Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-α, TGF-β and CD4+ and acinar IFN-γ, TNF-α, IL-1β and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-β. Significant positive correlations were found between portal CD4+ and TNF-α, portal CD8+ and TGF-β, portal CD45+RO and TNF-α, acinar CD45+RO and IFN-γ and acinar CD57+ and TGF-β. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Histomorphological and immunohistochemical characterization of 172 cutaneous round cell tumours in dogs

This paper describes the use of a panel of antibodies (CD117, CD3, CD79a, CD45, cytokeratin, vimentin and E-cadherin) on formalin-fixed, paraffin-embedded sections of canine cutaneous round cell tumours. Neoplastic tumours were diagnosed by histology and histochemical stains and included 107 mast cell tumours, 31 cutaneous histiocytomas, two localized histiocytic sarcomas, 21 cutaneous lymphomas, three plasma cell tumours, one transmissible venereal tumour and seven unclassified round cell tumours. The histologic diagnosis was modified in 39.5% of the total 172 neoplasms. The staining for CD45 and Ecadherin were variable, and therefore, the final diagnoses of cutaneous histiocytoma and localized histiocytic sarcoma were made based on histology in association with negative results for CD3, CD79a, CD117 and cytokeratin. The cellular origin of unclassified round cell tumours was defined in all cases. Cutaneous B-cell lymphoma and plasma cell tumours were CD79a-positive and could be distinguished from each other by the morphological characteristics. Mast cell tumours and T cell lymphoma were CD117 and CD3 positive, respectively. The positive staining for vimentin and the negative staining for CD3, CD79a, CD117 and cytokeratin favoured the diagnosis of transmissible venereal tumours. Thus, the final diagnosis of cutaneous round cell tumours should be based on the interpretation of immunohistochemical results together with the cellular morphology observed by histology. Therefore, more studies to optimize the specific markers in formalin-fixed, paraffinembedded tissues (especially for histiocytes) are required for definitive diagnosis of round cell tumours in dogs.

Isolation, expansion and differentiation of cellular progenitors obtained from dental pulp of agouti (Dasyprocta prymnolopha Wagler, 1831)

Abstract: The study aimed to isolate, expand, differentiate and characterize progenitor cells existent in the dental pulp of agouti. The material was washed with PBS solution and dissociated mechanically with the aid of a scalpel blade on plates containing culture medium D-MEM/F-12, and incubated at 5% CO2-37⁰C. The growth curve, CFU assay, osteogenic/adipogenic differentiation and characterization were obtained from the isolation. The cells began to be released from the explant tissue around the 7th day of culture. By day 22 of culture, cells reached 80% confluence. At the UFC test, 81 colonies were counted with 12 days of cultivation. The growth curves before and after freezing showed a regular growth with intense proliferation and clonogenic potential. The cell differentiation showed formation of osteoblasts and fat in culture, starting at 15 days of culture in a specific medium. Flow cytometry (FACs) was as follows: CD34 (positive), CD14 (negative), CD45 (negative), CD73 (positive), CD79 (negative), CD90 (positive), CD105 (positive), demonstrating high specificity and commitment of isolated cells with mesenchymal stem cells strains. These results suggest the existence of a cell population of stem cells with mesenchymal features from the isolated tissue in the explants of agouti dental pulp, a potential model for study of stem cell strains obtained from the pulp tissue.

Isolation and culture of umbilical vein mesenchymal stem cells

Bone marrow contains a population of stem cells that can support hematopoiesis and can differentiate into different cell lines including adipocytes, osteocytes, chondrocytes, myocytes, astrocytes, and tenocytes. These cells have been denoted mesenchymal stem cells. In the present study we isolated a cell population derived from the endothelium and subendothelium of the umbilical cord vein which possesses morphological, immunophenotypical and cell differentiation characteristics similar to those of mesenchymal stem cells isolated from bone marrow. The cells were isolated from three umbilical cords after treatment of the umbilical vein lumen with collagenase. The cell population isolated consisted of adherent cells with fibroblastoid morphology which, when properly stimulated, gave origin to adipocytes and osteocytes in culture. Immunophenotypically, this cell population was found to be positive for the CD29, CD13, CD44, CD49e, CD54, CD90 and HLA-class 1 markers and negative for CD45, CD14, glycophorin A, HLA-DR, CD51/61, CD106, and CD49d. The characteristics described are the same as those presented by bone marrow mesenchymal stem cells. Taken together, these findings indicate that the umbilical cord obtained from term deliveries is an important source of mesenchymal stem cells that could be used in cell therapy protocols.

Leptospira interrogans activation of peripheral blood monocyte glycolipoprotein demonstrated in whole blood by the release of IL-6

Glycolipoprotein (GLP) from pathogenic serovars of Leptospira has been implicated in the pathogenesis of leptospirosis by its presence in tissues of experimental animals with leptospirosis, the inhibition of the Na,K-ATPase pump activity, and induced production of cytokines. The aims of the present study were to investigate the induction of IL-6 by GLP in peripheral blood mononuclear cells (PBMC) and to demonstrate monocyte stimulation at the cellular level in whole blood from healthy volunteers. PBMC were stimulated with increasing concentrations (5 to 2500 ng/ml) of GLP extracted from the pathogenic L. interrogans serovar Copenhageni, lipopolysaccharide (positive control) or medium (negative control), and supernatants were collected after 6, 20/24, and 48 h, and kept at -80ºC until use. Whole blood was diluted 1:1 in RPMI medium and cultivated for 6 h, with medium, GLP and lipopolysaccharide as described above. Monensin was added after the first hour of culture. Supernatant cytokine levels from PBMC were measured by ELISA and intracellular IL-6 was detected in monocytes in whole blood cultures by flow-cytometry. Monocytes were identified in whole blood on the basis of forward versus side scatter parameters and positive reactions with CD45 and CD14 antibodies. GLP ( > or = 50 ng/ml)-induced IL-6 levels in supernatants were detected after 6-h incubation, reaching a peak after 20/24 h. The percentage of monocytes staining for IL-6 increased with increasing GLP concentration. Thus, our findings show a GLP-induced cellular activation by demonstrating the ability of GLP to induce IL-6 and the occurrence of monocyte activation in whole blood at the cellular level.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.