Biomedical subjects

D Mauri

Publications and source records attributed to D Mauri.

At least 19 recordsLinked to original sources

PubMed1998

Capillary zone electrophoresis of ds-DNA in isoelectric buffers: effect of adding of competing, nonamphoteric ions.

When separating ds-DNA in isoelectric His buffer (pH=pI=7.6), in the 50-250 mM concentration range, some unique phenomena were observed: improved resolution for smaller DNA fragments, up to ca. 150 bp, and a rapid deterioration of resolution above this critical length (which corresponds to the persistence length). Such phenomenon depended also on voltage and concentration of sieving liquid polymer. Direct binding of His to the DNA helix was hypothesized, with resultant stiffening and an increment of diameter of the DNA fragments, thus inducing an early onset of reptation at the applied voltage in the 100-300 V/cm range. In order to prove this hypothesis, "competing ions" (notably NaCl and KBr) were added to the His background electrolyte: a partial reversal of the His effect was already apparent at low concentrations of such ions (10 mM) and was complete at higher concentrations (30 and 50 mM). By molecular modeling, it was found that His could be docking on the negatively charged oxygen (bound to the phosphate) by offering both charged (primary and tertiary amino) groups to simultaneous binding, thus forming a salt and neutralizing the negative charge borne by the oxygen. The following characteristic bond distances were found: 0.34 nm between the N (imidazolic) and O; 0.32 nm between the primary N and O; 0.36 nm between the two nitrogens engaged in salt formation with the oxygen. In addition, for complexation to occur, the distance between the noncharged nitrogen in the imidazole ring and the nearest phosphate oxygen (engaged in the phosphodiester bridge) should be 0.44 nm. Under these conditions, the two rings present (a six-membered, ideal one, salt-linked with the oxygen and rather highly elongated, and the imidazole) will not be precisely coplanar, since the primary and tertiary nitrogens will be one slightly above and one slightly below the plane of the drawing. Upon extensive binding, occupying every available phosphate site, pi-pi interactions could occur among the stacks of bound His residues, thus further stabilizing the complex.

Buffers↗

PubMed1997

Plasma mitomycin C concentrations determined by HPLC coupled to solid-phase extraction.

The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer.

Antibiotics, Antineoplastic↗

PubMed1996

Cross-reactivity of T cell lines and clones to beta-lactam antibiotics.

To clarify on a molecular level the specific T cell response to haptens like penicillin G, we generated T cell lines and clones from penicillin-allergic patients. Two types of beta-lactam reactivity of T cells could be delineated: one group of patients showed a rather restricted specificity, as the penicillin-elicited T cell lines generated from such donors proliferated only to the stimulating penicillin, but not to other beta-lactam antibiotics nor to cephalosporines, even if the side chain was identical. This indicates that the penicilloyl structure together with the side chain was recognized by these T cells. The second group comprised patients with more broadly reactive T cells, as they were restimulated by penicillin G as well as by related penicillins like amoxicillin or ampicillin, but not cephalosporines. This indicates that the penicilloyl structure, a common motif of penicillins, was important for T cell recognition. Clones generated from a broadly reactive patient confirmed this heterogeneity, as either monospecific or broadly specific T cell clones could be identified. This broad or very restricted pattern of T cell reactivity was reflected in the use of TCR Vbeta-chains: while the broadly reactive T cell lines showed a heterogenous TCR usage, the highly restricted T cell lines showed an up-regulation of one TCR Vbeta-chain. Thus, our data suggest that the outgrowth of T cells bearing a certain TCR Vbeta may be a sign of a limited cross-reactivity.

Anti-Bacterial Agents↗

PubMed1996

Involvement of CD80 in the generation of CD4+ cytotoxic T cells.

