Elastic three-dimensional poly (ε-caprolactone) nanofibre scaffold enhances migration, proliferation and osteogenic differentiation of mesenchymal stem cells.

OBJECTIVES: We prepared 3D poly (ε-caprolactone) (PCL) nanofibre scaffolds and tested their use for seeding, proliferation, differentiation and migration of mesenchymal stem cell (MSCs). MATERIALS AND METHODS: 3D nanofibres were prepared using a special collector for common electrospinning; simultaneously, a 2D PCL nanofibre layer was prepared using a classic plain collector. Both scaffolds were seeded with MSCs and biologically tested. MSC adhesion, migration, proliferation and osteogenic differentiation were investigated. RESULTS: The 3D PCL scaffold was characterized by having better biomechanical properties, namely greater elasticity and resistance against stress and strain, thus this scaffold will be able to find broad applications in tissue engineering. Clearly, while nanofibre layers of the 2D scaffold prevented MSCs from migrating through the conformation, cells infiltrated freely through the 3D scaffold. MSC adhesion to the 3D nanofibre PCL layer was also statistically more common than to the 2D scaffold (P < 0.05), and proliferation and viability of MSCs 2 or 3 weeks post-seeding, were also greater on the 3D scaffold. In addition, the 3D PCL scaffold was also characterized by displaying enhanced MSC osteogenic differentiation. CONCLUSIONS: We draw the conclusion that all positive effects observed using the 3D PCL nanofibre scaffold are related to the larger fibre surface area available to the cells. Thus, the proposed 3D structure of the nanofibre layer will find a wide array of applications in tissue engineering and regenerative medicine.

Comparison of the IHC, FISH, SISH and qPCR methods for the molecular diagnosis of breast cancer.

Her2 proto-oncogene amplification and protein overexpression is observed in 20-40% of patients with breast cancer and plays a crucial role in invasive breast cancer and its treatment. In the present study, we investigated samples from 131 patients with invasive breast carcinoma. In all cases, the overexpression/amplification level of Her2 was determined using manual immunohistochemistry (IHC) and/or automatic IHC, fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH) and quantitative polymerase chain reaction (qPCR). Using various methods, we demonstrated candidate methods for Her2 detection and their dependability. Our results demonstrate that these methods are highly comparable for the detection of Her2 overexpression/amplification. It was also revealed that qPCR is a valuable tool for the evaluation of Her2 gene overexpression/amplification. The results from pPCR analysis positively correlated with the results from IHC and FISH analysis. Moreover, in contrast to IHC or SISH/FISH, the results obtained by qPCR were not encumbered with any subjective error on the part of the evaluator.

Study of the effect of neoadjuvant chemotherapy on the status of Her2/neu.

Her2/neu proto-oncogene amplification and protein over-expression is observed in 20-40 % of patients with breast cancer and plays a crucial role in invasive breast cancer and its treatment. A number of studies postulated the stability of Her2/neu gene expression, showing that in most patients the status of expression had not significantly changed after the neoadjuvant treatment. In the present study, we investigated samples from 20 patients with invasive breast carcinoma who had undergone neoadjuvant chemotherapy and subsequent surgery. In all cases, the expression level of Her2/neu was evaluated in both pre-therapeutically obtained tumour tissue by core needle biopsy and from specimens obtained during final surgery using immunohistochemistry. Fluorescence in situ hybridization and quantitative reverse transcription polymerase chain reaction methods were used for verifying the results obtained by immunohistochemistry. Her2/neu status determined by immunohistochemistry remained unchanged in 12 of 20 (60%) patients after neoadjuvant treatment. In six cases (30%) minor changes were observed after the treatment. However, in two cases (10%) we found altered Her2/neu expression from strongly positive in the pre-treatment biopsy to negative in the post-treatment surgery specimen. Moreover, this is the first report describing the changes in Her2/neu status at all protein, RNA and DNA levels by using immunohistochemistry, quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization, respectively. By using variable methods we demonstrated possible new ways for Her2/neu detection and their dependability. Improvement in specific molecule detection can prevent the use of tailored targeted therapy in an untargeted manner.

