Wound stimulation by growth-arrested human keratinocytes and fibroblasts: HP802-247, a new-generation allogeneic tissue engineering product

HP802-247 is a new-generation, allogeneic tissue engineering product consisting of growth-arrested, human keratinocytes (K) and fibroblasts (F) delivered in a fibrin matrix by a spray device.

Antifibrotic effects of tocotrienols on human Tenon's fibroblasts

Antifibrotic effects of α- (40, 60, 80, 100, and 120 μM), γ- (10, 20, 30, and 40 μM) and δ-tocotrienol (10, 20, 30, and 40 μM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7 days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100 μg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. Results All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for α-tocotrienol 80 μM with 36.7% and at day 7 for α-tocotrienol 80 μM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80 μM for α- and above 30 μM for γ- and δ-tocotrienol. The highest collagen synthesis inhibition has been found with 80 µM α-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 µM α- and 30 µM γ-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. Conclusion In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon’s fibroblasts and therefore may have the potential as an anti-scarring agent in filtrating glaucoma surger

Effect of low doses and high homeopathic potencies in normal and cancerous human lymphocytes: an in vitro isopathic study

OBJECTIVES: Biologic effects of high homeopathic potencies can be studied in cell cultures using cell lines or primary cells. We hypothesized that primary cells would be more apt to respond to high potencies than cell lines, especially cancer cell lines. We set out to investigate the effects of low doses and high homeopathic potencies of cadmium chloride, respectively, in an intoxication model with human primary lymphocytes compared to a human leukemia cell line (Jurkat). DESIGN: Cells were pretreated with either low concentrations (nM-microM) or high potencies (pool 15-20c) of cadmium for 120 hours, following which they were exposed to a toxic treatment with a range of cadmium concentrations (8-80 microM) during 24 hours. Cell viability was eventually assessed by use of the MTS/PES assay. Controls included a vehicle (NaCl 0.9%) for the low concentrations of cadmium or water 15-20c for cadmium 15-20c. A total of 34 experiments were conducted, 23 with low concentrations and 11 with high potencies of cadmium. Data were analyzed by analysis of variance. RESULTS: Pretreatment with low concentrations or high potencies of cadmium significantly increased cell viability in primary lymphocytes after toxic challenge, compared to control cells (mean effect +/- standard error = 19% +/- 0.9% for low concentrations respectively 8% +/- 0.6% for high potencies of cadmium; p < 0.001 in both cases). The pretreatment effect of low doses was significant also in cancerous lymphocytes (4% +/- 0.5%; p < 0.001), albeit weaker than in normal lymphocytes. However, high homeopathic potencies had no effect on cancerous lymphocytes (1% +/- 1.9%; p = 0.45). CONCLUSIONS: High homeopathic potencies exhibit a biologic effect on cell cultures of normal primary lymphocytes. Cancerous lymphocytes (Jurkat), having lost the ability to respond to regulatory signals, seem to be fairly unresponsive to high homeopathic potencies.

A comparative analysis of phenotype expression in human osteoblasts from heterotopic ossification and normal bone

BACKGROUND AND AIMS: Heterotopic ossification (HO) is a pathological bone formation process in which ectopic bone is formed in soft tissue. The formation of bone depends on the expression of the osteoblast phenotype. Earlier studies have shown conflicting results on the expression of phenotype markers of cells originating from HO and normal bone. The hypothesis of the present study is that cells from HO show an altered expression of osteoblast-specific phenotype markers compared to normal osteoblasts. The aims of the study were to further characterize the expression of osteoblast phenotypemarkers and to provide a comparison with other study results. PATIENTS AND METHODS: Using an in vitro technique, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and immunohistochemistry, we compared the phenotype gene expression (type I collagen, alkaline phosphatase, Cbfa-1, osteocalcin) of osteoblasts from resected HO and normal bone (iliac crest). RESULTS: Cells from HO expressed the osteoblast phenotype (type I collagen, alkaline phosphatase) but were characterized by a depleted osteocalcin expression. The expression of Cbfa-1 (osteocalcin transcription gene) showed a large variety in our study. Preoperative radiotherapy had no effect on phenotype expression in cells from HO. CONCLUSION: Our results provide a characterization of cells originating from HO and support the thesis of an impaired osteoblast differentiation underlying the formation of HO. The transcription axis from Cbfa-1 to osteocalcin could be involved in the pathogenesis of HO.

