Roberto Di Primio

Melatonin provokes cell death in human B‐lymphoma cells by mitochondrial‐dependent apoptotic pathway activation

Abstract:  Apoptosis is an important cell suicide programme involved in physiological and pathological processes. Apoptosis can be induced in different ways depending on cell type and acquired signal. Melatonin, the major secretory product of the pineal gland, participates in many important physiological functions and displays a remarkable functional versatility exhibiting antioxidant, oncostatic, anti‐aging, and immunomodulatory properties. Recently, it has been shown that, in addition to pineal gland, human lymphoid cells are an important physiological source of melatonin and that may be involved in the regulation of the immune system. In this work, we examine the effect of melatonin on RAMOS‐1 human leukaemic cells. Cell growth and viability, DNA fragmentation and JC‐1, and annexin V expression have been determined. To elucidate the mechanism of action of melatonin, Western blot analyses for Bcl‐2 and caspase‐3 expression, and cytochrome c release were carried out. The results suggest that the apoptotic effect of melatonin is associated with cell‐cycle arrest, downregulation of Bcl‐2, mitochondrial membrane depolarization, cytochrome c release and activation of caspase‐3. The intrinsic (mitochondrial dependent) pathway of caspase activation is the ‘point of no return’ commitment to cell death. Taken together, our study indicates that melatonin may play a role as potential therapeutic drug in specific lymphoproliferative diseases.

Functional interleukin-7/interleukin-7Rα, and SDF-1α/CXCR4 are expressed by human periodontal ligament derived mesenchymal stem cells

Hematopoiesis in the bone marrow (BM) is maintained by specific interactions between both hematopoietic and non‐hematopoietic stromal cells, which are mesenchymal stem cells (MSCs) capable of giving rise to several cell types. The human periodontal ligament (PDL), a tissue of ectomesenchymal origin, has been shown to also be a source of MSCs. We have investigated whether MSCs expanded from the PDL of healthy volunteers express characteristics similar to BM‐derived stem cells using structural, immunocytochemical and molecular approaches. Their ability to support the growth of hematopoietic progenitors was also analyzed. The PDL‐MSCs exhibited a fibroblast‐like morphology and their chromatin was dispersed, indicating active gene transcription. The mesenchymal‐related antigens CD90, CD29, CD166, CD105, and CD44 were homogeneously detected by cytofluorimetric analysis, whereas membrane CXCR4 was expressed only by a minority of cells. The PDL‐MSCs differentiated in vitro into osteogenic and adipogenic cells. Immunolocalization of IL‐7, IL‐7Rα, SDF‐1α, and CXCR4 resulted in a diffuse but specific labeling. RT‐PCR analysis confirmed the expression of the above‐mentioned transcripts. The cells spontaneously produced high levels of IL‐7 and SDF‐1α and were able to support the development and long‐term maintenance of BM precursor cells more efficiently than murine stromal cells and similarly to normal BM human stromal cells. We examined IL‐7 and SDF‐1α secretion pathway during adipogenic and osteogenic differentiation. IL‐7 increased during osteogenic and adipogenic differentiation, while the SDF‐1α secretion was downregulated during osteogenic differentiation but increased during adipogenic induction. Our study provides evidence that in human PDL there is an accessible niche of MSCs showing the features of BM‐derived MSCs. J. Cell. Physiol. 214: 706–713, 2008.

Melatonin regulates the respiratory burst of human neutrophils and their depolarization

Pieri C, Recchioni R, Moroni F, Marcheselli F, Marra M, Marinoni Silvia, Di Primio R. Melatonin regulates the respiratory burst of human neutrophils and their depolarization. J. Pineal Res. 1998; 24:43–49.

