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Biomedical subjects

R Longhi

Publications and source records attributed to R Longhi.

At least 55 records · Page 3Linked to original sources
PubMed1998

Thromboembolic events in beta thalassemia major: an Italian multicenter study.

Thromboembolic (TE) events have been frequently reported in beta-thalassemic patients in association with known risk factors such as diabetes, complex cardiopulmonary abnormalities, hypothyroidism, liver function anomalies, and postsplenectomy thrombocytosis. In a recent survey involving 9 Italian thalassemic centers, we identified 32 patients with TE episodes in a total of 735 subjects, of whom 683 had thalassemia major and 52 thalassemia intermedia, corresponding to 3.95 and 9.61%, respectively. There was a great variation in localization: the main one (16/32) was CNS, with a clinical picture of headache, seizures and hemiparesis. Other localizations were the pulmonary (3 patients), mesenteric (1 patient) and portal (2 patients) sites. There were 6 cases of deep venous thrombosis (2 in the upper limbs, 4 in the lower ones). Intracardiac thrombosis was found in 2 subjects and clinical and laboratory signs of DIC were observed in 2 others during pregnancy. Since our patients with TE events present a statistically significantly higher incidence of associated dysfunction (cardiomyopathy, diabetes, liver function anomalies, hypothyroidism) than those without TE events (50 vs. 13.8%), we suggest close monitoring of those patients who are at higher risk of developing TE events because of the presence of one or more of these predisposing factors.

Adolescent↗
PubMed1997

Biochemical and immunological characterization of recombinant allergen Lol p 1.

Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

Allergens↗
PubMed1997

Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria.

Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDS/PAGE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 microM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.

Antibodies, Monoclonal↗
PubMed1997

Chromogranin A fragments modulate cell adhesion. Identification and characterization of a pro-adhesive domain.

Although several functions have been suggested for chromogranin A, a glycoprotein secreted by many neuroendocrine cells, the physiological role of this protein and of its proteolytic fragments has not been established. We have found that mixtures of chromogranin A fragments can inhibit fibroblast adhesion. The anti-adhesive activity was converted into pro-adhesive activity by limited trypsin treatment. Pro-adhesive effects were observed also with recombinant N-terminal fragments corresponding to residues 1-78 and 1-115 and with a synthetic peptide encompassing the residues 7-57. These fragments induced adhesion and spreading of fibroblasts on plates coated with collagen I or IV, laminin, fetal calf serum (FCS) but not on bovine serum albumin. The long incubation time required for adhesion assays (4 h) and the FCS requirements for optimal adhesion suggest that the adhesive activity is likely indirect and requires other proteins present in the FCS or made by the cells. These findings suggest that chromogranin A and its fragments could play a role in the regulation of cell adhesion. Since chromogranin A is concentrated and stored within granules and rapidly released by neuroendocrine cells and neurons after an appropriate stimulus, this protein could be important for the local control of cell adhesion by stimulated cells.

3T3 Cells↗
PubMed1997

Antigen-driven C-C chemokine-mediated HIV-1 suppression by CD4(+) T cells from exposed uninfected individuals expressing the wild-type CCR-5 allele.

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1-specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32-base pair deletion in the C-C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Delta32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C-C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell-tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development.

Alleles↗
PubMed1997

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells.

The gamma-aminobutyric acid transporter (GAT-1) isoform of the gamma-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DeltaC-GAT) maintained the original functional activity and apical localization, either the removal (DeltaC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.

Amino Acid Sequence↗
PubMed1997

HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals.