Optimized conditions for mesenchymal stem cells to differentiate into osteoblasts on a collagen/hydroxyapatite matrix.

Collagen/hydroxyapatite (HA) composite scaffolds are known to be suitable scaffolds for seeding with mesenchymal stem cells (MSCs) differentiated into osteoblasts and for the in vitro production of artificial bones. However, the optimal collagen/HA ratio remains unclear. Our study confirmed that a higher collagen content increased scaffold stiffness but that a greater stiffness was not sufficient for bone tissue formation, a complex process evidently also dependent on scaffold porosity. We found that the scaffold pore diameter was dependent on the concentration of collagen and HA and that it could play a key role in cell seeding. In conclusion, the optimal scaffold for new bone formation and cell proliferation was found to be a composite scaffold formed from 50 wt % HA in 0.5 wt % collagen I solution.

Immobilization of thrombocytes on PCL nanofibres enhances chondrocyte proliferation in vitro.

OBJECTIVES: The aim of this study was to develop functionalized nanofibres as a simple delivery system for growth factors (GFs) and make nanofibre cell-seeded scaffold implants a one-step intervention. MATERIALS AND METHODS: We have functionalized polycaprolactone (PCL) nanofibres with thrombocytes adherent on them. Immobilized, these thrombocytes attached to nanofibre scaffolds were used as a nanoscale delivery system for native (autologous) proliferation and differentiation factors, in vitro. Pig chondrocytes were seeded on the thrombocyte-coated scaffolds and levels of proliferation and differentiation of these cells were compared with those seeded on non-coated scaffolds. RESULTS: Immobilized thrombocytes on PCL nanofibres effectively enhanced chondrocyte proliferation due to time-dependent degradation of thrombocytes and release of their GFs. CONCLUSIONS: These simply functionalized scaffolds present new possibilities for nanofibre applications, as smart cell scaffolds equipped with a GF delivery tool.

A comparision of RT-PCR and FISH techniques in molecular diagnosis of Ewing's sarcoma in paraffin-embedded tissue.

Ewing's sarcoma is relatively uncommon tumor representing 6-8 percent of malignant bone tumors with variable morphology. Cytogenetically, Ewing's sarcomas are characterized by a specific reciprocal chromosomal translocation t(11;22)(q24;q12). The presence of this chromosomal translocation has been detected in approximately 85 percent of the cases. The translocation results in the fusion of EWS gene from chromosome 22 to FLI1 gene at 11q24 which is a member of ETS family of transcription factors. In this study we performed a comparison of two molecular diagnostic strategies, namely RT-PCR and FISH, in fresh, frozen and formalin-fixed paraffin-embedded tissues. We conclude that FISH is a more sensitive technique than RT-PCR for the diagnosis of Ewing's tumors in formalin-fixed paraffin-embedded tissue. In conclusion, molecular pathology techniques, using reverse transcription-polymerase chain reaction (RT-PCR) and/or fluorescence in situ hybridization (FISH) are valuable diagnostic tools for evaluation of undifferentiated small round-cell tumors like Ewing's sarcoma.

[Primary synovial sarcoma of the kidney]. / Primární synoviální sarkom ledviny--popis dvou prípadu.

The authors present two cases of primary synovial sarcoma of the kidney. Both patients had a tumor mass in the kidney with vascular invasion of the inferior vena cava and right atrium of the heart in case no. 1. In case no. 2 retroperitoneal lymph node metastasis and multiple metastases to both lungs were observed. Radical nephrectomies were performed in both patients. Histologically, the tumor in case no. 1 was monophasic and in case no. 2 poorly differentiated. Immunohistochemically, vimentin was diffusely positive and a few tumor cells were positive for epithelial membrane antigen. The tumor cells were negative for keratins, S- 100 protein, CD 34, smooth muscle actin, and desmin. In both cases, reverse transcription-polymerase chain reaction using ribonucleic acid extracted from formalin-fixed, paraffin-embedded tissues detected SYT-SSX 1 fusion gene transcripts, which are characteristic molecular findings of synovial sarcoma.

Downregulation of myc promoter-binding protein 1 (MBP-1) in growth-arrested malignant B cells.