Collagen I membranes for tendon repair: Effect of collagen fiber orientation on cell behavior

Tendons have poor spontaneous regenerative capabilities, and complete regeneration is never achieved despite intensive remodeling. In this in vitro study, we characterized two multilamellar collagen I membranes differing in the arrangement of collagen fiber deposition (oriented vs. nonoriented) and compared their mechanical properties. Human dermal fibroblasts and tenocytes were seeded on the two membranes to evaluate the effect of fiber orientation on cell viability and cytoskeletal organization. Results demonstrate that the multilamellar collagen I membrane with oriented fibers has the better mechanical properties and affords optimum cell proliferation and adhesion. Its fiber arrangement provides an instructive pattern for cell growth and may serve to guide the alignment of cells migrating from the ends of a crushed or frayed tendon to obtain a strong, correctly structured tendon, thus providing a viable clinical option for tendon repair.

DIMETHYL SULFOXIDE INDUCES PROGRAMMED CELL DEATH AND REVERSIBLE G1 ARREST IN THE CELL CYCLE OF HUMAN LYMPHOID PRE-T CELL LINE

In human B- and T-differentiated lymphoid cell lines DMSO was found to arrest the proliferation at the G1 stage of the cell cycle, without any detectable differentiation and DMSO itself was found to prevent apoptosis. Programmed cell death, or apoptosis, is now thought to be an important regulatory process in normal hemopoiesis and in the lymphoid system this program is started in the immune process such as autoreactive T-cell elimination in the thymus, and antigen-driven B-cell selection in the terminal centre. For this purpose, we have analysed the effect of DMSO using undifferentiated pre-B (KM-3) and pre-T (RPMI-8402) human lymphoid cells. Results obtained by multiparametric analyses show that DMSO affect only the pre-T cell line inducing a reversible G1 arrest of the cell cycle with a significant presence of apoptotic cells and modification of terminal transferase (TdT) expression. Pre-B cell line is resistant to DMSO treatment. These data provide evidence of a new model for the study of the selective cell type depending effect of DMSO in the immune system.

Human skin-derived mesenchymal stem cells as a source of VEGF and nitric oxide

Researches on stem cells bring promise to functional skin repair. In particular, it has been recently suggested that mesenchymal stem cells (MSCs) could positively affect cutaneous wound healing through differentiation and paracrine action. The molecular mechanisms are not clear, even if there is increasing evidence for an important action of nitric oxide (NO), probably mediated by the regulation of the gene encoding for vascular endothelial growth factor (VEGF). The aim of our study was to investigate the immunohistochemical expression of VEGF and nitric oxide synthase (NOS) isoforms in human skin-derived MSCs, as well as the production of VEGF and NO, because these cells are less well characterized than bone marrow MSCs. MSCs were obtained from skin biopsies of healthy adult patients undergoing cosmetic plastic surgery, expanded and characterized for specific surface antigens. The cells were then evaluated for the immunohistochemical expression of VEGF, and NOS isoforms, as well as for VEGF and NO secretion in cell culture medium. Our immunohistochemical analysis showed that proliferating MSCs derived from human skin exhibit VEGF expression at cytoplasmic level as well as cytosolic and nuclear localization of all the three isoforms of NOS, even if with different patterns. In addition, our data evidenced the release of both VEGF and NO in cell culture supernatants. In conclusion, our results suggest that a therapeutic approach based on the human skin-derived MSCs may have a positive effect in wound healing conditions, through their ability to provide VEGF and NO to the damaged area.

Involvement of vascular endothelial growth factor, CD44 and CD133 in periodontal disease and diabetes: an immunohistochemical study.

AIM The aim of this study was to investigate the relationship between expression of angiogenic and regeneration markers and periodontal disease in subjects with/without diabetes mellitus. MATERIAL AND METHODS Immunohistochemical detection of vascular endothelial growth factor (VEGF), CD44 and CD133 was performed in 16 samples each of (1) healthy gingiva from non-diabetic subjects (controls), (2) gingiva from non-diabetic subjects with periodontitis, (3) gingiva from subjects with type 1 diabetes and periodontitis, (4) gingiva from subjects with type 2 diabetes and periodontitis. RESULTS Diseased gingivae from patients with diabetes and periodontitis had greater clinical measures of periodontal disease than those with periodontitis only. VEGF expression was significantly enhanced in epithelial and endothelial cells from patients with periodontitis compared with controls (p<0.05). Epithelial CD44 expression was strong in all groups, while CD44 was significantly enhanced (p<0.05) in connective tissue cells from both diabetic groups. Epithelial and endothelial CD133 expression was comparable in all patients except those with type 2 diabetes and periodontitis, where it was not detected. Stromal CD133 expression was significantly lower in patients with type 2 diabetes and periodontitis and was increased in periodontitis patients (p<0.05). CONCLUSIONS The involvement and high expression of VEGF, CD44 and CD133 in periodontal disease may predict a greater regeneration capacity of gingival tissue.