HIV-specific mucosal and cellular immunity was analyzed in heterosexual couples discordant for HIV status in serum and in HIV-unexposed controls. HIV-specific IgA but not IgG was present in urine and vaginal wash samples from HIV-exposed seronegative individuals (ESN), whereas both IgA and IgG were observed in their HIV-seropositive partners; antibodies were not detected in low-risk controls. Envelope protein (Env) peptide-stimulated interleukin-2 (IL-2) production by peripheral blood mononuclear cells (PBMCs) was detected in 9 out of 16 ESNs, 5 out of 16 HIV-infected patients and 1 out of 50 controls. Env peptide-stimulated PBMCs of ESNs produced more IL-2 and less IL-10 compared with those of HIV-infected individuals; no differences were observed in chemokine production or in CCR5 expression. These data demonstrate that a compartmentalized immune response to pathogens is possible in humans and raise the possibility of protective roles for cell-mediated immunity and mucosal IgA in HIV-seronegative individuals exposed to HIV.

Adult↗
PubMed1997

Pentoxifylline improves cell-mediated immunity and reduces human immunodeficiency virus (HIV) plasma viremia in asymptomatic HIV-seropositive persons.

The effects of pentoxifylline on immunologic and virologic parameters were evaluated in 10 human immunodeficiency virus-infected patients not receiving antiretroviral treatment. Patients were asymptomatic, had 300-500 CD4 cells/microL, and received pentoxifylline (1200 mg/day orally) for 4 months. Peripheral blood mononuclear cells were tested before and at five time points during therapy. A transient increase in CD4 cells was observed in 8 of 9 patients, and CD8 cells increased in 7 of 9 patients. These increases were negatively correlated with susceptibility to in vitro mitogen-stimulated apoptotic cell death. Pentoxifylline had a temporary effect on mitogen-stimulated cytokine production; thus, interferon-gamma, interleukin (IL)-2, tumor necrosis factor-alpha, and lymphotoxin increased more than IL-10. Pentoxifylline also potentiated antigen-stimulated IL-2 production and proliferation in 8 of 9 patients and induced significant but transient decreases in plasma viremia in 7 of 9 patients. These preliminary findings suggest that pentoxifylline in vivo has an interesting but temporary influence on both immunologic and virologic parameters.

Adjuvants, Immunologic↗
PubMed1997

Alpha7 and alpha8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties.

Nicotinic receptors are present in the chick retina, but their structure and functional characteristics are still unclear. Using anti-alpha7 and anti-alpha8 subunit-specific antibodies, we immunopurified the alpha7 and alpha8 subtypes of chick retina neuronal nicotinic receptors. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the two purified subtypes consistently showed a similar peptide composition characterized by the presence of two major peptides of M(r) 58 +/- 1 and 54 +/- 1 kDa, and two minor peptides of 67 and 61 +/- 1 kDa. In the alpha7 subtype, the 58 kDa peptide was recognized by anti-alpha7 but not by anti-alpha8 antibodies; in the alpha8 subtype, the 58 kDa peptide was recognized only by anti-alpha8 antibodies. The alpha7 subtype had a single class of [125I]alpha-bungarotoxin binding sites with a K(D) value of 1.2 nM, whereas the purified alpha8 subtype had two classes of binding sites, one with a K(D) of 5.5 nM and the other with very high affinity (KD 52 pM), but present in only 8% of the receptors. Competition binding experiments also showed the presence on the alpha8 subtype of high- and low-affinity classes of binding sites; the affinity for cholinergic drugs of the former was greater than that of the single class present on the alpha7 subtype. When reconstituted in planar lipid bilayers, both subtypes formed ligand-gated cation channels with major conductance levels of 42 and 52 pS but with different lifetimes; the two channels were activated by agonists and blocked by d-tubocurarine and the glycinergic antagonist strychnine. In line with the binding data, the reconstituted alpha8 subtype had greater agonist sensitivity than the alpha7 subtype.

Animals↗
PubMed1996

Role of HLA class I in HIV type 1-induced syncytium formation.

