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A A Manfredi

Publications and source records attributed to A A Manfredi.

At least 55 records · Page 3Linked to original sources
PubMed1996

Etoposide at different concentrations may open different apoptotic pathways in thymocytes.

Apoptosis of thymocytes has been investigated by morphological, biochemical and cytometric techniques, both in non-perturbed conditions and after exposure to the topoisomerase-II inhibitor etoposide in vitro. Apoptotic thymocytes were already present in untreated samples, and their frequency increased with increasing drug concentrations. Using flow cytometry, we demonstrated that the frequency of apoptotic thymocytes in S and G2 phase of the cell cycle was higher in etoposide-treated samples, consistent with the phase-preferential action proposed for this topoisomerase-II inhibitor. At electron microscopy, the sequence of nuclear events was apparently independent from the apoptogenic trigger, so that similar features of chromatin margination and nuclear fragmentation were observed in thymocytes undergoing apoptosis both spontaneously and after etoposide treatment. The morphology and function of plasma membrane were still preserved in apoptotic cells. At low (0.1 to 10 microM) etoposide concentrations apoptotic cells had cytoplasm patterns similar to naturally occurring apoptotic thymocytes, whereas at high (50 to 100 microM) concentrations extensive organelle damage took place. Damaged cytoplasm was sometimes polarized into discrete portions, which were eventually extruded within apoptotic bodies. This evidence suggests that, in the same cell type, different apoptotic pathways are opened by this agent, depending on the concentration used.

Animals↗
PubMed1995

Daudi lymphoma killing triggers the programmed death of cytotoxic V gamma 9/V delta 2 T lymphocytes.

A common feature of human V gamma 9/V delta 2 gamma delta T lymphocytes is their ability to kill Daudi lymphoma cells. We show here that during this killing a substantial fraction of cytotoxic gamma delta T cells dies as well. Their death has morphologic, cytometric, and biochemical features of apoptosis and depends on TCR triggering. We suggest that interaction with the target physiologically induces the programmed death of the cytotoxic gamma delta effector. The death of activated gamma delta T cells upon challenge with the relevant Ag may represent a negative feedback mechanism and contribute to explain the limited time span of gamma delta T cell expansions during infectious diseases, even when pathogens are not eliminated and persist in the host.

Apoptosis↗
PubMed1994

Constitutive expression of the heat shock protein 72 kDa in human melanoma cells.

Heat shock proteins (hsp) are a family of proteins characteristically produced under stress conditions in normal cells. Overexpression of hsp 70 kDa protects tumoral cells from tumor necrosis factor cytotoxicity and is related to drug resistance. In this study we investigated whether hsp are abnormally expressed in melanoma cells in vivo. Antibodies directed against hsp 72 and hsp 90 were used to identify hsp on non-stressed neoplastic cells derived from surgical specimens of two primary and four metastatic melanomas. Hsp 72 and hsp 90 were always present at high level in the cytoplasm of melanoma cells. It is possible that the activation of hsp genes during neoplastic transformation confers a selective advantage to tumoral cells in vivo, allowing them to escape the immunological surveillance.

Cells, Cultured↗
PubMed1994

Human acetylcholine receptor presentation in myasthenia gravis. DR restriction of autoimmune T epitopes and binding of synthetic receptor sequences to DR molecules.

Autoimmune Th cells in myasthenia gravis recognize several sequence regions of the human muscle acetylcholine receptor (AChR). Most AChR Th epitopes are presented by HLA class II DR molecules (DR). Four sequence regions of the AChR alpha-subunit form Th epitopes recognized by most myasthenic patients, irrespective of their DR haplotype. In this study we first identified in five myasthenic patients the DR molecule(s) likely to be involved in presentation of T immunodominant AChR sequences. We then investigated the binding to the affinity purified DR molecules thus identified (DR2/w51, DR4/w53, and DR7/w53) and to the DR1 molecule, of a panel of overlapping synthetic peptides screening the human alpha-subunit sequence, previously used to identify AChR Th epitopes in myasthenic patients. The AChR peptides that stimulated anti-AChR autoimmune Th cells all bound the relevant DR molecules. Some AChR peptides never recognized by Th cells of myasthenic patients also bound well to one or more DR molecules. The relative ability to bind to DR molecules of different sequence regions of the AChR, i.e., an autoantigen, agrees well with the results of previous studies on the DR binding of synthetic sequences of exogenous antigens. Some peptide sequences uniquely bound one DR molecule, others bound several DR molecules, and others did not bind any of the DR molecules tested.

Amino Acid Sequence↗
PubMed1994

In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen-presenting cells.

