Biomedical subjects

L Nencioni

Publications and source records attributed to L Nencioni.

At least 19 recordsLinked to original sources

PubMed2001

Nerve growth factor inhibits apoptosis in memory B lymphocytes via inactivation of p38 MAPK, prevention of Bcl-2 phosphorylation, and cytochrome c release.

Survival of memory B lymphocytes is tightly linked to the integrity of the Bcl-2 protein and is regulated by a nerve growth factor (NGF) autocrine circuit. In factor-starved memory B cells, the addition of exogenous NGF promptly induced p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK), dephosphorylation. Conversely, withdrawal of endogenous NGF was followed by p38 MAPK activation and translocation onto mitochondria, whereby it combined with and phosphorylated Bcl-2, as assessed by co-immunoprecipitation and kinase assays in vivo and in vitro. Mitochondria isolated from human memory B cells, then exposed to recombinant p38 MAPK, released cytochrome c, as did mitochondria from Bcl-2-negative MDCK cells loaded with recombinant Bcl-2. Apoptosis induced by NGF neutralization could be blocked by the specific p38 MAPK inhibitor SB203580 or by Bcl-2 mutations in Ser-87 or Thr-56. These data demonstrate that the molecular mechanisms underlying the survival factor function of NGF critically rely upon the continuous inactivation of p38 MAPK, a Bcl-2-modifying enzyme.

Animals↗

PubMed2000

Imbalance in corneal redox state during herpes simplex virus 1-induced keratitis in rabbits. Effectiveness of exogenous glutathione supply.

A significant decrease in the antioxidant glutathione (GSH) was found in the corneal tissue of rabbits with Herpes Simplex 1 (HSV-1)-induced keratitis. Such a decrease was due to a loss of the reduced species, since no increase in its oxidized form was observed. Topical administration of purified GSH was able to reduce the virus titre in corneal tissue and, at the same time, was effective in reducing the severity and progression of keratitis and conjunctivitis. This effect was paralleled by a partial recovery in the corneal GSH content. In vitro experiments performed on HSV-1 infected corneal-derived rabbit cells showed that exogenous GSH reduced virus titre in the supernatant of infected cells. These results are in agreement with our previous findings that an oxidative environment, due to GSH depletion, is necessary for virus replication and suggest that topical GSH treatment could be considered as complementary therapy in HSV-1-induced keratitis.

Animals↗

PubMed1999

Interferon-alpha-induced inhibition of B16 melanoma cell proliferation: interference with the bFGF autocrine growth circuit.

The molecular mechanisms underlying the growth inhibition induced by interferon-alpha (IFN-alpha) in B16 murine melanoma cells were investigated. IFN-alpha did not induce cell apoptosis, but strongly interfered with the synthesis of basic fibroblast growth factor (bFGF), which acts as an autocrine growth factor in this system. Inhibition of bFGF synthesis was observed at the same concentrations (50-500 pM, 10-100 U/ml) of IFN-alpha able to induce growth arrest of B16 melanoma cells. Although the synthesis of acidic (a)FGF was only slightly affected by IFN-alpha, the cytokine induced release of an aFGF-related low-molecular-weight peptide, which was able to interfere with bFGF binding to surface receptors. Thus, the molecular mechanisms of IFN-alpha activity on melanoma cells include a specific modulation of the bFGF autocrine circuit.

Animals↗

PubMed1996

Nerve growth factor is an autocrine survival factor for memory B lymphocytes.

Production of nerve growth factor (NGF) was assessed in cultures of human T and B lymphocytes and macrophages. NGF was constitutively produced by B cells only, which also expressed surface p140trk-A and p75NGFR molecules and hence efficiently bound and internalized the cytokine. Neutralization of endogenous NGF caused disappearance of Bcl-2 protein and apoptotic death of resting lymphocytes bearing surface IgG or IgA, a population comprising memory cells, while surface IgM/IgD "virgin" B lymphocytes were not affected. In vivo administration of neutralizing anti-NGF antibodies caused strong reduction in the titer of specific IgG in mice immunized with tetanus toxoid, nitrophenol, or arsonate and reduced numbers of surface IgG or IgA B lymphocytes. Thus, NGF is an autocrine survival factor for memory B lymphocytes.

