Recruitment of monocytes and mature macrophages in mouse pubic symphysis relaxation during pregnancy and postpartum recovery†.

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages in mouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy.

Characterization of a novel decellularized bone marrow scaffold as an inductive environment for hematopoietic stem cells.

Due to the increasing demand for a bone marrow study model, we developed a natural scaffold from decellularized bovine bone marrow (DeBM). The obtained bioscaffold was analyzed after the decellularization process; histological staining, scanning and transmission electron microscopy confirmed the preservation of its native 3D-architecture; including blood vessels and cell niches as well as the integrity of important components of the extracellular matrix; Collagen III, IV and fibronectin. In addition to biochemical composition, physical properties of the bone marrow were also conserved. We evaluated the suitability of this bio-scaffold as a tridimensional culture platform. Seeding experiments with umbilical cord-derived hematopoietic stem cells and human bone marrow stromal cell line HS5 demonstrated that this scaffold is capable of supporting hematopoietic and stromal cell adhesion and proliferation without the need of exogenous factors. DeBM provided an inductive environment for the repopulation of the bone marrow inducing the expression of SDF-1, HGF and SCF by seeded stromal cells. The presence of these potent hematopoietic chemoattractants would be crucial for ex vivo long-term culture of HSCs, and for recreating the natural microenvironment of the bone marrow for bioengineering applications. We conclude that the decellularization process succeeded in preserving the 3D structure and mechanical properties of the bone marrow. The resulting scaffold is suitable for cell culture, representing an advantageous bone marrow experimental model, and potentially an effective platform for CD34+ HSC expansion and differentiation for clinical applications.

Growth factor stimulation improves the structure and properties of scaffold-free engineered auricular cartilage constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-ß1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.

Development of scaffold-free elastic cartilaginous constructs with structural similarities to auricular cartilage.

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

Temporal changes in matrix metalloproteinases, their inhibitors, and cathepsins in mouse pubic symphysis during pregnancy and postpartum.

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The involvement of endogenous proteins such as matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and cathepsins in these phenomena remains unclear. In this work, we used a combination of immunolocalization, protein expression/activity, and relative messenger RNA (mRNA) expression to examine the changes in selected MMPs (-2, -9, and -8), TIMPs (-1 and -2), and cathepsins (B and K) during pregnancy and postpartum in mice. Immunohistochemistry revealed the presence of all of these proteins in the cytoplasm of chondrocytes, fibrochondrocytes, and fibroblast-like cells in the interpubic tissues. Zymography showed increases in the active forms of MMP-2 and -9 primarily on days 15 to 19 of pregnancy. Western blotting showed enhanced expression of MMP-8 on days 12 to 15 of pregnancy, with no changes in cathepsins B and K. Matrix metalloproteinases 2, TIMP-1 and -2, and cathepsin B had significant relative gene expression throughout pregnancy. These findings indicate that during pregnancy and postpartum there are variations in the expression and activity of proteins that may have an important role in remodeling the pubic symphysis during these events.

Histological and functional renal alterations caused by Bothrops alternatus snake venom: expression and activity of Na+/K+-ATPase.

BACKGROUND: Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na+/K+-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na+/K+-ATPase expression and activity in rats injected with Bothrops alternatus snake venom. METHODS: Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na+/K+-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively. RESULTS: Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na+/K+-ATPase α1 subunit were increased 6h post-venom, whereas Na+/K+-ATPase activity increased 6 h and 24 h post-venom. CONCLUSIONS: Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na+/K+-ATPase expression and activity in the early phase of renal damage. GENERAL SIGNIFICANCE: Enhanced Na+/K+-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.

ARHGAP21 associates with FAK and PKCzeta and is redistributed after cardiac pressure overload.

ARHGAP21 is highly expressed in the heart, which demonstrates activity over Cdc42 and interacts with proteins of the cytoskeleton and adherent junctions. The main cause of cardiac hypertrophy is mechanical stimulus; therefore we analyzed ARHGAP21 expression after acute mechanical stress in the myocardium and its association with FAK and PKCzeta. We demonstrated that ARHGAP21 is relocated to Z-lines and costameres after pressure overload, and interacts with PKCzeta and FAK in control rats (sham), rats submitted to aortic clamping and spontaneously hypertensive rats (SHR). Co-transfection using ARHGAP21 and PKCzeta constructions demonstrated that ARHGAP21 associates with PKCzeta-GST and endogenous FAK. Pulldown assay showed that ARHGAP21 binds to the C-terminal region of FAK. Moreover, ARHGAP21 binds to PKCzeta phosphorylated on Thr410 in sham and SHR. However, ARHGAP21 only binds to FAK phosphorylated on Tyr925 of SHR. Additionally, PKCzeta is phosphorylated by mechanical stimuli. These results suggest that ARHGAP21 may act as a signaling or scaffold protein of FAK and PKCzeta signaling pathways, developing an important function during cardiac stress.

Hyaluronan involvement in the changes of mouse interpubic tissue during late pregnancy and post-partum.

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th-18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-14.6 microg/mg dry weight). This was followed by a decrease beginning on D19 (12.4 microg/mg), reaching close to virgin levels (2.2 microg/mg) on the 5th day post-partum. The same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. In addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition.

Increased response of Vero cells to PHBV matrices treated by plasma.

The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is known that poly 3-hydroxybutyric acids (PHBs) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materials and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120 h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment decreased the contact angle and increased the surface roughness; it also changed surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. SEM analysis of Vero cells adhered to PHBV treated by plasma showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare an appropriate surface for tissue engineering application.