CD4+ T cells can exert different effector functions, which are partly distinguishable by the secretion of different cytokines, namely by either IFN-gamma, IL-2 and lymphotoxins for Th1-like or IL-4, IL-5, IL-10 and IL-13 for Th2-like T cells. Th1-like T cells can exert cytotoxic functions, too. The cytokinetic phenotype of an activated T cell clone (TCC) is mainly influenced by the cytokinetic pattern of the microenvironment where it was activated. However, the interaction between certain adhesion molecules (i.e. CD28-CD80 and CD28-CD86) may also have an influence on the functionality of the reactive T cell. On the contrary, the requirements for the induction of CD4+ cytotoxic T cells (CD4+ CTLs) are not well understood. We have focused this review on studies investigating the development of CD4+ T cells with cytotoxic effector functions. In particular, we discuss here whether the type of antigen-presenting cells (APCs) and the distinct expression of important adhesion molecules like CD80 and CD86 may influence the generation of CD4+ CTLs. Among a large panel of APCs only dendritic cells and TCCs are able to induce cytotoxicity. The level of CD80, but not of CD86, present on the APCs appears to be crucial for the induction of CD4+ CTLs.

Animals↗

PubMed1996

Successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous outer root sheath cells.

The outer root sheath cells of hair follicles can substitute for interfollicular epidermal keratinocytes, as during healing of skin wounds when these cells migrate onto the denuded area and contribute to epidermal regeneration. Using improved culture techniques, we generated epidermal equivalents from cultured outer root sheath cells of patients suffering from recalcitrant chronic leg ulcers, primarily of vascular origin. In such epidermal equivalents, tissue organization as well as immunolocalization of epidermal differentiation products (keratin 10, involucrin, filaggrin) and integrins were indistinguishable from normal epidermis. As determined by the number of bromodeoxyuridine-incorporating cells, the basal layer contained a large compartment of proliferative cells irrespective of donor age. FACS analysis of the outer root sheath cells, used to prepare the epidermal equivalents, disclosed a fraction of small cells with enhanced expression of beta1-integrin, a potential stem cell marker. in contrast to acute wounds, a major definitive take of grafted cultured autologous keratinocytes has not been convincingly demonstrated in chronic wounds. In a pilot study, grafting of epidermal equivalents generated in vitro from autologous outer root sheath cells on 11 ulcers in five patients resulted in a definitive take rate of about 80%, with subsequent complete healing within 2 to 3 wk of five out of seven ulcers grafted with densely arranged cultures. This improvement in the treatment of chronic leg ulcers with cultured autologous keratinocytes probably depends on the large compartment of proliferative cells as well as on a well-developed horny layer which prevents disintegration of the grafts. Practical advantages of the new technique are its noninvasiveness, the lack of need for surgical facilities or anesthesia, and a short immobilization period after grafting.

Aged↗

PubMed1995

Antigen-presenting T cells induce the development of cytotoxic CD4+ T cells. I. Involvement of the CD80-CD28 adhesion molecules.

The development of cytotoxic CD4+ T lymphocytes that can kill target cells in a MHC class II-restricted manner was evaluated by comparing different APCs. B-lymphoblasts (B-LCL) pulsed with the superantigen staphylococcus enterotoxin B or allogeneic B-lymphoblasts induce CD4+ T cells without cytotoxic activity. In contrast, superantigen-pulsed, MHC class II+ T cell blasts or allogeneic T cell blasts preferentially induce the development of specific, MHC class II-restricted CD4+ cytotoxic effector cells. CD4+ T cell clones generated with T or B cell blasts as APCs (T- or B-APCs) differ in their cytolytic potential, but secrete a similar cytokine pattern. Our data implicate that activated T-APCs preferentially induce a cytotoxic, CD8+ and CD4+ T cell response. Because the density of CD80 expression is lower on activated T-APCs than on B-APCs, we studied the involvement of CD28 and CD80 adhesion molecules in the generation of CD4+ CTLs. Partial blockade of the CD80 molecule with a CTLA4-Ig fusion protein and with specific anti-CD80 mAbs on B-APCs enhanced the generation of CD4+ CTLs. Specific anti-CD86 mAbs, on the contrary, had no effect on the generation of CD4+ CTLs. In contrast, stimulation of CD28, the CD80 counter-receptor, with a cross-linked B7-Ig fusion protein or with an anti-CD28 mAb, inhibited the generation of CD4+ CTLs. Thus, a reduced interaction between CD80 and CD28 may be relevant for the induction of CD4+ CTLs. This shows a new and not yet described function of these adhesion molecules. This induction of a cytotoxic immune response by T cells as APCs may be relevant for the anticlonotypic regulation of T cells and for the depletion of CD4+ T cells in HIV infection.

