Dentin slice model of dental stem cells in a fibrin-agarose construct for dental pulp regeneration / Modelo de trozo de dentina de células madre dentales en una construcción de fibrina-agarosa para la regeneración de la pulpa dental

Objectives: To implement a dentin slice model of mesenchymal stem cells derived from dental tissues in a fibrin-agarose construct for dental pulp regeneration. Material and Methods: MSCs derived from different oral cavity tissues were combined with a fibrin-agarose construct at standard culture conditions. Cell viability and proliferation tests were assayed using a fluorescent cell dye Calcein/Am and WST-1 kit. The proliferation assay was evaluated at 24, 48, 72, and 96 hours. Also, we assessed the dental pulp stem cells (DPSCs) cell morphology inside the construct with histological stains such as Hematoxylin and Eosin, Masson's trichrome, and Periodic acid­Schiff. In addition, we elaborated a tooth dentin slice model using a culture of DPSC in the fibrin­agarose constructs co-adhered to dentin walls. Results: The fibrin-agarose construct was a biocompatible material for MSCs derived from dental tissues. It provided good conditions for MSCs' viability and proliferation. DPSCs proliferated better than the other MSCs, but the data did not show significant differences. The morphology of DPSCs inside the construct was like free cells. The dentin slice model was suitable for DPSCs in the fibrin-agarose construct. Conclusion: Our findings support the dentin slice model for future biological use of fibrin-agarose matrix in combination with DPSCs and their potential use in dental regeneration. The multipotency, high proliferation rates, and easy obtaining of the DPSCs make them an attractive source of MSCs for tissue regeneration.

Objetivos: Implementar un modelo de dentina con células madre mesenquimales derivadas de tejidos dentales en una constructo de fibrina-agarosa para la regeneración de la pulpa dental. Material y Métodos: Las MSC derivadas de diferentes tejidos de la cavidad oral se combinaron con una construcción de fibrina-agarosa en condiciones de cultivo estándar. Las pruebas de viabilidad y proliferación celular se ensayaron utilizando un kit de colorante celular fluorescente Calcein/Am y WST-1. El ensayo de proliferación se evaluó a las 24, 48, 72 y 96 horas. Además, evaluamos la morfología celular de las células madre de la pulpa dental (DPSC) dentro de la construcción con tinciones histológicas como hematoxilina y eosina, tricrómico de Masson y ácido peryódico de Schiff. Además, elaboramos un modelo de rebanadas de dentina dental utilizando un cultivo de DPSC en las construcciones de fibrina-agarosa coadheridas a las paredes de la dentina. Resultados: La construcción de fibrina-agarosa fue un material biocompatible para las MSC derivadas de tejidos dentales. Proporcionó buenas condiciones para la viabilidad y proliferación de las MSC. Las DPSC proliferaron mejor que las otras MSC, pero los datos no mostraron diferencias significativas. La morfología de las DPSC dentro de la construcción era como la de las células libres. El modelo de corte de dentina fue adecuado para DPSC en la construcción de fibrina-agarosa.Conclusión: Nuestros hallazgos respaldan el modelo de corte de dentina para el futuro uso biológico de la matriz de fibrina-agarosa en combinación con DPSC y su uso potencial en la regeneración dental. El multipotencial, las altas tasas de proliferación y la fácil obtención de las DPSC las convierten en una fuente atractiva de MSC para la regeneración de tejidos.

