Biomedical subjects

D Rosa

Publications and source records attributed to D Rosa.

At least 19 recordsLinked to original sources

PubMed2001

Nonneutralizing human antibody fragments against hepatitis C virus E2 glycoprotein modulate neutralization of binding activity of human recombinant Fabs.

Evidence from clinical and experimental studies indicates that hepatitis C virus E2 (HCV/E2) glycoprotein is the major target of a putatively protective immune response. However, even in the presence of a vigorous production of anti-HCV/E2 antibodies, reinfection can occur. Dissection of the human immune response against HCV/E2 indicated that blocking of binding of HCV/E2 to target cells [neutralization of binding (NOB) activity] varies widely among antibody clones. Moreover, in vivo, simultaneous binding of antibodies to distinct epitopes can induce conformational changes and synergies that may be relevant to understanding the anti-HCV immune response. In this study, human recombinant Fabs were generated by affinity-selecting a phage display repertoire library with antibody-coated HCV/E2. These Fabs, which share the same complementarity-determining region DNA sequences, had higher affinity than other anti-HCV/E2 Fabs but showed no NOB activity even at the highest concentrations. Binding of Fabs to HCV/E2 caused conformational changes modifying Fab-binding patterns and reducing, with a negative synergistic effect, Fab-mediated NOB activity. These data suggest that some antibody clones have the potential to modify HCV/E2 conformation and that, in this state, binding of this glycoprotein to its cellular target is less prone to inhibition by some antibody clones. This can explain why high anti-HCV/E2 antibody titers do not directly correlate with protection from infection. Information on the interactions among different antibody clones can contribute to understanding virus-host interplay and developing more effective vaccines.

Amino Acid Sequence↗

PubMed2001

Behavior of ambulatory blood pressure surrounding episodes of headache in mildly hypertensive patients.

BACKGROUND: Headache is usually associated with high blood pressure (BP) despite the lack of evidence of such an association in most observational studies. Ambulatory BP monitoring provides an opportunity to analyze this relation because it permits measurement of BP before, during, and after episodes of headache. METHODS: We evaluated 76 patients with mild hypertension who underwent clinical evaluation, ambulatory BP monitoring, and questioning about the occurrence of headache and its characteristics during monitoring. The 24-hour BP curves of patients with and without headache during monitoring were compared using analysis of variance for multiple factors and repeated measurements. Hourly averages of BP surrounding the episode and 24-hour mean BP of patients with headache were compared using paired sample t tests. RESULTS: Twenty-five participants (33%) experienced headache during monitoring. Their 24-hour BP curves did not differ from those of participants without headache. Mean 24-hour BP was not different from BP registered during the episode of headache (mean +/- SD systolic BP: 137.0 +/- 17.3 vs 139.4 +/- 21.1; P =.13; diastolic BP: 83.3 +/- 12.8 vs 85.0 +/- 18.2; P =.30). Blood pressure values registered during the episode of headache and in the hours before and after the episode were not different from each other. Analysis restricted to 8 patients with migrainelike headache showed a similar pattern. CONCLUSIONS: In patients with mild hypertension, there is no association between the occurrence of headache and variation of BP. Health professionals must discourage patients with hypertension from believing that they can rely on the presence of such a symptom to know about their BP levels.

Blood Pressure↗

PubMed2000

Structure-function analysis of hepatitis C virus envelope-CD81 binding.

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease. We have recently found that the large extracellular loop (LEL) of human CD81 binds HCV. This finding prompted us to assess the structure-function features of HCV-CD81 interaction by using recombinant E2 protein and a recombinant soluble form of CD81 LEL. We have found that HCV-E2 binds CD81 LEL with a K(d) of 1.8 nM; CD81 can mediate attachment of E2 on hepatocytes; engagement of CD81 mediates internalization of only 30% of CD81 molecules even after 12 h; and the four cysteines of CD81 LEL form two disulfide bridges, the integrity of which is necessary for CD81-HCV interaction. Altogether our data suggest that neutralizing antibodies aimed at interfering with HCV binding to human cells should have an affinity higher than 10(-9) M, that HCV binding to hepatocytes may not entirely depend on CD81, that CD81 is an attachment receptor with poor capacity to mediate virus entry, and that reducing environments do not favor CD81-HCV interaction. These studies provide a better understanding of the CD81-HCV interaction and should thus help to elucidate the viral life cycle and to develop new strategies aimed at interfering with HCV binding to human cells.

