Biomedical subjects

D Ponzin

Publications and source records attributed to D Ponzin.

12 recordsLinked to original sources

PubMed2001

p63 identifies keratinocyte stem cells.

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

3T3 Cells↗

PubMed2001

Further evaluation of amniotic membrane banking for transplantation in ocular surface diseases.

Objective: To define the best conditions for amniotic membrane preparation, storage and banking in its use for corneal reconstruction. Methods: Amniotic membrane pieces were prepared under sterile conditions from placentas selected on the basis of donor medical and social history, serology, microbiological tests and histology. The pieces were kept at -140 degrees C but before grafting they were thawed and stored at 4 degrees C in RPMI medium, to have a preparation usable within 72 h. This procedure was validated by testing its therapeutic effectiveness in 25 patients 13 of which had corneal ulcers of various origin, 3 had sequelae of herpes simplex keratitis, 3 band keratopathy and 6 corneal stem cell deficiency due to chemical or thermal burns. Results: The preparation showed appreciable anti-inflammatory and analgesic effects. In the absence of corneal stem cell deficiency a stable re-epithelialisation was achieved in 15 out of 19 patients. When the limbus was lesioned, the amniotic membrane decreased vascularization and increased the number of corneal epithelial cells only in 1 of the 6 patients. No adverse reactions attributable to the tissue were recorded. Conclusions: A ready-to-use amniotic membrane preparation stored at 4 degrees C after cryopreservation has been tested in corneal reconstruction. Like the amniotic membrane thawed immediately before grafting, this preparation displayed full therapeutic effect in epithelial defects with stromal ulceration but without severe limbal stem cell deficiency. In two years banking activity 463 pieces of the preparation were successfully distributed to 90 Italian hospitals.

Journal Article↗

PubMed1995

Antigen presentation is inhibited in vivo by betamethasone.

Aim of the present study was to assess whether betamethasone, a synthetic glucocorticoid used as immunosuppressant, could modify in vivo the antigen presentation by antigen presenting cells (APC). Interleukin-2 (IL-2) production by a T cell hybridoma specific for the hen egg white lysozyme (HEL) cultured in the presence of HEL and APC from treated or control mice was utilized as read out. Betamethasone induced a dose-dependent inhibition of antigen presentation. Fifty percent maximal response was observed with 1152 (95% confidence interval: 948-1419) resident peritoneal macrophages from untreated animals, and 5843 (4700-7445, 21, 235 (12,857-43,705), 28,313 (20,847-40,955) macrophages from mice injected for 3 days with betamethasone 10, 25, and 50 mg/kg respectively. Similar findings were obtained with spleen cells. When given for 3 days at 25 mg/kg, betamethasone reduced the number of cells recovered from the peritoneum by approximately half and from the spleen by one order of magnitude. One day vs. 3 days treatment resulted in similar recovery of cells but lower inhibition of APC function. In the experimental conditions utilized, no carryover of betamethasone with APC could be demonstrated and no reversal of inhibition was observed by increasing the antigen concentration. The data here presented demonstrate that short curses of high dose betamethasone specifically impair antigen presentation. Thus, this mechanism appears to be involved in the immunosuppressant activity of betamethasone.

Animals↗

PubMed1995

Inhibition by cholesterylphosphorylserine of T-cell-mediated immune responses in mice.

The synthetic analogue of phosphatidylserine, cholesterylphosphorylserine (CPHS) inhibits T-cell-mediated immune responses in mice. Tested in cultured mouse spleen cells, CPHS inhibits concanavalin A-induced activation of DNA synthesis (IC50, 3.5 microM). Injected i.p. during the efferent phase, CPHS (25-100 mg/kg) inhibits the manifestations of delayed-type of hypersensitivity. The compound (25 mg/kg i.p., daily) reduces the acute graft-versus-host reaction when given for 5 days to donor mice before the isolation of spleen cells used for the inoculum. These data suggest that the addition of a phosphorylserine group to a steroid ring may produce immunoregulatory compounds.

Adjuvants, Immunologic↗

PubMed1993

Production of tumor necrosis factor and other proinflammatory cytokines by human mononuclear phagocytes stimulated with myelin P2 protein.