Amino Acid Supplementation Improves the Production of Extracellular Peptidases by Aspergillus Section Flavi and their Ionic Immobilization

Abstract Bioprocess studies have been highlighted due to the importance of physiological processes and industrial applications of enzymes. The potential of peptidase production from Aspergillus section Flavi using different amino acids as a supplemental nitrogen source was investigated. A production profile revealed that amino acids had positive effects on peptidase production when compared to the control without amino acids. Optimal production (100 U/mL) was obtained with Arginine amino acid in 96 h of fermentation. Extracellular peptidase from Aspergillus section Flavi was identified in submerged bioprocesses by in situ activity. Biochemical studies revealed that the maximum activities of the enzyme extract were obtained at pH 6.5 and a temperature of 55°C. The inhibition by EDTA and PMSF suggests the presence of more than one peptidase while the Ni2+ and Cu2+ had a negative influence on the enzyme activity. When the crude extract was reversibly immobilized on ionic supports, DEAE-Agarose and MANAE-Agarose the derivative showed different profiles of thermal and pH stabilities. Hence, this study revealed the basic properties and biochemical characteristics that allowed the production improvement of this class of enzyme. Moreover, with known properties stabilization and immobilization process is required to further explore its biotechnological capacities.

Non-small cell lung cancer 95D cells co-cultured with 3D-bioprinted scaffold to construct a lung cancer model in vitro / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To fabricate an innovative scaffold for lung cancer cell culture and establish a three-dimensional lung cancer model in vitro, and to reveal the differences in biological functions of lung cancer cells under the two-dimensional and three-dimensional culture conditions.</p><p><b>METHODS</b>We chose agarose and alginate as the scaffold materials, and 3D printing technique was applied to construct cell culture scaffold. 95D cells were co-cultured with this scaffold. The differences of cell morphology, proliferation ability, protein expression, etc. in the cells cultured under 2D and 3D cultural conditions were evaluated by light microscopy using HE staining, MTT assay, scanning electron microscopy, and Western blot analysis.</p><p><b>RESULTS</b>Cells cultured in 2D wells displayed a spindle and polygonal morphology, whereas those grown in the 3D culture aggregated into spheroids, which invaded, migrated and disseminated into the surrounding scaffold. MTT assay showed that the proliferation rates of the 3D-cultured cells for 2-6 days were significantly lower than, but those cultured for 8-9 days were significantly higher than that of the 2D-cultured cells, indicating that proliferative activity of the cells grown in 2D cultures for 8-9 days was inhibited. In contrast, cells grown on 3D scaffolds still maintained a higher proliferation. The Western blot assay showed that the expression of Cdc42, p53, mTOR were significantly down-regulated in 3D scaffold-cultured group (0.529±0.103, 0.820±0.038 vs. 1.967±0.066), compared with that of the 2D-cultured group (3.063±0.139, 1.738±0.122 vs. 2.472±0.151) (P<0.05 for all), while the expression of MMP-2 was up-regulated in the 3D-cultured cells (1.110±0.029), significantly higher than that of the 2D-cultured cells (0.017±0.001) (P<0.05).</p><p><b>CONCLUSIONS</b>The cell morphology, proliferation and associated protein expression of lung cancer cells in 3D-culture systems are distinctively different as compared to those of the 2D-cultural cells. 3D-bioprinted agarose-alginate scaffold can better mimic the growth microenvironment of lung cancer in vivo and may provide a promising model for lung cancer research in vitro.</p>

Preparation of Compact Agarose Cell Blocks from the Residues of Liquid-Based Cytology Samples

BACKGROUND: Inevitable loss of diagnostic material should be minimized during cell block preparation. We introduce a modified agarose cell block technique that enables the synthesis of compact cell blocks by using the entirety of a cell pellet without the loss of diagnostic material during cell block preparations. The feasibility of this technique is illustrated by high-throughput immunocytochemistry using high-density cell block microarray (CMA). METHODS: The cell pellets of Sure- Path residues were pre-embedded in ultra-low gelling temperature agarose gel and re-embedded in standard agarose gel. They were fixed, processed, and embedded in paraffin using the same method as tissue sample processing. The resulting agarose cell blocks were trimmed and represented on a CMA for high-throughput analysis using immunocytochemical staining. RESULTS: The SurePath residues were effectively and entirely incorporated into compact agarose cell buttons and embedded in paraffin. Sections of the agarose cell blocks revealed cellularities that correlated well with corresponding SurePath smears and had immunocytochemical features that were sufficient for diagnosis of difficult cases. CONCLUSIONS: This agarose-based compact cell block technique enables preparation of high-quality cell blocks by using up the residual SurePath samples without loss of diagnostic material during cell block preparation.

