Claudio Torresani

Clarithromycin versus doxycycline in the treatment of rosacea

Forty patients with rosacea were entered into a study comparing clarithromycin with doxycycline in the systemic treatment of mild and severe rosacea. The patients, 25 women and 15 men, aged from 26 to 62 years, were subdivided into two homogeneous groups with regard to age, sex, and disease seventy. The first group of 23 patients, 14 women and 9 men, was treated with 250 mg of clarithromycin for 4 weeks twice daily, and then with 250 mg once daily for the following 4 weeks. The second group of 17 patients, 11 women and 6 men was treated with 100 mg of doxycycline for 4 weeks twice daily, and then with 100 mg once a day for the following 4 weeks. Both objective and subjective evaluations of the dermatosis were performed prior to therapy and after 4, 6, and 8 weeks of treatment.

Hairy cell leukemia cells express CD1A antigen

Five patients with hairy cell leukemia (HCL) were studied. Peripheral blood leukocytes, rosette‐forming cells (T) and non‐T‐cells were stained in immunofluorescence by a panel of monoclonal antibodies to investigate the phenotype of HCL cells (HCLC). In all patients HCLC showed B‐lymphocyte phenotype, although they were not stained by antibodies reactive for monocytes, natural killer cells, or T‐cells. However, in all instances the large majority of HCLC were unexpectedly stained with an antibody (anti‐CD1a) usually detectable only in early thymocytes and on Langerhans cells. This finding was further confirmed by immunoelectron microscopy. This type of ambiguity in the lineage of HCL could imply that HCLC might arise from cells differentiated towards the B‐cell lineage, still sustaining an early antigen of a different (T) lineage. These results, moreover, extend the range of the known distribution of the CD1a antigen, which could be useful in diagnosing HCL.

The S-100β protein in normal human peripheral blood is uniquely present within a discrete suppressor-T-cell compartment

The S-100-positive T lymphocytes, and, particularly, the S-100 beta subunit, are restricted, as demonstrated by quantitative subset analysis and double-labeling (gold-peroxidase) immunoelectron microscopy of T-cell subpopulations, to an unique T8-positive cell subset which interestingly was 9.3-negative and CD11b-positive. Since both the T8-positive, 9.3-negative and the T8-positive, CD11b-positive subpopulations have been demonstrated to show suppressive activities, the S-100-positive T cells seem to be closely restricted to a small T-suppressor-cell compartment. Although functional studies on viable isolated S-100 beta-positive cells are impossible to achieve, due to the lack of this protein on the cell membrane, its presence in a discrete T-suppressor compartment might suggest a possible role for the S-100 beta-positive T cells in the regulation of the immune system.

Contact urticaria syndrome due to phyenylmercuric acetate

Case no. 2 A 32-year-old male had a !-month history of eczema on the hands, neck and face (Fig. 2). He had started work as a parquet layer a short time before its onset. Lesions improved greatly when the patient stopped work. Patch tests were performed with the GIRDCA standard series, their glue (Pelpren PL/6) and its constituents. Both patients reacted to Pelpren PL/ 6 as is ( + + + ), epoxy resin ( + + + ), and benzyl alcohol ( + + + ), plus isophoronediamine ( + + +) in patient no. 1. After leaving their jobs, both patients made a definite and long-lasting recovery.

Identification of Fas-L-Expressing Apoptotic T Lymphocytes in Normal Human Peripheral Blood: In Vivo Suicide

Fas-L molecules expressed by in vitro stimulated T cells may be critically involved in suicidal activation-induced cell death (AICD) of such cells through engagement of their Fas receptors. A similar suicide of T cells was postulated to occur even in vivo, to eliminate dangerous activated lymphocytes; however, the demonstration of suicidal AICD of T cells in healthy humans in vivo is still lacking. We therefore investigated the possible occurrence of Fas-L-linked suicidal apoptosis of T cells in normal human peripheral blood. For this purpose, we took advantage of immunoelectron microscopy, which allows simultaneous visualization of the morphological apoptotic cellular changes together with surface expression of Fas-L molecules. Very few T lymphocytes were observed showing the ultrastructural features of apoptotic lymphocytes; these occasional apoptotic T cells, together with the majority of the normal T cell population, expressed the Fas molecule on the plasma membrane, as expected. Interestingly, the apoptotic cells were also Fas-L-positive, whereas normal T cells were Fas-L-negative. Such Fas-L-associated T cell suicide operating in vivo in healthy individuals is presumably able to suppress immune responses and prevent autoreactivity, thus maintaining the homeostasis of human blood.