Amino Acid Sequence↗

PubMed2000

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates.

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.

Animals↗

PubMed1998

Binding of hepatitis C virus to CD81.

Chronic hepatitis C virus (HCV) infection occurs in about 3 percent of the world's population and is a major cause of liver disease. HCV infection is also associated with cryoglobulinemia, a B lymphocyte proliferative disorder. Virus tropism is controversial, and the mechanisms of cell entry remain unknown. The HCV envelope protein E2 binds human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Binding of E2 was mapped to the major extracellular loop of CD81. Recombinant molecules containing this loop bound HCV and antibodies that neutralize HCV infection in vivo inhibited virus binding to CD81 in vitro.

Amino Acid Sequence↗

PubMed1998

Characterization of human monoclonal antibodies specific to the hepatitis C virus glycoprotein E2 with in vitro binding neutralization properties.

Both linear and conformational determinants of hepatitis C virus (HCV) are believed to be involved in viral neutralization. After immortalization of B cells from HCV chronically infected patients with Epstein-Barr virus, we obtained two polyclonal lymphoblastoid cell lines (LCL) secreting human monoclonal antibodies (HMabs). One clone was derived from a patient infected with a genotype 4 isolate while the second was isolated from a genotype 1b-infected patient. Immunoprecipitation studies, Western blot, and immunofluorescence analysis, peptide scanning, and ELISA studies indicated that the HMabs (1) recognized conformation-dependent determinant(s), (2) were capable of recognizing genotype 1a and 1b derived antigens, and (3) were able to precipitate noncovalently associated E1E2 complexes believed to exist on the surface of virion particles. The HMab derived from the genotype 4-infected patient was in addition shown to neutralize the in vitro binding of recombinant E2 protein onto susceptible cells suggesting a potential for in vivo neutralization. These data indicate that anti-E2 antibodies directed at conserved conformational-dependent determinant(s) exist in chronic HCV infection.

Antibodies, Monoclonal↗

PubMed1998

Perspectives for a hepatitis C virus vaccine.

BACKGROUND: Natural hepatitis C virus (HCV) infection elicits poor immunity. Although HCV proteins elicit immune responses in virtually all cases of infection, the great majority of HCV infections become chronic. Currently, no vaccine is available for HCV despite an estimated incidence of approximately 50000 new cases per year in the USA alone. OBJECTIVES: To discuss how the problems associated with developing a vaccine against HCV infection may be overcome and describe recent progress made towards overcoming these problems and developing a vaccine. STUDY DESIGN: A cytofluorimetric assay that can assess the ability of a serum sample to neutralise the binding of the HCV-envelope glycoprotein E2 to human cells (neutralisation of binding or NOB assay) was developed. The assay was used to assess the levels of antibodies capable of neutralising E2 binding in the sera of vaccinated and carrier chimpanzees. RESULTS: Low titres of NOB antibodies were found in the majority of chimpanzees challenged with HCV infection. Chimpanzees immunised with the E1/E2 heterodimer developed NOB antibodies and high levels of neutralising antibodies. These chimpanzees were not protected from challenge with heterologous virus but were protected from subsequent chronic infection. CONCLUSIONS: A subunit vaccine composed of recombinant HCV proteins may protect from infection or chronic infection by different HCV genotypes.

Animals↗

PubMed1998

Bactericidal monoclonal antibodies that define unique meningococcal B polysaccharide epitopes that do not cross-react with human polysialic acid.