Producción de bloques de agarosa para visualización de hongos / Agarose block preparation for fungi visualization

Establecer un protocolo para la obtención de bloques de hongos utilizando agarosa como matriz. Métodos: los hongos se incluyeron en agarosa, el proceso se estandarizó y se probaron los protocolos en horno microondas, convencional y en el procesador automático para la obtención de láminas siguiendo la técnica histológica usual. Conclusiones: el protocolo del procesador para obtener múltiples láminas es reproducible y las preparaciones permitieron la visualización fácil de los hongos con coloraciones de H&E y Gomori...

To establish a protocol to obtain fungi blocks in an agarose matrix. Methods: fungi were embedded in agarose, the process was standardized and protocols were tested using a microwave oven, a conventional oven and an automated processor to obtain slides following the usual histological technique. Conclusions: the multiple slides processor protocol is reproducible and preparations allowed easy visualization of fungi with H&E and Gomori stains. Key words: agarose cell block technique, fungi, hematoxylin and eosin, Gomori...

In-house Manual Construction of High-Density and High-Quality Tissue Microarrays by Using Homemade Recipient Agarose-Paraffin Blocks

BACKGROUND: Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. METHODS: Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. RESULTS: The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. CONCLUSIONS: Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks.

Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05). The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05), indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

The ferric aerobactin receptor IutA, a protein isolated on agarose column, is not essential for uropathogenic Escherichia coli infection / O receptor de aerobactina IutA, uma proteína isolada em coluna de agarose, não é essencial para a infecção por Escherichia coli uropatogênica / El receptor de aerobactina IutA, una proteína aislada en columna de agarosa, no es esencial para la infección por Escherichia coli uropatógena

Although many proteins have been described involved in Escherichia coli colonization and infection, only few reports have shown lectins as important components in these processes. Because the mechanisms underlying E. coli colonization process involving lectins are not fully understood, we sought to identify the presence of other non-described lectins in E. coli. Here, we isolated a 75-kDa protein from E. coli on Sepharose column and identified it as ferric aerobactin receptor (IutA). Since IutA is controversially associated with virulence of some E. coli strains, mainly in uropathogenic E. coli (UPEC), we evaluated the presence of iutA gene in UPEC isolated from patients with urinary infection. This gene was present in only 38% of the isolates, suggesting a weak association with virulence. Because there is a redundancy in the siderophore-mediated uptake systems, we suggest that IutA can be advantageous but not essential for UPEC.

Apenas alguns relatos na literatura demonstram que lectinas são importantes nos processos de colonização e infecção por Escherichia coli. A falta de compreensão clara dos mecanismos envolvendo lectinas, no processo de colonização por E. coli, motivou a realização deste estudo para se identificar a presença de outras lectinas não descritas em E. coli. Neste trabalho, isolou-se uma proteína de 75kDa de E. coli em coluna de Sepharose, correspondente ao receptor de aerobactina férrica (IutA). A associação de IutA com virulência de cepas de E. coli é controversa, principalmente em E. coli uropatogênica (UPEC), o que levou a se avaliar a presença do gene iutA em UPECs isoladas de pacientes com infecção urinária. O gene estava presente em 38% dos isolados, sugerindo fraca associação com virulência. Devido à existência de redundância nos sistemas de captura de ferro, sugere-se, aqui, que IutA possa ser vantajosa, mas não essencial para UPEC.