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

INTRODUCTION: Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage. METHODS: Expression of the markers was analyzed by immunohistochemistry (IHC) and flow cytometry. Hoechst 33342 dye was used to identify and sort the cartilage side population (SP). Multilineage differentiation assays including chondrogenesis, osteogenesis and adipogenesis were performed on SP and non-SP (NSP) cells. RESULTS: A surprisingly high number (>45%) of cells were positive for Notch-1, Stro-1 and VCAM-1 throughout normal cartilage. Expression of these markers was higher in the superficial zone (SZ) of normal cartilage as compared to the middle zone (MZ) and deep zone (DZ). Non-fibrillated OA cartilage SZ showed reduced Notch-1 and Sox9 staining frequency, while Notch-1, Stro-1 and VCAM-1 positive cells were increased in the MZ. Most cells in OA clusters were positive for each molecule tested. The frequency of SP cells in cartilage was 0.14 +/- 0.05% and no difference was found between normal and OA. SP cells displayed chondrogenic and osteogenic but not adipogenic differentiation potential. CONCLUSIONS: These results show a surprisingly high number of cells that express putative progenitor cell markers in human cartilage. In contrast, the percentage of SP cells is much lower and within the range of expected stem cell frequency. Thus, markers such as Notch-1, Stro-1 or VCAM-1 may not be useful to identify progenitors in cartilage. Instead, their increased expression in OA cartilage implicates involvement in the abnormal cell activation and differentiation process characteristic of OA.

Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

Immunohistochemical evaluation of heat shock protein expression in normal canine nerve and peripheral nerve sheath tumours

Abnormal expression of heat shock proteins (HSPs) has been observed in many human neoplasms and such expression has prognostic, predictive and therapeutic implications. The aim of this study was to evaluate immunohistochemically the expression of HSP 27, HSP 32 and HSP 90 in normal canine peripheral nerves and in four benign and 15 malignant canine peripheral nerve sheath tumours (PNSTs). In normal nerve, all of the HSPs were detected in axons, epineurial fibroblasts and scattered Schwann cell bodies. Cytoplasmic expression of HSP 27 was more widespread and intense in benign PNSTs compared with malignant PNSTs (P <0.05). Widespread and intense nuclear expression of HSP 32 was also associated with benign tumours (P <0.01), while high HSP 90 immunoreactivity was detected in all tumours, suggesting that HSP 90 might represent a new therapeutic target.

Molecular analysis of TNF sensitivity in normal and Ha-{\it ras\/} transformed murine fibroblasts

Tumor necrosis factor (TNF) is known to have antiproliferative effects on a wide variety of tumor cells but proliferative effects on normal cells. However, the molecular basis for such differences in the action of TNF are unknown. The overall objectives of my research are to investigate the role of oncogenes in TNF sensitivity and delineate some of the molecular mechanisms involved in TNF sensitivity and resistance. To accomplish these objectives, I transfected TNF-resistant C3H mouse embryo fibroblasts (10T1/2) with an activated Ha-ras oncogene and determined whether these cells exhibit altered sensitivity to TNF. The results indicated that 10T1/2 cells transfected with an activated Ha-ras oncogene (10T-EJ) not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. In contrast, 10T1/2 cells transfected with the pSV2-neo gene alone were resistant to the cytotoxic effects of TNF. I also found that TNF-induced cell death was mediated through apoptosis. The differential sensitivity of 10T1/2 and 10T-EJ cell lines to TNF was not due to differences in the number of TNF receptors on their cell surface. In addition, TNF-resistant revertants isolated from Ha-ras-transformed, TNF-sensitive cells still expressed the same amount of p21 as TNF-sensitive cells and were still tumorigenic, suggesting that Ha-ras-induced transformation and TNF sensitivity may follow different pathways. Interestingly, TNF-resistant but not sensitive cells expressed higher levels of bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. However, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. Based on these results I can conclude that (i) Ha-ras oncogene induces both transformation and TNF sensitivity, (ii) TNF-induced cytotoxicity involves apoptosis, and (iii) TNF-induced upregulation of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts. ^

Regulation of NAMPT in human gingival fibroblasts and biopsies

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas.

Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting.Modern Pathology advance online publication, 12 September 2014; doi:10.1038/modpathol.2014.113.