Tigecycline accelerates staphylococcal-infected burn wound healing through matrix metalloproteinase-9 modulation

OBJECTIVES We investigated the in vivo efficacy of tigecycline, a new glycylcycline (a tetracycline derivative), in the management of methicillin-resistant Staphylococcus aureus (MRSA)-infected experimental surgical wounds in rats. The main outcome measures were quantitative bacterial culture, histological examination and immunohistochemical expression of matrix metalloproteinase-9 (MMP-9) and collagen IV. METHODS An animal model was used to compare the in vivo efficacy of teicoplanin and tigecycline in the treatment of burn wound infections by S. aureus. A copper bar, heated in boiling water, was placed on the paraspinal site of each rat, resulting in full thickness burns. A small gauze was placed over each burn and then inoculated with 5 × 10(7) cfu of S. aureus ATCC 43300. To mimic the clinical situation in burn patients, surgical debridement was performed 48 h after the injury. The wounds were left to heal by secondary intention. The study included an uninfected control group that did not receive any treatment, a contaminated group that did not receive any treatment, and two contaminated groups treated with intraperitoneal tigecycline (2 mg/kg) and teicoplanin (7 mg/kg), respectively. RESULTS All antibiotic treatments were significantly effective. Tigecycline showed the highest antimicrobial activity, with a better impact on histological results. Infected rats treated with tigecycline showed a significant decrease in MMP-9 expression both in epithelium and in dermis compared with rats treated with teicoplanin. CONCLUSIONS Tigecycline, besides its antimicrobial activity, exerts an important modulatory effect on MMP-9, accelerating wound healing in staphylococcal-infected burns.

Nitric oxide synthase and tyrosine nitration in idiopathic asthenozoospermia: an immunohistochemical study

OBJECTIVE To characterize the expression pattern of constitutive and inducible nitric oxide synthase (NOS) in spermatozoa isolated from normospermic fertile donors and asthenozoospermic infertile patients, by means of immunohistochemistry. Also to evaluate the immunohistochemical expression of citrulline, a marker of NOS activity, and nitrotyrosine, which indicates the formation of peroxynitrite, which may affect sperm functionality through its cytotoxic action. DESIGN Prospective study. SETTING Academic male infertility center. PATIENT(S) Twenty-nine infertile patients affected by idiopathic asthenozoospermia and 26 age-matched normospermic fertile donors. INTERVENTION(S) Sperm parameters were evaluated, and immunohistochemical analysis was performed in isolated spermatozoa. MAIN OUTCOME MEASURE(S) Semen analyses, to ascertain volume, sperm count, motility, and morphology. Immunohistochemical expression of NOS isoforms, citrulline, and nitrotyrosine. RESULT(S) Constitutive NOS expression was greater in spermatozoa isolated from normospermic fertile donors. In contrast, the immunohistochemical expression of inducible NOS and nitrotyrosine was higher in asthenozoospermic samples. Our data concerning citrulline indicated enhanced NOS activity in sperm from idiopathic asthenozoospermic patients. CONCLUSION(S) Our results support the hypothesis that increased NOS activity and an excess of tyrosine nitration may affect the functional characteristics of spermatozoa in idiopathic asthenozoospermia.

Skin-derived mesenchymal stem cells (S-MSCs) induce endothelial cell activation by paracrine mechanisms

Please cite this paper as: Skin‐derived mesenchymal stem cells (S‐MSCs) induce endothelial cell activation by paracrine mechanisms. Experimental Dermatology 2010; 19: 848–850.