La falta de una clara comprensión de los mecanismos de participación de las lectinas en el proceso de colonización por Escherichia coli, nos motivó a identificar la presencia de otras lectinas que no han sido descritas en E. coli. En este estudio, se aisló una proteína de 75kDa de E. coli en una columna de Sepharosa, correspondiente al receptor de aerobactina (IutA). La asociación de IutA con cepas virulentas de E coli es controvertido, especialmente en E. coli uropatógena (UPEC), lo que nos llevó a evaluar la presencia del gen iutA en UPECs aisladas de pacientes con infección urinaria. El gen estaba presente en 38% de los aislamientos, lo que sugiere una débil asociación con la virulencia. Debido a la existencia de redundancia en los sistemas de captura de hierro, se sugiere que IutA puede ser una ventaja, sin embargo no es esencial para la UPEC.

Purification and characterization of phytase with a wide pH adaptation from common edible mushroom Volvariella volvacea (Straw mushroom).

A novel phytase with a molecular mass of 14 kDa was isolated from fresh fruiting bodies of the common edible mushroom Volvariella volvacea (Straw mushroom). The isolation procedure involved successive chromatography on DEAE-cellulose, CM-cellulose, Affi-gel blue gel, Q-Sepharose and Superdex-75. The enzyme was a monomeric protein and was unadsorbed on DEAE-cellulose, CM-cellulose and Affi-gel blue gel, but was adsorbed on Q-Sepharose. The enzyme was purified 51.6-fold from the crude extract with 25.9% yield. Its N-terminal amino acid sequence GEDNEHDTQA exhibited low homology to the other reported phytases. The optimal pH and temperature of the purified enzyme was 5 and 45oC, respectively. The enzyme was quite stable over the pH range of 3.0 to 9.0 with less than 30% change in its activity, suggesting that it can be used in a very wide pH range. The enzyme exhibited broad substrate selectivity towards various phosphorylated compounds, but lacked antifungal activity against tested plant pathogens.

Functional analysis of glycin rich- RNA binding protein, a suppressor of trehalose-6-phosphate mediating growth arrest in Arabidopsis thaliana

Metabolism of the alpha-1,1 glucose disaccharide, trehalose, is indispensable in plants. In the Murashige and Skoog [MS] medium, trehalose inhibits plant growth and allocation of carbon to roots. A suppressor of trehalose-6-phosphate [T6P] mediated growth arrest, GR-RBP2, is characterized in more detail. Phylogenetic analysis revealed that GR-RBP2 is a protein of likely prokaryotic origin. A knockout mutant of GR-RBP2 was identified in the T-DNA insertion line SALK-059714, yet plants of this line were not altered with regard to growth on different carbon sources and on trehalose compared to WT. GUS expression analysis showed that GR-RBP2 was detected in adult leaves, flowers and siliques. Expression was particularly high in root tips. GR-RBP2 expression also is insensitive to 100 mM trehalose. TAP-tagged versions of this protein showed that GR-RBP2 is part of a protein complex in planta

Association of Alanine-Valine Manganese Superoxide Dismutase Gene Polymorphism and Microheterogeneity Manganese Superoxide Dismutase Activity in Breast Cancer and Benign Breast Tissue / 한국유방암학회지

PURPOSE: Although the etiology of breast cancer is multifactorial, oxidative stress plays an important role in carcinogenesis. In this study, manganese superoxide dismutase (MnSOD) gene polymorphism and activity were evaluated in benign and breast cancer tissue. METHODS: One hundred and one females were enrolled in this study, 65 who were histopathologically diagnosed with breast cancer and 46 who were benign patients. MnSOD enzyme activity was determined using an indirect competitive inhibition assay and MnSOD gene polymorphism using poly merase chain reaction and agarose gel electrophoresis. RESULTS: MnSOD enzymatic activity (79.83+/-42.14) was lower in breast cancer tissue compared to benign tumors (236.18+/-46.37). At the same time, MnSOD enzymatic activity among Ala/Val patients was significantly lower in breast cancer tissue (39.19+/-7.33) than in Val/Val malignant breast tumors tissue (96.9+/-22.9). MnSOD enzymatic activity was significantly lower in Val/Val cancer tissue (96.9+/-22.9) than in benign tissue (255.44+/-42.7). CONCLUSION: Breast cancer tumors contain less MnSOD than benign breast samples. Patients with Ala/Val polymorphism had reduced MnSOD activity compared to patients with Val/Val breast cancer. Ala/Val gene polymorphism may be a risk factor associated with more advanced breast cancer stage. MnSOD gene polymorphism Ala/Val may be a risk factor associated with more advanced breast cancer stage, and reduction of MnSOD activity may be a mechanism of the progression of benign to malignant tumors. Further investigations are needed to evaluate the role of MnSOD in breast cancer progression.