Neutralization of HIV-1 in vitro by anti-HLA class I antibodies suggests that class I molecules are involved in HIV-1 infection. HIV-infected cells can fuse with uninfected cells in a process that leads to the formation of multinucleated syncytia, involving an interaction between host and viral antigens expressed at the cell surfaces. We used a syncytium assay between the 8E5 cell line chronically infected with a pol-defective variant of LAV IIIb, and the CD4-positive cell line MOLT3, to study the role of HLA class I in HIV-1-induced cell fusion. By probing cells with a panel of anti-HLA monoclonal antibodies (MABs) we demonstrated that the fusion process is modulated specifically by C alleles of HLA class I expressed on uninfected cells but not by that on already infected cells. Addition of beta 2-microglobulin to the cocultures resulted in a dose-dependent enhancement in both the number and size of syncytia, whereas exogenous HLA-C-restricted peptides inhibited syncytium formation, implying that only certain conformational states of HLA class I are permissive for syncytium formation. Treatment of cocultures with HLA-Cw4-restricted peptides containing amino acid substitutions in the anchor residues showed that syncytium inhibition was dependent on conventional binding of the peptide inside the groove. The data indicate that HLA class I, in a conformation free of peptide but associated with beta 2-microglobulin, can directly influence virus-induced cell fusion.

Antibodies, Monoclonal↗
PubMed1996

Antigenic regions of human chromogranin A and their topographic relationships with structural/functional domains.

Chromogranin A is a protein contained in the secretory granules of many neuroendocrine cells. The linear antigenic sites of human chromogranin A were studied by examining the cross-reaction of polyclonal and monoclonal anti-chromogranin A antibodies with native chromogranin A and with synthetic peptides encompassing most of the chromogranin A sequence. Chromogranin A residues 1-20, 47-67, 107-158, 254-297, 331-375, and 395-419 were found to be poorly or not antigenic, while residues 25-46, 163-210, 231-253, 298-314 and 68-106, 222-230, 315-330, 376-394 were found to contain weak and strong antigenic sites, respectively. Residues 68-70 (GAK) and 81-90 (GFEDELSEVL) were strongly recognized by two mouse mAbs (B4E11 and A11, respectively). Since mAb A11 has been previously used for immunohistochemical analysis of chromogranin-A-producing tissues from different species and for in vivo imaging of chromogranin-A-positive endocrine tumors, these results imply that at least part of the 81-90 region is surface-exposed in cryostat tissue sections as well as in vivo. The results may help in selecting new antibodies with improved affinity and immunogenicity for in vivo targeting of chromogranin-A-producing tumors.

Amino Acid Sequence↗
PubMed1996

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41.

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577-599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization.

Amino Acid Sequence↗
PubMed1996

CD and NMR structural characterization of ceratotoxins, natural peptides with antimicrobial activity.

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8-25 region of the peptide. A ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10-19 is always helical and differences refer to residues 8-9 and 19-23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization.

Amino Acid Sequence↗
PubMed1996

Human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications.

One of the major allergens from the pollen of perennial rye grass (Lolium perenne), Lol pII, was used to isolate specific antibody fragments from a random combinatorial library displaying a large repertoire of human Fab on filamentous phages. After five panning cycles on recombinant Lol pII immunotubes, phage binders were isolated and the antibody fragments expressed as soluble Fab molecules in the Escherichia coli periplasm. The DNA sequencing of the clones producing antibodies with the highest binding activity showed three of them to be identical, while one differed by two amino acid substitutions in the heavy chain. The antibody fragments were produced in milligram amounts, affinity-purified and further characterized. They bound the natural allergen as well as the recombinant one, with no cross-reactivity with other allergens contained in the pollen extract of L. perenne. One antibody bound the allergen with Kd = 2.63 x 10(-9) M, as demonstrated by the surface plasmon resonance technique, and was able to compete with a fraction of serum IgE. Epitope mapping using synthetic peptides revealed that antigenic domains, located between amino acids 39 and 51 of Lol pII, are recognized by Fab and polyclonal IgE from sera of allergic donors. The Fab fragments inhibited the binding of serum IgE to the allergen. In vitro experiments on whole blood from allergic subjects showed that recombinant Fab fragments had a blocking activity on histamine release from cells challenged with recombinant Lol pII allergen. Thus, serum IgE and recombinant Fab fragments recognize common epitopes, although they represent the outcome of different maturation and/or selection processes. Our molecular and functional findings altogether indicate that allergen-specific human antibodies may be useful for the characterization of the antigenic structure of allergens. We conclude that a phage library is a powerful source of anti-allergen human antibodies with high affinity and high specificity. Moreover, these molecules may be potentially innovative reagents for the treatment of atopic allergy.