CD95-ligand (Fas-L)-expressing epidermal lymphocytes

Sir, The functional role played by T lymphocytes within normal human epidermis are still a matter of debate. A major function of T lymphocytes is cytotoxicity against a number of targets, including infected/neoplastic cells, autoreactive B cells and antigen-bearing dendritic cells. One of the most important such cytotoxic mechanisms is the CD95 (Fas)/ CD95-ligand (Fas-L) system. In particular, Fas-L expression is induced in cytotoxic T lymphocytes (CTL) on antigen-specific recognition of the target cell; engagement of Fas-L on the activated CTL and Fas on the target cell, leads to signalling of a death programme in the target cell. We therefore investigated whether epidermal T lymphocytes express Fas-L. We used an immunoelectronmicroscopy (IEM) technique on epidermal cells suspended from normal human skin. This technique was chosen because it is ideally suited to detect even very scarce amounts of surface antigenic moieties of epidermal cells. Isolation of epidermal cells from specimens of normal human skin, obtained by plastic surgery operations, was performed according to an enzymatic procedure previously described. Suspended epidermal cells were then enriched in lymphocytes by utilizing a Ficoll±Hypaque gradient centrifugation. The enriched epidermal cell suspension was subjected to a previously reported immunogold labelling technique, using, after a `blocking preincubation, the mouse purified monoclonal antibody antihuman Fas-L G247±4 (PharMingen International, San Diego, CA, U.S.A.), and, thereafter, a F(ab)2 fragment of goat-antimouse IgG conjugated to 6 nm gold particles (Aurion, Wageningen, the Netherlands). The epidermal suspension was then subjected to a silver-enhancement method, detailed previously, with the only difference that the Aurion R-Gent reagents (Aurion, Wageningen, the Netherlands) were used, according to the manufacturers instructions. Finally, a standard ultrastructural examination was performed. Three controls for method specificity were carried out, as previously reported. Although epidermal lymphocytes constitute only a minority of epidermal cells, a discrete number of lymphocytes, recognizable on the basis of their ultrastructural characters, was detected, because of the enrichment technique. It was therefore possible to observe that a subset of such cells was indeed immunolabelled. In fact, these Fas-l-positive lymphocytes showed deposits of metallic silver on the cell surface (Fig. 1). The three types of controls gave negative results. The present study shows by IEM that a proportion of epidermal lymphocytes freshly isolated from normal human skin expresses the Fas-L molecule on its plasma membrane. In the last decade it has been clearly shown that a proportion of epidermal T lymphocytes freshly isolated from normal human skin expresses recent activation markers such as HLA-DR and CD25, consistent with a predominance of memory T lymphocytes in human epidermis, which reflects the chronicity of antigenic exposure in this tissue. This may also be the case for Fas-L expression by a proportion of epidermal lymphocytes freshly isolated from normal human skin, since Fas-L is also an activation marker for T cells. Fas-l-positive epidermal lymphocytes are obviously T lymphocytes, since virtually all epidermal lymphocytes are known to be T cells. Further investigations, including double-and triple-staining procedures, will be necessary to determine to which T cell subset(s) Fas-l-positive epidermal lymphocytes belong. Epidermal T cells predominantly belong to three subsets, namely, the CD4-positive subset, the CD8positive subset either aa type or ab type, and the CD4negative CD8-negative subset. CD4-positive lymphocytes which show cytotoxicity are known to preferentially use the Fas-L molecule to exert such a function. CD8-positive lymphocytes, which are professional CTL, can use not only the perforin/granzyme pathway, but also the Fas-L cytolytic molecule. Finally, CD4-negative CD8-negative lymphocytes exert CD1-restricted cytotoxicity, which is indeed typically mediated by the expression of the Fas-L molecule. In conclusion, the present investigation demonstrates by IEM that a subpopulation of epidermal lymphocytes freshly isolated from normal human skin expresses the cytolytic molecule Fas-L, and may participate in skin immunosurveillance as a result of cytolytic potential against a variety of epidermal Fas-expressing target cells bearing different antigens.