The voice test for newborn hearing screening

OBJECTIVES. The goal of the study is to find a reasonable aIternative test that can be utilized in the Philippine setting to operationalize the Universal Newborn Hearing Screening Act. Thus the components of the Voice Test were studied. The objectives of the study are to determine: (1) which of the two words "Baah" and "Psst" is better for newborn hearing screening rocedure as far as their physical characteristics are concerned, ~) how do the two words "Baah" and Psst" differ between genders and distance from sound source, (3) to determine the proportion of the participants who could recite the words at intensity of 80db or louder. METHODS. Frequency characteristics and sound intensity differences of two words "Baah" and "Psst" were determined and ompared. RESULTS. The word "Baah" exhibited more favorable physical attributes over the word 'Psst" for purposes of being a screening tool for newborn hearing assessment. CONCLUSION. This study reports the results of an initial step in the search for an inexpensive, feasible, and valid tool for neonatal earing screening. Correlation studies with speech developmental milestones may eventually enhance the usefulness of the voice test.

Padronização da técnica de eletroforese de isoenzimas para certificação de linhagens celulares / Standardization of isoenzymes electrophoresis technique for cell lines certification

A comprovação da espécie de origem é um dos itens da certificação de linhagens celulares, que pode ser feita por meio de técnica de eletroforese de isoenzimas. Com o objetivo de padronizar essa técnica, o grupo de estudo do Núcleo de Cultura de Células do IAL utilizou extratos celulares de diferentes espécies animais, que foram submetidos à eletroforese horizontal ou vertical em géis de poliacrilamida ou agarose, 100 V e4 oC. A revelação das bandas para a enzima glicose 6-fosfato desidrogenase foi realizada com o auxílio de sais de tetrazólio. Observou-se a presença de uma única banda para cada linhagem celular testada, com os padrões de migração esperados para as diferentes espécies utilizadas, com exceção da linhagem bovina MDBK, que não apresentou uma das bandas, provavelmente em função de menor expressão dessa enzima. Após a avaliação dos resultados obtidos com os diferentes sistemas de eletroforese e géis testados, optou-se pelo uso de eletroforese horizontal em géis de agarose a 2. A padronização e a implantação dessa técnica permitirão que o laboratório forneça linhagens celulares com o devido controle de qualidade.

Modification of seaweed polysaccharide-agarose and its application as skin dressing (III)--skin regeneration with agarose grafting hyaluronic acid sponge / 生物医学工程学杂志

In this paper, a kind of skin dressing, agarose- grafting- hyaluronic acid (Ag-g-HA) sponge was applied to test the modified agarose based scaffold for skin regeneration. The bFGF loading agarose-grafting hyaluronan scaffold had homogenous porosities, and the loaded bFGF was bioactive in 2 weeks. The Ag-g-HA sponge was applied into skin of mice, and it was found that the dressing promoted skin regeneration and no infection and leakage in lesion site took place. H&E staining results showed that the repaired skin was similar to autologous skin. These demonstrate that Ag-g-HA sponge has a promise in skin regeneration.