Cytotoxicity of Lachesis muta muta snake (bushmaster) venom and its purified basic phospholipase A2 (LmTX-I) in cultured cells.

Human envenoming by Lachesis muta muta venom, although infrequent, is rather severe, being characterized by pronounced local tissue damage and systemic dysfunctions. Studies on the pharmacological actions of L. m. muta venom are relatively scant and the direct actions of the crude venom and its purified phospholipase A(2) (PLA(2)) have not been addressed using in vitro models. In this work, we investigated the cytotoxicity of L. m. muta venom and its purified PLA(2) isoform LmTX-I in cultured Madin-Darby canine kidney (MDCK) and in a skeletal muscle (C2C12) cell lines. As revealed by neutral red dye uptake assay, the crude venom (10 or 100 microg/ml) induced a significant decrease in cell viability of MDCK cells. LmTX-I at the concentrations tested (70-270 microg/ml or 5-20 microM) displayed no cytotoxicity in both MDCK and C2C12 cell lines. Morphometric analysis of Feulgen nuclear reaction revealed a significant increase in chromatin condensation (pyknosis), apparent reduction in the number of mitotic nuclei and nuclear fragmentation of some MDCK cells after incubation with L. m. muta venom. Monolayer exposure to crude venom resulted in morphological changes as assessed by scanning electron microscopy. The staining with TRITC-labelled phalloidin showed a marked disarray of the actin stress fiber following L. m. muta venom exposure. In contrast, LmTX-I had no effect on nucleus and cell morphologies as well as on stress fiber organization. These results indicate that L. m. muta venom exerts toxic effects on cultured MDCK cells. The LmTX-I probably does not contribute per se to the direct venom cytotoxicity, these effects are mediated by metalloproteinases/disintegrins and other components of the venom.

An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis.

Acquired somatic mutations in exon 2 of the hematopoietic transcription factor GATA-1 have been found in individuals with Down syndrome with both transient myeloproliferative disorder and acute megakaryoblastic leukemia. These mutations prevent the synthesis of the full-length protein but allow the synthesis of its short isoform, GATA-1s. Experiments in mice suggest that GATA-1s supports normal adult megakaryopoiesis, platelet formation and erythropoiesis. Here we report a mutation, 332G --> C, in exon 2 of GATA1, leading to the synthesis of only the short isoform in seven affected males from two generations of a family. Hematological profiles of affected males demonstrate macrocytic anemia, normal platelet counts and neutropenia in most cases. Altogether, data suggest that GATA-1s alone, produced in low or normal levels, is not sufficient to support normal erythropoiesis. Moreover, this is the first study to indicate that a germline splicing mutation does not lead to leukemia in the absence of other cooperating events, such as Down syndrome.

Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom.

A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.

Biological and structural characterization of a new PLA2 from the Crotalus durissus collilineatus venom.

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A2 (PLA2) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA2s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA2s such as from the Bothrops snake venom. Also, this PLA2 induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and p-BPB showed a similar reduction. The p-BPB did not reduce significantly the myotoxic activity induced by the PLA2, but the anhydrous acetic acid treatment and the pre-incubation of PLA2 with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiforae (Gram-negative), which was drastically reduced by incubation of this PLA2 with p-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or beta-wing region may be involved in the binding of this PLA2 to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Identification and characterization of an antigen recognized by monoclonal antibody TRA 54 in mouse epididymis and vas deferens.

Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.

Ultrastructural and immunohistochemical analysis of proteoglycans in mouse pubic symphysis.

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase. The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.

Hemagglutinating factor (HAF) associated with adhesiveness in enteroinvasive Escherichia coli (EIEC).

A cell-associated mannose-resistant hemagglutinating factor (HAF) was extracted from enteroinvasive Escherichia coli (EIEC) serotype O124:H- by sonication. Ultrastructural analysis of EIEC and immunocytochemical assays with the cell-free HAF and EIEC bacterial cells on HeLa cells, suggested that the HAF is a non-fimbrial putative adhesive factor that mediates in vivo adherence of EIEC to human epithelial cells.

Diagnóstico rápido de vírus por microscopia eletrônica / Rapid virus diagnosis by electron microscopy

A Seçäo de Microscopia Eletrônica(ME) do Instituto Adolfo Lutz(IAL) realiza diagnósticos rápidos de: Herpesvírus, Poxvírus, Papilomavírus, Rotavírus, Adenovírus, Calcivírus, "Small Round Vírus Particles-SRVP", agente de Norwalk, Parvovírus e bacteriófagos em diversas amostras biológicas. Contribui também no controle de qualidade de antígenos virais como Sarampo, Influenza, Rocio, Dengue, Hepatite A e B, HIV e outros. A coloraçäo negativa é uma das técnicas utilizadas no diagnóstico rápido de vários vírus associados às doenças humanas. A morfologia, a subestrutura e o diâmetro säo características importantes para diferenciar esses vírus. Apesar da impossibilidade de se realizar exames em larga escala, do alto custo e da necessidade de técnicos especializados, é uma técnica de fácil execuçäo, com obtençäo rápida do resultado. É útil para detectar vírus de difícil cultivo e é o único método que permite observar mais de um agente numa mesma amostra. Embora existam vários laboratórios de ME em Universidades e em Instituiçöes de Pesquisa, cada laboratório está empenhado em sua linha de pesquisa e säo poucos os que se dedicam à área de diagnóstico rápido de vírus

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay: ultrastructural aspects

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, wich corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories