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Biomedical subjects

M Bellone

Publications and source records attributed to M Bellone.

At least 37 records · Page 2Linked to original sources
PubMed1996

Heterogeneous effects of B7-1 and B7-2 in the induction of both protective and therapeutic anti-tumor immunity against different mouse tumors.

Experimental mouse tumors are classified as intrinsically immunogenic when, after a single injection into syngeneic mice as nonreplicating cell vaccines, they elicit a protective immune response against a subsequent lethal challenge. Tumors that do not retain this residual immunogenicity are defined as poorly immunogenic or nonimmunogenic. The expression of the B7-1 co-stimulatory molecule on immunogenic tumors can further increase their capacity to induce a T cell-dependent anti-tumor immunity, whereas it has limited effects on nonimmunogenic tumors. Recently, B7-2, a second molecule with an apparently similar co-stimulatory activity, has been cloned. In this report, we compare the efficiency of nonreplicating cells from one immunogenic and two nonimmunogenic mouse tumors transfected with B7-1 or B7-2 in the induction of protective and curative anti-tumor immunity. Immunogenic lymphoma cells expressing B7-1 or B7-2 are equally effective in both protecting against a subsequent challenge and curing established tumors. By contrast, nonimmunogenic adenocarcinoma and melanoma cells expressing B7-2 provide superior protective immunity, and only B7-2+ adenocarcinoma cells induce an efficient curative immunity. CD8+ and polymorphonuclear cells, but not CD4+ T cells, are critically involved in the rejection of the adenocarcinoma elicited by both B7-1+ and B7-2+ vaccines. These data indicate that B7-1 and B7-2 are not redundant co-stimulatory molecules and that, in these experimental models, B7-2 is superior to B7-1 in the induction of an efficient immunity when the immunogenicity of a tumor is a limiting factor.

Adenocarcinoma↗
PubMed1995

Co-expression of B7-1 and ICAM-1 on tumors is required for rejection and the establishment of a memory response.

Although the transfection of B7-1 cDNA into a few mouse tumor cell lines can induce anti-tumor T cell immunity, its expression alone is ineffective in many other tumor cell lines tested. We were interested to study what factors limit B7-1 co-stimulatory activity, and decided to investigate whether B7-1 requires the cooperation of ICAM-1 to provide the minimal co-stimulatory signal for establishing an efficient anti-tumor immunity. We show that the transfection of B7-1 cDNA into three ICAM-1+ (plasmocytoma J558L, T lymphomas EL-4 and RMA), but not into two ICAM-1- tumors cell lines (adenocarcinoma TS/A and melanoma B16.F1), is sufficient to induce their complete rejection in syngeneic mice. The expression of ICAM-1 is necessary for the rejection of the B7 expressing tumors, since the primary response elicited by B7-1+ EL-4 and RMA clones expressing reduced levels of ICAM-1 is severely reduced. Furthermore, super-transfection of ICAM-1 cDNA into B7-1+ adenocarcinoma and melanoma clones optimizes their primary rejection. Histologic examination of transfected tumors reveals that B7-1 and ICAM-1 exert a potent pro-inflammatory activity. The intra-tumor infiltration is composed of both eosinophils and lymphomonocytes, and is already massive 5 days after the tumor challenge. The primary rejection of the B7-1+ ICAM-1+ tumors depends critically on CD8+ T cells, natural killer cells and granulocytes, but is independent of CD4+ T cells. Remarkably, in addition to its effects on the early phases of the immune response, the co-expression of ICAM-1 and B7-1 on tumors is also necessary for the efficient induction of a memory response. In fact, only the primary challenge with B7-1+, ICAM-1+ tumor cells protects the majority of the mice from a second injection of parental tumor cells. Collectively, our findings indicate that B7-1 and ICAM-1 are fundamental components for triggering the primary rejection of tumors and establishing a protective memory response. These findings may help to define new strategies for the rational application of co-stimulation in tumor immunotherapy.

Adenocarcinoma↗
PubMed1995

Clustering of B and T epitopes within short sequence regions of the nicotinic acetylcholine receptor.

The epitope repertoire of B cells, due to their selective ability to process their specific antigen and the potential bias imposed on the resulting peptides by the surface immunoglobulins bound to the antigen, may influence the T-helper repertoire. Immunization of C57B1/6 mice with Torpedo acetylcholine receptor (TAChR) causes experimental autoimmune myasthenia gravis (EAMG). Anti-TAChR CD4+ cells recognize epitopes within three sequence regions of the TAChR alpha subunit ('dominant epitopes'). Immunization of mice with denatured or synthetic TAChR antigens sensitizes CD4+ cells to other TAChR sequence regions ('cryptic epitopes'). We investigated here whether clustering of B and T epitopes within the same short sequence segments occurs during the anti-TAChR response, as previously described for the response to hexogenous antigens unrelated to homologous self proteins. Twelve 19-20 residue synthetic sequences of the TAChR alpha, gamma and delta subunits, containing dominant or cryptic CD4+ epitopes for C57B1/6 mice, were tested for ability to induce anti-peptide antibody production. C57B1/6 mice were immunized with the individual peptides. Ten peptides stimulated antibody production. Therefore > 80% of these short TAChR sequences also contain B epitopes. Therefore also in the anti-TAChR response leading to EAMG T and B cell epitopes frequently reside within the same short sequence segment.

Amino Acid Sequence↗
PubMed1995

Mechanisms by which the I-ABM12 mutation influences susceptibility to experimental myasthenia gravis: a study in homozygous and heterozygous mice.

The I-Abm12 mutation in C57B1/6 (B6) mice yields the B6.C-H-2bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4+ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR alpha subunit which dominate the CD4+ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12 x B6)F1 mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4+ cells recognized the same TAChR alpha subunit peptide sequences (T alpha 150-169, T alpha 181-200 and T alpha 360-378). CD4+ cells from TAChR-sensitized F1 mice were challenged with TAChR and alpha subunit epitope peptides, using F1, B6 or bm12 APC. B6 and F1 APC presented all these Ag efficiently, while bm12 APC presented TAChR and peptide T alpha 150-169 poorly and erratically. Anti-TAChR and anti-alpha subunit epitope CD4+ lines propagated from F1 and B6 mice had similar TcR V beta usage. All lines but those specific for the sequence T alpha 150-169 had unrestricted V beta usage. Anti-T alpha 150-169 lines from both B6 and F1 mice had a strong preferential usage of V beta 6. Anti-T alpha 150-169 lines from F1 mice had also a slightly higher V beta 14 usage. B6, bm12 and F1 mice developed similar anti-TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti-AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4+ cells sensitized to epitopes within the sequence T alpha 150-160, consequent to the inability of the I-Abm12 molecule to present this sequence.

Animals↗
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

Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes.

T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope. However, when the T epitope peptide T alpha 360-378 was co-injected with the B epitope, Ab were also produced against the B epitope peptide. Injection of the individual peptides T alpha 360-378 and T alpha 63-80 at different and distant sites along the back of mice elicited sensitization of CD4+ cells and Ab production only against peptide T alpha 360-378. Therefore, when optimal cooperation between T and B cells occurs, spatial proximity but not covalent association of the B and the CD4+ epitope is necessary for production of Ab against the B epitope.

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↗
PubMed1994

Residues within the alpha subunit sequence 304-322 of muscle acetylcholine receptor forming autoimmune CD4+ epitopes in BALB/c mice.

BALB/c mice develop myasthenic symptoms after immunization with rodent acetylcholine receptor (AChR). After immunization with Torpedo AChR (TAChR), their CD4+ cells become strongly sensitized against a conserved region of the TAChR alpha subunit sequence (residues alpha 304-322), and cross-react vigorously with the homologous sequences of mouse and human AChR, which are almost identical. Therefore AChR-specific potentially autoreactive CD4+ cells exist in this strain. We immunized BALB/c mice with the synthetic TAChR sequence alpha 304-322. The CD4+ cells thus sensitized responded to TAChR, indicating that they recognize an epitope(s) produced upon TAChR processing. They recognized peptide alpha 304-322 in association with the I-Ad molecule. Anti-alpha 304-322 CD4+ cells cross-reacted well with the corresponding murine and human synthetic sequences. To identify residues involved in formation of an autoimmune epitope(s), CD4+ cells from mice immunized with peptide alpha 304-322 were challenged in vitro with single residue glycine-substituted analogues of this sequence. Substitution of residue W311, and of any residue within the sequence alpha 313-319 (RKVFIDT), consistently and, in some cases, strongly affected the CD4+ cells response. Substitution of residues in the region alpha 311-319 had variable effects in different experiments, and in general affected moderately the CD4+ response. These results suggest that anti-alpha 304-322 CD4+ cells comprise several clones, recognizing overlapping epitopes which share residues alpha 311-319. The importance of the sequence region alpha 311-319 for formation of CD4+ cell epitope(s) was verified by testing CD4+ cells sensitized to T alpha 304-322 with analogues of this sequence, carrying non-conservative substitutions at positions Q310, K314 and D318. Substitution of Q310 had minimal or no effects, while those of K314 or D318 strongly affected the CD4+ cell response.

Amino Acid Sequence↗
PubMed1994

Need to expand microvascular investigation of patients with Raynaud's phenomenon of different etiology: clinical patterns of 106 consecutive patients.

The differential diagnosis of Raynaud's Phenomenon (RP) is still problematic because of the wide variety of underlying etiological possibilities. Therefore, the diagnostic screening of patients requires easily reproducible, rapid and reliable tests to find those cases of RP secondary to a connective tissue disorder. The present study of 106 consecutive RP patients was carried out by using a combination of clinical examination, biomicroscopy of fingernail folds and bulbar conjunctiva (with scoring of vascular damage), plus the assessment of antinuclear antibodies on HEP2 cells. On the basis of the reported findings, it can be concluded that patients with RP secondary to collagen disease or so suspected also show abnormalities of the conjunctival microcirculatory bed. Patients with both primary and suspected secondary RP are significantly younger at the time of first diagnosis than patients with collagen disease-associated RP.

Adult↗
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

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↗
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↗
PubMed1991

[Cancer of the appendix. A report of 2 cases].

The paper reports two cases of adenocarcinoma of the appendix. Following a review of the literature on this neoplasia, it concludes that prognosis is poor on account of the early spread of the disease, the low ratio of suspected disease, and difficulties of diagnosis prior to surgery. The preferred surgical treatment is right hemicolectomy which consents a 5-year survival rate of 50% in comparison to 20% achieved with appendectomy alone. The authors also stress the importance of carrying out histological tests after any type of appendectomy.

Adenocarcinoma, Mucinous↗
PubMed1991

The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H-2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones.

Animals↗
PubMed1991

Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the alpha subunits of Torpedo californica and murine acetylcholine receptors.

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.

Amino Acid Sequence↗