Immunoelectron microscopic characterization of a subpopulation of freshly isolated epidermal Langerhans cells that reacts with anti-CD23 monoclonal antibody

Large subsets of leucocytes were recently shown to express the low affinity receptor for the Fc portion of IgE. Because Langerhans cells (LC) are epidermal leucocytes, we investigated whether LC of normal human subjects might express this receptor. Whereas conventional immunofluorescence on epidermal sheets gave negative results, highly sensitive immunoelectron microscopy revealed that a subset (about one‐third) of freshly isolated LC express the CD23 molecule.

The tolerogenic molecule CD95-L is expressed on the plasma membrane of human activated, but not resting, Langerhans' cells.

Abstract:u2002 Although dendritic cells (DC) are well known for their immunogenic capacities, they may even induce peripheral T‐cell tolerance, and such a tolerogenic potential can be exerted in mouse through the expression on the DC plasma membrane of the CD95‐ligand (CD95‐L) molecule, which is able to trigger apoptosis of CD95‐expressing antigen‐specific T cells. We therefore asked whether epidermal DC, namely Langerhans cells (LC), either resting (i.e. within the epidermis, ‘in situ’) or activated (i.e. suspended from the epidermis) or both, could express the CD95‐L molecule on the plasma membrane. For such a purpose, two colloidal gold‐immunoelectron microscopy (IEM) double‐step procedures were carried out: an ‘in situ’ method, able to investigate resting LC, was performed on ultrathin frozen sections obtained by ultracryomicrotomy (UCMT) of normal skin biopsies; a pre‐embedding (P‐E) method, able to investigate suspended LC, was performed on epidermal cells (EC) suspended from normal skin specimens. In UCMT/IEM sections, resting LC showed gold particles within the cytoplasm but very rarely within organelles and never along the plasma membrane: resting LC are therefore capable of synthesizing CD95‐L but not of expressing it in a functional location, thus autoreactive phenomena against CD95‐expressing EC being avoided in normal epidermis. On the other hand, in P‐E/IEM preparations, suspended LC showed several gold particles along the plasma membrane: activated LC are therefore capable of expressing CD95‐L in a functional location, thus bearing the potential to exert tolerogenic capabilities against CD95‐expressing T cells, e.g. to prevent inflammatory/autoimmune cutaneous disorders and/or favor the resolution thereof.

The immunogold-silver staining approach in the study of lymphocyte subpopulations in transmission electron microscopy

The potential of immunogold-silver staining has been evaluated in immunoelectron microscopic studies of human normal peripheral blood lymphocyte subpopulations. The cells were labeled, before being embedded in resin, using 5 nm colloidal gold particles and this was followed by silver enhancement. The use of colloidal gold particles permits detection of small amounts of antigen; the silver intensification forms a sphere of heavy metal around the gold granule giving rise to an ultrastructural marker which can be easily seen even at low magnification. The ultrastructural details of the cells were well preserved and there was no significant background staining. The major advantage of the present IGS technique is that it permits a rapid and simultaneous evaluation of both the immunophenotype and the ultrastructural characteristics of cells.

Morphometric evaluation of CD16-positive cells with respect to CD2 antigen coexpression

We examined morphometric as well as functional characteristics of CD16-positive human peripheral blood lymphocytes on the basis of the coexpression of the CD2 antigen. For morphometric analyses, nuclear area and cellular area were determined by counting line cross-points of a superimposed quadratic lattice test system overlying nuclei and the whole cell, respectively. Moreover, to evaluate the cellular villousity degree, the maximum inscrible circle and an irregular polygon were inscribed within cell profiles. The cytoplasm fraction included between the plasmalemma and the traced irregular polygon was considered as the villous portion of the cell. Finally, the NK capability was measured in a 6-hr 51Cr-release assay with human K-562 myeloid cells as targets. Within the CD16-positive cell population, the CD16-positive/CD2-negative cells seem to represent the most efficient NK cell subset. To the higher NK capability correspond a higher villousity degree and a lower nuclear area/cellular area ratio of the CD2-negative/CD16-positive subset, when compared with CD2-positive/CD16-positive cells.