The poor immunogenicity of the Neisseria meningitidis group B polysaccharide capsule, a homopolymer of alpha(2-->8) sialic acid, has been attributed to immunologic tolerance induced by prenatal exposure to host polysialyated glycoproteins. Substitution of N-propionyl (N-Pr) for N-acetyl groups on the meningococcal B polysaccharide, and conjugation of the resulting polysaccharide to a protein carrier, have been reported to yield a conjugate vaccine that elicits protective Abs with minimal autoantibody activity. To characterize the protective epitopes on the derivatized polysaccharide, we isolated 30 anti-N-Pr meningococcal B polysaccharide mAbs. These Abs were heterogeneous with respect to complement-mediated bactericidal activity, fine antigenic specificity, and autoantibody activity as defined by binding to the neuroblastoma cell line, CHP-134, which expresses long-chain a(2-->8)-linked polysialic acid. Eighteen of the Abs could activate complement-mediated bacteriolysis. Seven of these 18 Abs cross-reacted with N-acetyl meningococcal B polysaccharide by ELISA and had strong autoantibody activity. Thus, N-Pr meningococcal B polysaccharide conjugate vaccine has the potential to elicit autoantibodies. However, 7 of the 18 bactericidal mAbs had no detectable autoantibody activity. These Abs may be useful for the identification of molecular mimetics capable of eliciting protective Abs specific to the bacteria, without the risk of evoking autoimmune disease.

Animals↗

PubMed1998

Dynamics of intra-hepatic lymphocytes in chronic hepatitis C: enrichment for Valpha24+ T cells and rapid elimination of effector cells by apoptosis.

Chronic viral hepatitis is characterized by a dramatic lymphocyte infiltrate in the liver. Although it is one of the most common chronic inflammatory diseases in humans, little information is available on the functional state of these intra-hepatic lymphocytes (IHL). To address this issue, we have optimized cytofluorimetric techniques to assess directly ex vivo the functions, dynamics and repertoires of IHL isolated from biopsies of patients with chronic hepatitis C. We estimate that 1% of the total body lymphocytes infiltrate the inflamed liver and find that, at variance with peripheral blood lymphocytes (PBL) isolated from the same patients, most IHL display an activated phenotype and produce Th1 type lymphokines when stimulated in vitro. Virtually all IHL are found in the G0/G1 state of the cell cycle, while a sizeable percentage of them is undergoing programmed cell death in vivo, as detected by the TUNEL assay performed on freshly isolated cells. In contrast again to PBL from the same patients, IHL show a preferential compartmentalization of NK and TCRgamma/delta+ cells, and a remarkable (up to 20-fold) enrichment for Valpha24+ T cells. Together our data suggest that in a liver injured by chronic hepatitis C, most IHL are pro-inflammatory activated cells which are highly enriched for effectors of innate resistance. These IHL do not undergo clonal expansion in the liver but rather display effector function and die in situ at a high rate, suggesting that maintenance of the IHL pool is dependent on continuous migration from extra-hepatic sites.

Antigens, CD1↗

PubMed1998

Dissection of human humoral immune response against hepatitis C virus E2 glycoprotein by repertoire cloning and generation of recombinant Fab fragments.

Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 microg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 microg/mL.

Amino Acid Sequence↗

PubMed1998

High titers of antibodies inhibiting the binding of envelope to human cells correlate with natural resolution of chronic hepatitis C.