Normal human B lymphocytes are sensitive to the growth-inhibitory action of TGF-beta1 whereas malignant B lymphoma cells are mostly resistant to TGF-beta1 effects. We have shown in our previous work that, TGF-beta1 treatment resulted in significant growth inhibition of the DoHH2 cell line. In the present study we showed that TGF-beta1-induced growth arrest was associated with notable downregulation of the myc-binding protein-1 (MBP-1). Moreover, our results indicated that c-Myc overexpression in TGF-beta1-arrested malignant B cells is mediated by binding of MBP-1, as a transcription repressor, to the (+118/+153) element of the promoter region of the myc gene.

Expression of actin isoforms in human auricular cartilage.

The objective of this study was to determine whether human auricular chondrocytes can also express alpha-- -smooth muscle actin. Immunohistochemistry using monoclonal antibodies for alpha-smooth actin, muscle-specific actin, beta-actin, S-100 protein, CD34, and desmin was performed on samples of human ear cartilage obtained from 20 individuals during a partial resection of the ear for different reasons. Moreover, the RT-PCR analysis of actin isoforms in auricular chondrocytes was performed. Approximately 60 % of the chondrocytes of the ear cartilage expressed alpha-smooth muscle actin as demonstrated by immunohistochemistry in all the examined samples. Actin-positive chondrocytes occurred in both external subperichondrial layers of the auricular cartilage. This finding was confirmed by the RT-PCR technique. The knowledge of this fact could help us to better understand the chondrocyte changes occurring during the healing and transplantation of auricular cartilage. The question of whether it is necessary to refer to these predominating cells in ear cartilage as myochondrocytes is considered. This is the first report of an unusual immunophenotype and contractile potential for human auricular chondrocytes.

Leiomyoma of the gastrointestinal tract with intracytoplasmic inclusion bodies. Report of three cases.

We report three cases of leiomyoma of the gastrointestinal tract with intracytoplasmic inclusion bodies similar to those characteristic of inclusion body fibromatosis (IBF). The first two cases represent leiomyoma of the stomach: one in a 70-year-old female and the other in a 72-year-old female. In both instances inclusion bodies were present in a large amount. In the third case the leiomyoma was located in the esophagus of a 63-year-old male and inclusion bodies in this case were rare. In all three cases an immunohistochemical analysis showed positivity of the tumor cells for muscle specific actin HHF35 (MSA), alpha-smooth muscle actin (SMA), h-caldesmon and desmin. The first case showed some inclusion bodies with positivity for cytokeratin CAM 5.2 and focal weak positivity for cytokeratin 18. In the second case the inclusion bodies were positive at the periphery with antibodies directed against MSA and SMA. In the third case the inclusion bodies were immunohistochemically entirely negative. Ultrastructurally, the inclusion bodies in the first case were composed of aggregated filaments, some with entrapped cytoplasmic organels and others with finely granular dense cores.

Desmin-positive and alpha-smooth muscle actin positive chondrocytes in human defective articular cartilage--preliminary report.

We studied the results of immunostaining for S-100 protein, alpha-smooth muscle actin, muscle specific actin and desmin in articular cartilage specimens obtained during an arthroscopy from eight patients with different degrees of osteoarthritis of the knee joint. In all cases, most of the cartilage cells were strongly positive for S-100 protein. Actin positive chondrocytes were present in four samples showing repair cartilage changes with occurrence of fibrocartilage tissue. Moreover, in one case, we observed typical desmin-positive chondrocytes in the layer of cartilage filling the defect of the articular cartilage surface. The expression of desmin can be regarded as a reaction to trauma or the indication of an inherent abnormality. The chondrocytes probably switched on smooth muscle features during the healing process, because desmin is to a great extent a typical muscle cell marker. This fact could probably support our previous supposition that cartilage cells expressing muscle markers could be designated as myochondroblasts and myochondrocytes analogously to the terminology of myofibroblasts. It is possible that during the healing of the cartilage defects, such a transformation of the immunophenotype of the cartilage cells is quite frequent, but it could also be only transient nature only.

Cyclin D-cdk6 complex is targeted by p21(WAF) in growth-arrested lymphoma cells.