Simple and Novel Three Dimensional Neuronal Cell Culture Using a Micro Mesh Scaffold

Conventional method of cell culture studies has been performed on two-dimensional substrates. Recently, three-dimensional (3D) cell culture platforms have been a subject of interest as cells in 3D has significant differences in cell differentiation and behavior. Here we report a novel approach of 3D cell culture using a nylon micro mesh (NMM) as a cell culture scaffold. NMM is commonly used in cell culture laboratory, which eliminates the requirement of special technicality for biological laboratories. Furthermore, it is made of a micro-meter thick nylon fibers, which was adequate to engineer in cellular scales. We demonstrate the feasibility of the NMM as a 3D scaffold using E18 rat hippocampal neurons. NMM could be coated with cell adhesive coatings (polylysine or polyelectrolyte) and neurons showed good viability. Cells were also encapsulated in an agarose hydrogel and cultured in 3D using NMM. In addition, the 3D pattern of NMM could be used as a guidance cue for neurite outgrowth. The flexible and elastic properties of NMMs made it easier to handle the scaffold and also readily applicable for large-scale tissue engineering applications.

Fractionation of DNases Specific to Haemonchus contortus Intestine by Phenyl Sepharose Column / 한국실험동물학회지

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.

Fractionation of DNases Specific to Haemonchus contortus Intestine by Phenyl Sepharose Column / 한국실험동물학회지

Multiple DNases were identified from Haemonchus contortus intestine based on previous studies. The DNases detected at 34, 36 and 38.5 kDa had diverse characteristics. Some of them had characteristics similar to those of mammalians and others had unusual characteristics. This study was carried out to fractionate worm intestinal DNases from other proteins using phenyl Sepharose chromatographic methods. All DNases detected from Haemonchus contortus intestine were fractionated in the flowthrough of phenyl Sepharose, indicating the worm DNases are hydrophilic. The DNases were enriched five-fold in the flowthrough fraction while additional steps are required for isolation of the worm DNases. Thus, fractionation with phenyl Sepharose could be used as a good initial step to enrich and separate DNases from other proteins.

Magnolol Inhibits LPS-induced NF-kappaB/Rel Activation by Blocking p38 Kinase in Murine Macrophages

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-kappaB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-kappaB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-kappaB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-kappaB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-kappaB/Rel and p38 kinase signaling.

Molecular imaging of thrombus with microbubbles targeted to alphavbeta3-integrin using an agarose flow chamber model / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound.</p><p><b>METHODS</b>Targeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images.</p><p><b>RESULTS</b>The VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05).</p><p><b>CONCLUSION</b>MBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.</p>

Padronização de dosagem de produto de degradação fibrinogênio/fibrina humano / Human fibrin/fibrinogen degradation product dosage standardization

A hemostasia contribui para integridade e fluidez sanguinea. Após ativação de plaquetas, fatores da coagulação e células endoteliais ocorrem reações enzimáticas gerando trombina e fibrina, principal constituinte do trombo. A plasmina remove o coágulo degradando a fibrina em produtos solúveis (sistema fibrinolítico) e fragmentados (Produtos de Degradação da Fibrina - PDF) onde o Dímero D é o mais estável. Por isso torna-se um importante indicador do risco vascular. Realizaram-se 2 protocolos: LAMB F (Fibrinogênio) e Lamb D (Fragmentado D) produzindo 2786 hibridos com atividade anti PDF. Os hibridos selecionados foram submetidos à técnica de immunoblotting onde foram utilizadas as fontes de antigenos diferentes (Fib comercial, Fib purificado in house, Fragmento D, líquido de drenagem rico em PDF. Com base no reconhecimento das frações protéicas, selecionou-se 2 híbridos (LAMB F1 - 120 e LAMB D3-6), que foram clonados e expandidos em cultura (in vitro) e ascite (in vivo) para obtenção de altas concentrações de AcMm. O líquido ascítico foi purificado (coluna de afinidade)e os anticorpos acoplados em esferas (beads) de agarose. Testes qualitativos foram realizados em paralelo com o produto comercial (Dimer Test Dade Behring) utilizando 20 amostras de pacientes (15 PDF normal e 05 PDF alterado). Os resultados mostraram 100% de concordância entre o produto comercial e os anticorpos anti-PDF produzido no laboratório.