In this study we examined the effect of myelin P2 protein on some proinflammatory functions exerted by human mononuclear phagocytes. Northern blot analysis demonstrated that P2 protein selectively induced in monocytes and macrophages mRNA accumulation of tumor necrosis factor (TNF), interleukin 1 beta (IL-1 beta), and interleukin 8 (IL-8) in a time-dependent manner. Natural killer stimulating factor (IL-12) mRNA and protein secretion was strongly induced by lipopolysaccharide but not by P2 protein. Supernatants harvested from P2-stimulated monocytes contained significant amounts of TNF, IL-1 beta, and IL-8, whereas those from macrophages contained only TNF and IL-8. The effect of the P2 protein on TNF and IL-8 mRNA accumulation and secretion was not affected by polymyxin B, which, on the other hand, almost completely abolished the effect of lipopolysaccharide. Finally, P2 protein did not directly trigger hydrogen peroxide release but, through the induced release of TNF, potentiated monocyte respiratory burst capability. Since P2 protein is the antigen responsible for the induction of experimental allergic neuritis, these findings identify a potential mechanism involved in the inflammatory reaction and myelin damage during experimental allergic neuritis.

Cytokines↗

PubMed1991

Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system.

To test whether gangliosides (GA) might exert neuritogenic effects in vivo, experimental allergic neuritis (EAN) was studied clinically, neuropathologically, and immunologically in Lewis rats immunized with bovine peripheral nerve, P2 myelin protein, P2 myelin protein plus two different doses of GA, P2 with galactocerebroside (GC), and GA alone, each emulsified in adjuvant. All except the GA-treated group developed signs of EAN between days 11 and 14 after the injection. Rats immunized with P2 alone were the most severely affected. Rats given P2 plus GA and those given P2 plus GC displayed a significantly lower clinical score. Histological analysis revealed a comparable degree of inflammation of the peripheral nervous system and demyelination in the spinal nerve roots of bovine peripheral nerve- and P2-immunized rats. The P2 plus GA and P2 plus GC groups revealed similar degrees of pathology in the spinal nerve roots but the latter group stood apart from the rest in that it showed widespread peripheral nervous system changes extending distally into the sciatic nerve. Serological analysis demonstrated that P2 and GC, but not GA, elicited antibody (IgG) responses, but there was no correlation between antibody titer and clinical or histological involvement. The present data fail to support an enhancing role for gangliosides in the expression of EAN and, by extrapolation, in the Guillain-Barré syndrome, for which EAN serves as the laboratory model, and in which suggestions have been made that antibodies to GA may have pathogenetic significance.

Animals↗

PubMed1986

Characterization of macrophages elicited by intraperitoneal injection of hyaluronate.

Hyaluronate of 120,000 molecular weight has been injected in the peritoneal cavity of mice to study its effect on migration of inflammatory cells in vivo. After one day a dose-dependent granulocyte migration is observed. Three days later the number of granulocytes is greatly reduced and macrophages form about half of the total cell population. Hyaluronate-elicited macrophages show a decreased 5'-nucleotidase and an increased acid phosphatase activity as compared to resident macrophages. The production of superoxide anion in response to the phorbol ester tetradecanoyl-phorbolacetate, and the phagocytic activity are also enhanced. Macrophages elicited by hyaluronate secrete growth factor(s) for non-lymphoid mesenchymal cells. It is concluded that hyaluronate in vivo stimulates the migration of inflammatory cells, thus causing the recruitment of a population of stimulating macrophages. These effects may explain previous reports on the acceleration of wound healing by hyaluronate.

5'-Nucleotidase↗

PubMedDate not supplied

Phosphatidylserine-induced modulation of the immune response in mice: effect of intravenous administration.

The action of phosphatidylserine on the immune response has been examined in mice after the intravenous administration of phospholipid or exposing cultured splenocytes to the action of phosphatidylserine vesicles. Phosphatidylserine (5-25 mg/kg) reduces the T-dependent and the T-independent antibody production. This effect is observed when the phospholipid is injected before (4 h) but not after (24 h) the immunization. The decreased influence of phosphatidylserine injected 24 h before the immunization indicates the reversibility of the action of phospholipid. The effect on the immune system may in part reflect a direct interaction with lymphocytes, since phosphatidylserine (12-60 microM) decreases the production of T-cell growth factors (mainly interleukin-2) elicited by mitogens in cultured spleen cells and reduces the expression of growth factor receptors in the same cells activated by mitogens. In addition, the activity of T-helper cells is found to be reduced in mice receiving the injection of phosphatidylserine. By contrast, the antigen processing and presentation by macrophages is not affected. The data suggest that the intravenous injection of phosphatidylserine vesicles in mice is followed by a transient decrease of lymphocyte activity.