Allergens↗
PubMed1995

Peptide-based radioimmunoassay for the two isoforms of the human insulin receptor.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the COOH-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. In this study, we developed a radioimmunoassay for the two isoforms employing antibodies raised against two peptides, one (Pep-12) corresponding to residues encoded by exon 11, and the other (Pep-13) corresponding to a COOH-terminal domain of the alpha-subunit which is common to both HIR-A and HIR-B isoforms. These peptides were iodinated and used as both ligands and standards. The assay is specific, highly reproducible, and sensitive with a detection limit of 10 fmol of receptor. One mole of purified insulin receptor, measured by Scatchard analysis, is read as one mole of receptor in the radioimmunoassay with either Pep-12 or Pep-13 as standards. The radioimmunoassay is applicable to the measurement of total content and relative abundance of the two isoforms in extracts from various tissues. We applied the radioimmunoassay to measure the relative abundance of the two isoforms in fat and muscle from normal, obese non-diabetic and non-insulin-dependent diabetic (NIDDM) subjects. Results demonstrate that expression of the low-affinity HIR-B form is significantly increased in obese and NIDDM subjects compared with control subjects. In addition, the increased expression of the HIR-B isoform was significantly correlated with both body mass index (r = 0.52; p = 0.006) and fasting glucose levels (r = 0.59; p = 0.001).

3T3 Cells↗
PubMed1995

Direct interaction of complement factor H with the C1 domain of HIV type 1 glycoprotein 120.

A protein that binds specifically to Env 105-119 (HEDIISLWDQSLKPC) was found in pools of normal human plasma when this peptide was used in affinity chromatography procedures. These samples represented the negative control in experiments aimed at the purification of putative human antibodies to the Env 105-119 region from AIDS sera. In this article we describe the biochemical characterization of this protein, which turned out to be complement factor H (CFH). We propose a functional role for this protein in the complex, early steps of CD4-dependent HIV infection.

Amino Acid Sequence↗
PubMed1995

HIV glycoprotein 41 and complement factor H interact with each other and share functional as well as antigenic homology.

We have shown that complement factor H (CFH) interacts with HIV-1 at the level of the sequence Env 105-119, contained in the C1 domain of gp120. CFH interaction with HIV was evident only after dissociation of the Env complex induced by exposure to sCD4. We hypothesized that CFH could act as a gp41 analog in the interaction with Env 105-119. A panel of partially overlapping, synthetic peptides reproducing the extracellular portion of gp41 was therefore used to compete the binding of CFH to Env 105-119. Three sets of peptides that competed this interaction were identified. These peptides defined a region of functional homology between the gp41 molecule and CFH (Env 580-600), and two regions of interaction (Env 620-640 and Env 650-670). In addition to this, a monoclonal antibody directed against peptide Env 580-600 and a polyclonal mouse antiserum raised against recombinant gp41 were shown to recognize CFH in Western blots and ELISA, respectively, also defining a region of antigenic homology between gp41 and CFH. These data provide evidence for interaction and molecular mimicry between an HIV structural protein and a negative regulator of the complement pathway. We show here that CFH can interact with both HIV Env proteins, suggesting a possible and efficient mechanism of downregulation of the complement cascade at the surface of infected cells.

Amino Acid Sequence↗