Most cases of hepatitis C virus (HCV) infection result in chronic disease; however, a very small fraction of patients naturally clear the virus and resolve chronic hepatitis. In an attempt to correlate immune response with chronic disease resolution, we compared the antibody response in patients with different outcomes of the infection. Antibody responses to HCV structural proteins were assessed in 34 patients originally diagnosed with acute hepatitis. Five cases resolved acute infection, 22 developed chronic hepatitis, and 7 naturally resolved chronic hepatitis C. To estimate HCV neutralizing antibodies we used the neutralization of binding (NOB) assay, which evaluates inhibition of the envelope-2 protein binding to human cells. Enzyme-linked immunosorbent assay was used for the quantitative assessment of serum antibodies. The presence of HCV RNA was ascertained by reverse transcription-polymerase chain reaction. In 6 of 7 patients naturally recovered from chronic hepatitis C, the emergence and the persistence (for more than 3 months) of high serum titers (>1/600) of NOB antibodies coincided with virus clearance and clinical resolution of hepatitis. NOB antibody activity was observed in only 2 of 5 patients recovered from acute hepatitis C. Chronic patients who did not show any resolution during the course of the study developed low or no NOB antibodies. Because of the correlation between prolonged high NOB titers and natural resolution of chronic hepatitis C, vaccination or passive immunization aimed at high titers of NOB antibodies may be valuable new therapeutic approaches for chronic hepatitis C.

Adult↗

PubMed1998

Fatty acid profiles and non enzymatic lipid peroxidation of microsomes and mitochondria from bovine liver, kidney, lung and heart.

The light emission = chemiluminescence and fatty acid composition, two markers of lipid peroxidation process, was evaluated in bovine liver, kidney, lung and heart microsomes and mitochondria incubated in the presence of ascorbate-Fe2+ at 37 degrees C. Microsomal and mitochondrial fractions obtained from lung, kidney and heart were more resistant to lipid peroxidation than those isolated from the liver. The ascorbate-Fe2+ seemed to be totally ineffective in stimulating peroxidation of bovine lung, heart and kidney microsomes or mitochondria. Whereas bovine liver microsomes or mitochondria displayed the highest values of chemiluminescence when incubated in the presence of ascorbate-Fe2+. The peroxidizability index a parameter that indicate the maximal rate of oxidation of specific fatty acids showed statistically significant differences in mitochondria isolated from lung heart and kidney and microsomes isolated from lung. The fatty acid profile of the subcellular fractions obtained from those tissues do not appear to be responsible for their different susceptibility to free radical degradation. Other factor/s may be responsible for the protection to lipid peroxidation observed in microsomal or mitochondrial fractions obtained from bovine lung, heart and kidney.

Animals↗

PubMed1997

Sexual transmission of the hepatitis C virus and efficacy of prophylaxis with intramuscular immune serum globulin. A randomized controlled trial.

OBJECTIVE: To estimate the risk of sexual transmission of hepatitis C and to assess the value of prophylaxis with periodic intramuscular immune serum globulin administration. METHODS: Of 1102 steady heterosexual partners of patients with antibodies to the hepatitis C virus (HCV), 899 were enrolled in a single-blind, randomized, controlled trial. All the partners tested negative for antibodies to HCV and had normal baseline serum aminotransferase concentrations. The partners were assigned to receive 4 mL of 16% polyvalent immune serum globulin prepared from unscreened donors every 2 months (n = 450) or a placebo (n = 449). Tests for HCV infection were performed every 4 months. RESULTS: Eight hundred eighty-four partners completed the study. Seven partners became infected with HCV: 6 in the control group (incidence density, 12.00 per 1000 person-years; 95% confidence interval, 3.0 21.61) and 1 in the immune serum globulin group (incidence density, 1.98 per 1000 person-years; 95% confidence interval, 0-5.86). The risk of infection was significantly higher for partners in the control group (P = .03): for each year approximately 1% of the partners became infected. Sequence homology studies strongly suggest the sexual transmission of HCV. All immune serum globulin lots used had high enzyme-linked immunosorbent assay titers of neutralizing antibodies to HCV envelope glycoproteins and high neutralization titers in the neutralization of binding assay. CONCLUSIONS: Hepatitis C can be sexually transmitted. Immune serum globulin prepared from unscreened donors significantly reduced the risk. The treatment was safe and well tolerated. Because only immune serum globulin from unscreened donors (and not from those screened for HCV) contain anti-HCV neutralizing antibodies, hyperimmune anti-HCV immune serum globulin should be prepared from blood testing positive for antibodies to HCV, which is currently discarded.