Animals↗

PubMedDate not supplied

Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains.

The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.

Binding Sites↗

PubMedDate not supplied

Role of protein kinase C in the phosphatidylserine-induced inhibition of DNA synthesis in blood mononuclear cells.

The mechanism of immunosuppressant activity of phosphatidylserine has been studied in peripheral blood mononuclear cells depleted or not of monocytes. After the addition of phosphatidylserine, mass determinations and uptake of labeled compound demonstrate its transfer into the cells. Phosphatidylserine incorporation causes a 2.5-fold increase of membrane-bound protein kinase C activity. The activation of translocated enzyme is indicated by the inhibition of phosphoinositide hydrolysis, and early feedback effect induced by activated protein kinase C. This action of phosphatidylserine is reproduced by tetradecanoylphorbolacetate and is prevented by the protein kinase C inhibitor, staurosporine. Consistently, phosphatidylserine (8 nmol/10(6) cells) decreases by 46% the production of inositol phosphates in cells responding to phytohemagglutinin. The decrease of phosphoinositide signal pathway as well as the inhibition of mitogen-induced DNA synthesis are produced at the same phosphatidylserine concentration and are equally manifest in total mononuclear cells or in preparations depleted of monocytes. However, only in the presence of monocytes does tetradecanoylphorbolacetate enhance the action of phospholipid, decreasing its IC50 from 13-15 microM to 7 microM. Thus, the data suggest that a reaction driven by protein kinase-C and a factor released by activated monocytes are involved in the phosphatidylserine-induced inhibition of lymphocyte DNA synthesis.

Alkaloids↗

PubMedDate not supplied

Ab-interno trabeculo-canalectomy: surgical approach and histological examination.

PURPOSE: To evaluate, on eye bank eyes, a new surgical approach aimed at removing a quadrant of the trabecular meshwork (TM), with an ab interno approach. METHODS: Gonioscopically controlled ab interno removal of the TM was done with a subretinal forcep on six human bank eyes. Serial histological sections were obtained from the treated and untreated part of each globe to assess the effect of the technique on intraocular tissues. RESULTS: Under the gonioscope, the TM was easily removed in strings of varying length. Histological examination showed, unexpectedly, that this resulted in a well-defined deep furrow in the middle of the trabecular region involving both the TM and the inner wall of Schlemm's canal. The operation created a direct communication between the anterior chamber and Schlemm's canal lumen without any evident damage to the outer canal wall and adjacent ocular structures such as the iris base and corneal endothelium. CONCLUSIONS: Our small series on human bank eyes showed that the procedure involves both the TM and the inner wall of Schlemm's canal and is therefore called ab interno trabeculocanalectomy (AITC). The intraoperative findings and the histological evidence are encouraging, and suggest that the procecedure could have potential clinical application.

Cornea↗

PubMedDate not supplied

Improvement of human corneal endothelium in culture after prolonged hypothermic storage.

PURPOSE: To evaluate whether the organ culture method for human cornea preservation may be applied to corneas stored for several days at 4 degrees C. METHODS: The cell density, viability, and morphology of corneal endothelium were examined in 140 human corneas stored at 4 degrees C for the minimal time required for transport to the bank and for the preliminary controls of cornea status (1.6 +/- 1.1 days) and in 46 corneas preserved at 4 degrees C for 6.1 +/- 1.9 days in Optisol-GS. The evaluation was repeated after 19.7 +/- 9.1 days of incubation at 31 degrees C in a culture medium containing 2% newborn calf serum. RESULTS: After the hypothermic storage the corneal endothelium had a mean density of 2475 +/- 159 cells/mm2 without significant difference between the short and the long-term incubation. Several corneas of the two groups showed signs of endothelium degeneration and were positive to trypan blue test. After the incubation at 31 degrees C, the corneas with endothelial degeneration decreased by 52.2% and those positive to trypan blue decreased by 21.7%. Polymorphism (enlarged endothelial cells) increased from 9.6% to 14.5% of the corneas. The remodeling of the endothelium led to a 6.7% decrease in cell density. These results were similar after short-term and long-term storage at 4 degrees C. CONCLUSIONS: Organ culture was effective in improving corneal endothelium when the hypothermic storage was prolonged to the upper temporal limit for this procedure (7-10 days). These results may encourage the possibility of an eye bank to allocate the available cornea pool, thus decreasing the risk of discarding precious material.

Aged↗