Abatacept↗

PubMed1995

Activation of drug-specific CD4+ and CD8+ T cells in individuals allergic to sulfonamides, phenytoin, and carbamazepine.

To investigate how T cells are involved in hypersensitivity reactions to drugs that become immunogenic after metabolization, e.g., sulfonamides and antiepileptics, we analyzed in vitro the drug-induced activation of CD4+ and CD8+ T cell subsets, cytokine secretion, TCR V beta distribution, and proliferation of T cells from four drug-allergic individuals. In addition, the activation parameters CD25 and HLA-DR were analyzed in vivo on CD4+ and CD8+ T cells from five patients with acute drug allergies, some of them with anticonvulsant hypersensitivity syndrome with hepatitis. Our results show that, in vitro, drug-induced proliferation of PBMC from patients with allergy to sulfamethoxazole, phenytoin, or carbamazepine was specific and dose dependent. CD4+ as well as CD8+ T cells expressed elevated levels of CD25 and HLA-DR molecules after drug stimulation. Drug-activated lymphocytes secreted high amounts of IL-5 and normal or low levels of IL-2, IFN-gamma, IL-4, and TNF-alpha. An enhanced expansion of TCR V beta 17+ T cells 9 days after in vitro stimulation with sulfamethoxazole was observed in one patient with sulfamethoxazole allergy. The drug specificity of the in vitro-activated T cells was confirmed by generation of different sulfamethoxazole specific T cell lines and CD4+ and CD8+ T cell clones. T cell analysis of patients with acute drug allergy to carbamazepine, phenytoin, allopurinol, or paracetamol confirms the in vitro data, because all patients had activated CD4+ or CD8+ T cells in the circulation. Our data clearly show the involvement of drug-specific T cells in drug allergies.

Anticonvulsants↗

PubMed1995

Noncytotoxic human CD4+ T-cell clones presenting and simultaneously responding to an antigen die of apoptosis.

Activated T-cells expressing MHC class II surface antigens are able to present antigen and thus function as peptide-presenting cells (T-APCs). In this study we investigated whether antigen presentation by T-cells induced programmed cell death. As a model we used tetanus p30 peptide (aa 947-967)-specific, noncytotoxic CD4+ T-cell clones (C11 and C31). For experimental purposes these T-cell clones were stimulated (a) with p30 peptide-pulsed and fixed EBV-transformed antigen-presenting cells (B-APCs), (b) with p30-pulsed and fixed activated T-cells as APCs (as T-APCs we used either the T-cell clones themselves or an autologous T-cell clone (CT3) with p30 unrelated specificity), or (c) with soluble p30 peptide. The efficiency of antigen presentation was monitored by measuring proliferation as [3H]thymidine uptake. Apoptosis was measured by quantifying fragmented, cytoplasm DNA with the fluorescent dye 4,6-diamidino-2-phenylindole or by visualizing fragmented DNA by gel electrophoresis. Stimulation with p30-pulsed and fixed B-APCs or T-APCs induced proliferation but no apoptosis of the responding T-cells. However, stimulation of cloned T-cells with soluble peptide induced up-regulation of the FAS surface molecules and apoptosis, which was dependent on the peptide doses. Because cloned T-cells express HLA class II molecules, they can theoretically exert both functions at once: antigen presentation and antigen response when they are stimulated with soluble peptide. Because death by apoptosis is only seen under such circumstances, we suggest that T-cells simultaneously presenting and responding to an antigen die of apoptosis and thus contribute to the down-regulation of the immune response. Such phenomena might occur in HIV infection when activated CD4+ T-cells take up gp120 via their CD4 molecules, present it on their HLA class II surface antigens, and are simultaneously stimulated via their TCR.

Antigen-Presenting Cells↗

PubMed1994

Improved sensitization of antigen-presenting cells with transferrin-bound peptides: advantages in competition for antigen presentation.