Adult↗

PubMed1996

A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development.

Cell Line↗

PubMed1994

Nonpeptide ligands for human gamma delta T cells.

gamma delta T cells respond to a variety of microbial pathogens and transformed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human gamma delta T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated gamma delta T cells and stimulated their proliferation in vitro. All gamma delta T cells stimulated by MEP expressed V gamma 2/V delta 2 receptors. The purified natural ligand of mycobacteria is chemically similar to, though distinct from, MEP and contains a phosphate residue that is critical for biological activity. Recognition and expansion of a specific T-cell receptor-bearing population to non-peptide ligands is unprecedented among T cells. We suggest that MEP mimics small natural ligands capable of expanding one subset of gamma delta T cells and that this recognition of nonpeptide antigens may play an important role in human immunity to pathogens.

Antibodies↗

PubMed1994

Immunocytochemical localization of plasminogen activators in carcinomas in the cervix and vulva.

The presence of plasminogen activators (PA) in a variety of solid tumours appears to correlate, in a number of instances, with enhanced invasive and/or metastatic ability. Urokinase and tissue-type plasminogen activators (u-PA and t-PA) in normal and neoplastic tissues of cervix and of vulva were immunohistochemically identified by means of polycyclonal antibodies. In addition, frozen sections were analysed for u-PA and t-PA activity by in-situ zymography technique. Data collected in our study showed that in invasive cancer u-PA increased more in malignant cells as compared to normal cells in both the inactive and active enzymatic forms. The t-PA distribution pattern was related to angiogenesis while it did not relate to the degree of tumor differentiation. A synergic interaction between proteolytic tumoral activity and proteolytic inflammatory action could be hypothesized.

Carcinoma, Adenosquamous↗

PubMed1991

The vitelline coat spikes: a new peculiar structure of Mytilus galloprovincialis eggs with a role in sperm-egg interaction.

In the course of this study we found that in Mytilus galloprovincialis eggs long filamentous protrusions never described before, which we have termed "vitelline coat spikes," could be clearly detected using the lectin from Dolichos biflorus, which recognizes the GalNAc residues. The spikes could be also observed by transmission electron microscope but only in some fortuitous sections could their origin in the vitelline coat be clearly observed. The spikes were also clearly visible using the scanning electron microscope. Observations of the sperm-egg interaction very few seconds after insemination or using fixed eggs suggested that the spikes could play a role in a primary binding to the unreacted sperm. Experiments have been done to test the effect of GalNAc on the sperm-egg binding and on the fertilization process which seem to confirm this hypothesis.

Acetylglucosamine↗

PubMed1991

Basement membrane components in normal, dysplastic, neoplastic laryngeal tissue and metastatic lymph nodes.

The immunohistochemical localization of the basement membrane (BM) components laminin, type IV collagen and fibronectin was analyzed in normal, dysplastic and neoplastic laryngeal specimens. The distribution of these macromolecules was also investigated in metastatic lymph nodes. A regular and continuous staining for laminin and type IV collagen was present in normal and mild dysplastic epithelium (LIN I); interruptions and reduplications were more evident in severe dysplasia (LIN III), together with an increased positivity for fibronectin in the subepithelial connective tissue. In squamous cell carcinomas the distribution of BM components was related to the degree of cellular differentiation, with a decreased immunostaining being evident in moderately and poorly differentiated carcinomas. Furthermore, the positivity for laminin and type IV collagen was influenced by the pattern of neoplastic growth, being continuous around the "pushing" border and discontinuous where the neoplastic front had an "invading" appearance. Similar changes were present in cervical metastatic lymph nodes. These observations tend to support the theory that the neoplastic growth is a cyclic process, with BM component synthesis and reabsorbtion related to the shifts of cellular metabolism.

Antibodies, Monoclonal↗