metabolism of estrogen, especially in estrogen-dependent breast cancer. The gene encoding 17beta-HSD type 2 is located at 16q24.1-2, and earlier studies have shown that allelic loss in this region is an early and frequent event in breast cancer progression. Recurrence of hormone-dependent breast cancer frequently occurs several years after the primary treatment. The aim of this study was to investigate whether the expression of 17beta-HSD types 1 and 2 differs in tumors from patients with late relapses (>5 years) compared with controls without recurrence after long-term follow-up. Using real-time reverse transcription-PCR, we found that the normal mammary gland expressed both 17beta-HSD types 1 and 2, whereas the tumors frequently lacked detectable levels of type 2. Only 10% of the estrogen receptor-positive tumors expressed type 2, whereas 31% of the ER-negative tumors did so (P = 0.031). In a case-control series of 84 patients, a high level of 17beta-HSD type 1 indicated increased risk to develop late relapse of breast cancer (odds ratio, 3.0; 95% confidence interval, 1.0-12.6; P = 0.041), whereas retained expression of type 2 indicated decreased risk (odds ratio, 0.25; 95% confidence interval, 0.05-1.2; P = 0.050). In multivariate analysis of the estrogen receptor-positive patients, the absence of 17beta-HSD type 2 combined with a high expression of type 1 showed prognostic significance (P = 0.016) in addition to DNA aneuploidy (P = 0.0058), whereas progesterone receptor status did not (P = 0.71). These findings suggest that abnormal expression of 17beta-HSD isoforms has prognostic significance in breast cancer and that altered expression of these enzymes may have importance in breast cancer progression.

Helicobacter pylori and neutrophils: sialic acid-dependent binding to various isolated glycoconjugates.

Helicobacter pylori has been shown to agglutinate erythrocytes in a sialic acid-dependent manner. However, very few studies have examined relevant target cells in the human stomach. Neutrophils are required for the onset of gastritis, and the inflammatory reaction may be induced on contact between bacteria and neutrophils. In the present work, glycolipids and glycoproteins were isolated from neutrophils and were studied for binding by overlay with radiolabeled bacteria on thin-layer chromatograms and on membrane blots. There was a complex pattern of binding bands. The only practical binding activity found was sialic acid dependent, since treatment of glycoconjugates with neuraminidase or mild periodate eliminated binding. As shown before for binding to erythrocytes and other glycoconjugates, bacterial cells grown on agar bound to many glycoconjugates, while growth in broth resulted in bacteria that would bind only to polyglycosylceramides, which are highly heterogeneous and branched poly-N-acetyllactosamine-containing glycolipids. Approximately seven positive bands were found for glycoproteins, and the traditional ganglioside fraction showed a complex, slow-moving interval with very strong sialic-acid-dependent binding, probably explained by Fuc substitutions on GlcNAc.

Mass spectrometric analysis of ceramide composition in mono-, di-, tri-, and tetraglycosylceramides from mouse kidney: an experimental model for uropathogenic Escherichia coli.

Many bacteria have been shown to bind to the carbohydrate part of glycosphingolipids, but also the lipid moieties of receptor-active glycolipids are of importance. To investigate the chemistry of the ceramides of kidney glycolipids to which the uropathogenic Escherichia coli bind, different mass spectrometric techniques were utilized. First, a mixture of glycolipids isolated from man and mice kidney was separated by thin-layer chromatography (TLC) and scanned by direct desorption from the plate by fast atom bombardment mass spectrometry (TLC/FAB-MS). Second, the glycolipids were purified by preparative TLC and analyzed by negative-ion FAB-MS. After methylation, further analyses were made with positive-ion FAB-MS, positive-ion electron ionization (EI)-MS, high-temperature capillary gas chromatography (GC/EI-MS) and positive-ion matrix-assisted laser desorption/ionization (MALDI)-MS. The ceramide compositions of the four glycolipids were determined using all these MS techniques and the reliability of the different methods for this type of analyses is discussed. Comparison of the mouse kidney glycolipids with the corresponding glycolipids from human kidney showed the same degree of hydroxylation of ceramides among mono- and disaccharide glycolipids, but a significantly higher degree of hydroxylation among mouse kidney glycolipids with three and four sugar residues. This result might be of relevance for the binding of P-fimbriated E. coli to the urinary tract tissues.

Glycosphingolipid binding specificities of Neisseria meningitidis and Haemophilus influenzae: detection, isolation, and characterization of a binding-active glycosphingolipid from human oropharyngeal epithelium.

The glycosphingolipid binding specificities of Haemophilus influenzae and Neisseria meningitidis were investigated as to the binding of radiolabeled bacteria to glycosphingolipids on thin-layer chromatograms. Thereby, similar binding profiles, for the binding of the two bacteria to lactosylceramide, isoglobotriaosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, lactotetraosylceramide, neolactotetraosylceramide, and sialylneolactohexaosylceramide, were obtained. On a closer view the binding preferences of the bacteria could be differentiated into three groups. The first specificity is recognition of lactosylceramide. The second specificity is binding to gangliotriaosylceramide and gangliotetraosylceramide, since conversion of the acetamido group of the N-acetylgalactosamine of gangliotriaosylceramide and gangliotetraosylceramide to an amine prevented the binding of the bacteria, and thus the binding to these two glycosphingolipids represents a separate specificity from lactosylceramide recognition. Preincubation of H. influenzae with neolactotetraose inhibited the binding to neolactotetraosylceramide, while the binding to lactosylceramide, gangliotetraosylceramide, or lactotetraosylceramide was unaffected. Thus, the third binding specificity is represented by neolactotetraosylceramide, and involves recognition of other neolacto series glycosphingolipids with linear N-acetyllactosamine chains, such as sialyl-neolactohexaosylceramide. The relevance of the detected binding specificities for adhesion to target cells was addressed as to the binding of the bacteria to glycosphingolipids from human granulocytes, epithelial cells of human nasopharyngeal tonsils and human plexus choroideus. Binding-active neolactotetraosylceramide was thereby detected in human granulocytes and the oropharyngeal epithelium.

The lactosylceramide binding specificity of Helicobacter pylori.

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

Glycoconjugate receptors for P-fimbriated Escherichia coli in the mouse. An animal model of urinary tract infection.

Glycosphingolipids were isolated from kidneys, urethers, and bladders (including urethrae) of C3H/HeN mice. Binding was studied of a clinical isolate and recombinant strains of uropathogenic P-fimbriated Escherichia coli to these glycolipids. A series of receptor-active glycolipids with Gal alpha 4Gal in common, previously shown to be recognized by these bacteria, was identified by use of specific monoclonal antibodies, fast-atom bombardment and electron-impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy: galabiosylceramide (Gal alpha 4Gal beta Cer), globotriaosylceramide (Gal alpha 4Gal beta 4Glc beta Cer), globoside (GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), the Forssman glycolipid (GalNAc alpha 3GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer), Gal beta 4GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer, and Gal beta 4(Fuc alpha 3)GlcNAc beta 6(Gal beta 3)GalNAc beta 3Gal alpha 4Gal beta 4Glc beta Cer. The binding pattern for mouse kidney glycolipids differed from that for kidney glycolipids of man and monkey. In particular, the dominant 8-sugar glycolipid in the mouse was not detected in the primates. A second difference was found in the binding of E. coli to kidney glycoproteins on blots after electrophoresis; the mouse showed distinct receptor-active bands while human and monkey did not. These differences may be of relevance when using the mouse as a model for clinical urinary tract infection of man.