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC-I complexes and the presence of co-stimulatory molecules on antigen-presenting cells (APC). The antigen-processing defective mutant cell line RMA-S, when cultured at low temperature, expresses high amounts of MHC-I molecules that do not contain endogenously derived peptides. These "empty" MHC-I molecules can be stabilized by addition of MHC-binding peptides. RMA-S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA-S cells do not express detectable amounts of B7 co-stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA-S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co-stimulation for CD8+ T lymphocytes. RMA-S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when "sub-optimal" antigen peptides were used. Most importantly, RMA-S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA-S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co-stimulation and relative occupancy of MHC-I by single peptide epitopes in CTL priming.

Amino Acid Sequence↗
PubMed1993

Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4+ cells.

Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits. Only three epitopes on the alpha subunit were recognized consistently. Mice immunized with large doses (nanomoles) of TAChR clearly recognized only the immunodominant sequence T alpha 150-169. Anti-TAChR CD4+ cells did not cross-react with murine alpha subunit sequences, or with any synthetic sequence of human gamma and delta subunits, which are very similar to the corresponding murine subunits. To facilitate recognition of cryptic epitopes, we injected mice with pools of synthetic peptides corresponding to the sequences of TAChR alpha, gamma, and delta subunits. In addition to the three immunodominant alpha subunit epitopes, other epitopes were recognized by CD4+ cells within the sequences T alpha 304-322, T gamma 105-124, T gamma 120-139, T gamma 401-420, T gamma 357-376, T delta 16-35, T delta 61-80, T delta 121-140, and T delta 301-320. CD4+ cells thus sensitized cross-reacted with the mammalian sequences alpha 304-322, gamma 105-124, gamma 120-139, and delta 301-320. Mice were immunized with large doses (approximately 40 nmol) of individual TAChR synthetic cryptic epitopes. CD4+ cells sensitized to five cryptic epitopes (the ones listed above plus delta 121-140) cross-reacted with autologous sequences. We determined the dose dependence of the sensitization of CD4+ cells in vivo to the strongly immunodominant epitope peptide T alpha 150-169 and to the cryptic epitope peptides T gamma 120-139 and T delta 301-320 by immunizing mice with increasing doses of peptide (approximately 1.2 to approximately 20 nmol), and testing the in vitro anti-peptide response of the CD4+ cells. No difference was found for the epitopes tested. Doses of 3 to 10 micrograms induced a strong CD4+ sensitization, and the dose dependence of the in vitro response of the sensitized cells to the relevant peptide was comparable. Production of cryptic epitopes upon in vitro TAChR processing was investigated by testing peptide-sensitized CD4+ cells with native TAChR: only two cryptic epitopes were produced.

Amino Acid Sequence↗
PubMed1993

T-helper epitopes on human nicotinic acetylcholine receptor in myasthenia gravis.

The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

An assay for simultaneous multiple determinations of peptide binding to MHC class II molecules.

Binding of positively charged radiolabeled synthetic peptides to human major histocompatibility complex class II DR molecules, purified by affinity chromatography from lymphoblastoid B cell lines of different haplotypes, is rapidly, quantitatively, and specifically assayed by selective adsorption of the complexes between peptide and DR molecules onto DEAE-cellulose paper disks. This assay can be used as a revealing system of the ability of unlabeled test peptides to competitively inhibit the binding between the radiolabeled peptide and the DR molecules, thus measuring the binding of the competitor peptides, irrespective of their charge properties, to different DR molecules.

Amino Acid Sequence↗
PubMed1993

Myasthenia gravis: recognition of a human autoantigen at the molecular level.

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.

Animals↗
PubMed1993

T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.

Adult↗
PubMed1993

The 'embryonic' gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle.

We have PCR-amplified cDNA sequence of the "embryonic" gamma subunit of muscle acetylcholine receptor (AChR) from adult bovine extraocular muscle (EOM). We cloned and sequenced this product and used it to probe Northern blots. We detected the gamma subunit in EOM mRNA, but not in control skeletal muscle. The presence of gamma subunit in EOMs may explain their preferential involvement in myasthenia gravis, where an autoimmune response to the gamma subunit is generally present.

Animals↗
PubMed1993

A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes.