Normal human B lymphocytes are sensitive to the growth-inhibitory action of transforming growth factor beta1 (TGFbeta1) whereas malignant B lymphoma cells are mostly resistant to TGFbeta1 effects. We examined the phosphorylation status of retinoblastoma protein and the activity of G(1) cyclin-dependent kinases (cdk) in TGFbeta1-sensitive malignant follicular lymphoma cells during the TGFbeta1 treatment. The kinase activity of cdk2, cdk4, and cdk6 was significantly reduced and hypophosphorylation of pRb on serine 795 (S795) and threonine 373 (T373) was observed. We examined the composition of cdk complexes and the level of cdk inhibitors to explain the inhibitory action of TGFbeta1 toward cdk activity. Both cdk4 and cdk6 were notably dissociated from cyclin D cofactors, while cyclin E-cdk2 complexes remained coupled in TGFbeta1-treated cells. TGFbeta1-induced growth arrest was associated with notably increased binding of p21(WAF1) to cdk4 and cdk6. No induction of cdk-inhibitor molecules of INK family was observed in TGFbeta1-treated DoHH2 cells. As shown, TGFbeta1-induced growth arrest of malignant B cells was associated with the activation of CIP/KIP family members of cdk inhibitors.

Cyclin A down-regulation in TGFbeta1-arrested follicular lymphoma cells.

Transforming growth factor beta1 (TGFbeta1) induces growth arrest in many cell types, including B lymphocytes. We examined the effect of TGF on cell cycle progression of a non-Hodgkin lymphoma cell line of follicular lymphoma subtype (FL). After 48 h of TGFbeta1 (10 ng/ml) treatment, a significantly increased number of DoHH2 cells was retained in G(0)/G(1) phase. We examined the level of cell cycle components, cyclins, cyclin-dependent kinases (cdk), and their inhibitors. We found that the expression of cyclin A and p21(WAF1) molecules was primarily modulated by TGFbeta1 treatment while the expression of other regulatory components, like cyclins D, cyclin E, cdk2, cdk4, and cdk6 or p15(INK4B), p16(INK4A), and p27(KIP1) was not significantly affected. We further examined expression and activity of CREB/ATF family members to examine their roles in cyclin A inhibition. The binding activity of CREB-1 and ATF-2 to the CRE region of the cyclin A promoter was almost completely abolished due to the treatment. The total level of CREB-1, ATF-2, and ATF-3 was notably reduced. Moreover, CREB-1 was dephosphorylated due to the treatment as revealed by immunoblotting. We assume that down-regulation of cyclin A was mediated by the absence of CREB/ATF activation dimers. The profound effect on the ATF family of transcription factors indicates the complexity of TGFbeta1 action on FL B malignant cells.

Expression of beta-catenins and cadherins by follicular dendritic cells in human lymph nodes.

In lymph nodes, dendritic cells form a complex meshwork and are linked by intercellular junctions. Intercellular junctions contribute to the integrity of lymphatic follicles and can potentially be affected by malignant processes in neighbouring B cells. We examined whether transmembrane molecules that constitute "adherens junctions" are present in follicular dendritic cells of normal human lymph nodes. We found that follicular dendritic cells but not interdigitating dendritic cells or sinus lining cells expressed cadherin molecules. Follicular dendritic cells also expressed beta-catenin but not vinculin. The cadherin molecules, which were identified in situ with the use of a monoclonal pan-cadherin antibody, were not recognized by antibodies to E-cadherin, N-cadherin or P-cadherin. Intrafollicularly, cadherins were clearly colocalized with beta-catenins, in a dot-like fashion. We also detected intrafollicular expression of desmogleins and desmosomal plaque proteins. These findings indicate the presence of desmosomes within the dendritic meshwork. However, pan-cadherin reactivity was not only colocalized with desmoglein immunoreactivity that was abundantly present. Immunoprecipitation showed that pan-cadherin reactivity was absent in fractions of desmosomal plaque proteins or pan-desmogleins. We speculate that complexes of cadherins of an unknown subclass and beta-catenins form non-desmosomal intercellular junctions in the intrafollicular dendritic meshwork.