T cells recognize peptides in association with major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). To sensitize APC for antigen presentation in vitro and in vivo, high concentrations of synthetic peptides can be added from the outside and bind to the MHC molecules, thereby mimicking naturally processed peptides. In this report we investigated whether the transferrin (Tf) molecule could be used as a carrier to introduce antigenic peptides into the antigen presentation pathway of APC. We coupled to Tf various MHC class II DR1 restricted peptides and compared the sensitization of DR1+ APC by the Tf-bound or by the soluble peptide, using peptide-specific T cell clones (TCC). The presentation of the Tf-bound peptides was MHC restricted and could be blocked by the fixation of the APC with glutaraldehyde or by the addition of an excess of Tf. Tf-bound peptides were more efficiently presented than soluble peptides, since smaller concentrations were required to sensitize APC. Moreover, they could compete with a soluble peptide for MHC restricted presentation with a very high efficiency if compared to soluble competing peptides. Tf peptide conjugates could even compete with the presentation of a native antigen like tetanus toxoid. Peptides bound to the transferrin molecule might be useful for immunization strategies, as the relevant bound peptides are efficiently presented to peptide-specific TCC.

Antigen Presentation↗

PubMed1994

Peptide-induced T cell clones: specificity, MHC restriction, proliferation and cytokine pattern as a function of different stimulations.

CD4+ T cell clones were generated to tetanus toxin or to two tetanus toxin-derived peptides p2 (AA 830-834) and p30 (AA 947-976). 11 of the 24 p30-specific clones reacted to shorter p30 subunits (p301 or p302), and only 14 of the p2 or p30-specific clones reacted with TT presented by EBV-transformed B cell lines (B-LCL). The p30-specific clones were HLA-DP4 restricted. In contrast to autologous B cell lines, the majority of allogeneic, but HLA-DP4-positive cell lines failed to present p30 to the specific clones. We concluded that T cell clones are highly specific and that both, small alterations of the peptide length as well as discrete differences of the HLA-molecule may abrogate recognition of the peptide HLA complex by T cells. Moreover, use of peptides as stimulators of T cells may recruit and activate T cells which fail the "original" peptide, derived from normal antigen processing. Clones could usually be maintained in culture for 4-6 months, but with the help of freezing and thawing some clones are now available for over 2 years and still specific. Comparison of different autologous antigen-presenting cells, namely B-LCL and activated MHC class II-positive T cells revealed that not all clones were able to mount a proliferative response to peptide presentation by T cells, while all clones proliferated to B cells as APC. If stimulated with peptide and B-LCL, the clone proliferating to T cells as APC (so-called T responder clones) secreted a broad spectrum of cytokines (Th0-like) and were easier to maintain in culture. In contrast, clones which were unable to proliferate to peptide presentation, so-called T-nonresponder clones, showed a more restricted cytokine pattern and elevated or very low IL4/IFN gamma ratio upon antigen specific stimulation. However, all clones secreted at least small amounts of IL2, IL4, IFN gamma and TNF alpha, if stimulated by PMA and ionomycin. Thus, both chemical and antigen-specific stimulations should be considered if T cell clones are classified as Th1 or Th2, whereby those clones, which secrete a limited cytokine pattern after antigen stimulation only, might be named Th1 or Th2 like clones, while clones which even after PMA/ionomycin do not secrete all cytokines, might represent "real" Th1 or Th2 clones.

Clone Cells↗

PubMed1993

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide.

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57-68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8+ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s,r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Antigen-presenting human T cells and antigen-presenting B cells induce a similar cytokine profile in specific T cell clones.

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen-presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM-1, LFA-3 and B7, they might play a role as APC and be involved in the regulation of T-Tcell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide- or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B-LCL) or HTLV-1-transformed T cells, or with non-classical APC, like activated keratinocytes or eosinophils. CD4+, DP-restricted T cell clones specific for a tetanus toxin peptide (amino acids 947-967) and CD4+, DR-restricted allospecific T cell clones produced interleukin (IL)-2, IL-4, tumor necrosis factor-alpha and interferon-gamma (IFN-gamma) after phorbol 12-myristate 13-acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose-response curves revealed that the antigen-presenting capacity of activated, MHC class II+, B7+ T cells was comparable to the one of B-LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN-gamma/IL-4 ratio. Cytokine-treated, MHC class II+ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM-1-positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM-1 and LFA3), provided a reduced signal resulting in a diminished IFN-gamma/IL-4 ratio. We conclude that non-classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM-1) expressed seems to determine the efficacy of this presentation.

Antigen-Presenting Cells↗

PubMed1991