In this study we describe a cytometric method to sort apoptotic cell fractions, suitable for biochemical and morphological analyses. Rat thymocytes were used as a model system, as apoptosis naturally occurs in the thymus, where the negative selection of the T cell repertoire takes place. Massive apoptosis was induced in vitro by the topoisomerase-II inhibitor, etoposide. After etoposide treatment, a large fraction of thymocytes showed the morphological and electrophoretic features of apoptotic cells. Unfixed thymocytes were stained for 30 min with propidium iodide (PI: 50 micrograms/ml containing RNase type A and detergent), and analyzed by flow cytometry. Apoptotic thymocytes typically showed sub-G1 DNA contents. Compared to non-apoptotic thymocytes, sub-G1 cells had lower values of low-angle (FSC) scatter and higher values of right-angle (SSC) scatter, so that, in dual parameter cytograms of FSC versus SSC, two distinct cell populations were apparent, and were separated by flow sorting. The purified cell fractions obtained by this procedure had a very well preserved ultrastructural morphology; both early and late apoptotic stages (until the onset of cytoplasm segmentation) were present in the sorted apoptotic fraction.

Animals↗
PubMed1992

T helper function of CD4+ cells specific for defined epitopes on the acetylcholine receptor in congenic mouse strains.

We previously identified sequence segments of Torpedo acetylcholine receptor (TAChR) alpha subunit recognized by CD4+ cells of congenic mouse strains of different H-2 haplotypes, susceptible to experimental autoimmune myasthenia gravis. CD4+ cells from BALB/c and CB17 mice (H-2d) recognized the peptide sequences alpha 1-20 and alpha 304-322, while C57BL/6 and BALB/b mice (H-2b) recognized alpha 150-169 and alpha 360-378. C57BL/6 mice recognized to a lesser extent also peptide alpha 181-200. In the present study we demonstrate that CD4+ cells which recognize these epitopes have T-helper function. CD4+ cells from TAChR immunized mice, stimulated in vitro with synthetic epitope peptides, induced proliferation in vitro of B cells via soluble factors which were not strain specific, and induced secretion in vitro of anti-AChR antibodies. Upon in vitro stimulation with T-epitope peptides, they secreted interleukin-2. Immunization of mice with synthetic T-epitope peptides caused sensitization of CD4+ cells, which responded in vitro both to the immunizing peptides and to TAChR, and appearance of anti-AChR antibodies in vivo, further identifying the epitope-specific CD4+ cells as AChR-specific T-helper cells.

Animals↗
PubMed1992

Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor.

In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients. We identified sequence segments of the human gamma subunit forming epitopes recognized by four embryonic AChR-specific CD4+ T cell lines, propagated from MG patients' blood by stimulation with synthetic peptides corresponding to the human gamma subunit sequence. Each line had an individual epitope repertoire, but two 20-residue sequence regions were recognized by three lines of different HLA haplotype. Most T epitope sequences were highly diverged between the gamma and the other AChR subunits, confirming the specificity of the T cells for embryonic AChR. These T cells may have been sensitized against AChR expressed by a tissue other than innervated skeletal muscle, possibly the thymus, which expresses an embryonic muscle AChR-like protein, containing a gamma subunit. Several sequence segments forming T epitopes are similar to regions of microbial and/or mammalian proteins unrelated to the AChR. These findings are consistent with the possibility that T cell cross-reactivity between unrelated proteins ("molecular mimicry"), proposed as a cause of autoimmune responses, is not a rare event.

Adult↗
PubMed1992

CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR alpha subunit. Several epitopes were identified. Only the most severely affected patients recognized alpha subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several alpha subunit peptide sequences could be detected when CD(8+)-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any alpha subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48-67, 101-137, 293-337, and 308-437).

Amino Acid Sequence↗
PubMed1991

Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor.

Autoimmune T cell lines specific for muscle nicotinic acetylcholine receptor (AChR) were propagated from the blood of three myasthenia gravis patients by the use of a pool of synthetic peptides (delta-pool) corresponding to the complete sequence of the delta-subunit of human muscle AChR. Propagation of AChR-specific T cell lines was attempted unsuccessfully from four other myasthenia gravis patients and from four healthy controls. The lines had CD3+, CD4+, CD8- phenotype, strongly recognized the delta-pool, and cross-reacted vigorously with non-denatured AChR purified from mammalian muscle. They did not cross-react detectably with pools of similar overlapping synthetic peptides corresponding to the complete sequences of the alpha- and gamma-subunits of human muscle AChR. The sequence segments of the delta-subunit that contain T epitopes were identified by investigating the response of the three CD4+ T cell lines to the individual synthetic peptides forming the delta-pool. Each line had an individual pattern of peptide recognition. Although no immunodominant region, recognized in association with different DR haplotypes, could be identified, the sequence segments most strongly recognized by the CD4+ T cell lines were clustered within residues 121-290. One of the peptides more strongly recognized by the T cells corresponded to a sequence segment with high predicted propensity to form an amphipathic alpha-helix, a structural motif proposed to be typical of T epitopes.

Amino Acid Sequence↗