Animals↗

PubMed1996

Interleukin-4 rapidly down-modulates the macrophage colony-stimulating factor receptor in murine macrophages.

The activation of macrophages interferes with their response to macrophage colony-stimulating factor (M-CSF), the main growth and differentiation factor for mononuclear phagocytes. We tested the rapid effects of interleukin-4 (IL-4), the alternative macrophage activator produced by Th2 helper lymphocytes, on the receptor for M-CSF (M-CSFR) expressed on the cell surface of murine macrophages. IL4 rapidly down-modulated M-CSFR in a dose-dependent fashion. This effect was unique to IL-4 among a number of Th2-produced cytokines, none of which, with the exception of IL4 itself, is able to activate macrophages. The down-modulation of M-CSFR by IL4 was partially prevented by the inhibition of the activity of phospholipase C or protein kinase C. The data are consistent with the hypothesis that the down-modulation of M-CSFR is a property common to, and exclusive of, macrophage activators, and is driven by different activators via a common mechanism.

Animals↗

PubMed1996

Interleukin 2 down-modulates the macrophage colony-stimulating factor receptor in murine macrophages.

Macrophage colony-stimulating factor (M-CSF) is the main growth factor for mononuclear phagocytes. Responsiveness to growth factors is reduced in the course of functional activation of macrophages. We studied the interference of the macrophage activator interleukin 2 (IL-2) with the response to M-CSF, in macrophages of the M-CSF-dependent murine line BAC-1.2F5. Long-term effects of IL-2 on cell growth were determined, showing that IL-2 reduces the M-CSF-dependent proliferation of macrophages. Short-term effects of IL-2 on the expression of the receptor for M-CSF (M-CSF.R) were characterized in more detail. IL-2 rapidly down-modulated M-CSF.R in a dose-dependent fashion, and interferon-gamma and lipopolysaccharides synergized with IL-2 in this modulation. The IL-2-induced down-modulation of M-CSF.R was shown to require the activity of protein-kinase-C and phospholipase-C. The data are consistent with the hypothesis that the down-modulation of M-CSF.R is a general property of macrophage activators.

Animals↗

PubMed1996

The reaction of bacterial toxins with formaldehyde and its use for antigen stabilization.

Since the discovery of diphtheria toxin inactivation in the early 1920s, formaldehyde has been used to inactivate bacterial toxins and viruses used as vaccine antigens. More recently, formaldehyde was used to inactivate pertussis toxin (PT), a component of the newly developed diphtheria-tetanus-acellular pertussis (DTaP) vaccine. This application however illustrated the complexity of the reaction. To eliminate the need for inactivation, the mutant PT-9K/129G was developed. This toxin analogue is irreversibly devoid of toxicity and is a more immunogenic antigen than chemically detoxified PT. Native antigens however proved less stable than detoxified antigens upon storage or heating. We investigated the use of low concentrations of formaldehyde as a stabilizing agent for PT-9K/129G. Under the conditions selected, its antigenic characteristics were retained. Enhanced immunogenicity compared to detoxified preparations was demonstrated in clinical trials in infants where DTaP vaccines containing formalin-stabilized PT-9K/129G were compared to other DTaP vaccines containing detoxified wild type PT. Additional studies with filamentous haemagglutinin (FHA), another component of acellular pertussis vaccines, showed how high formaldehyde concentrations could depress the presentation of epitopes to T-cells by limiting the antigen processing. In conclusion, mild formaldehyde treatment can be applied to stabilize vaccine antigens while retaining optimum antigenic activity.

Antigens, Bacterial↗

PubMed1993

Priming to heat shock proteins in infants vaccinated against pertussis.

To investigate whether and in which proportion normal individuals experience a priming to microbial heat shock proteins (hsp), the presence of antibodies to two mycobacterial hsp was tested in serum sample from 2- to 4-mo-old children before and at different times after vaccination with the trivalent vaccine against tetanus, diphtheria, and pertussis (DTP). We show that 88.9% of infants vaccinated with DTP developed antibody responses to mycobacterial hsp. Such a response was due to the whole-cell pertussis component of the vaccine, because it was not observed in infants receiving an acellular pertussis vaccine. Antibodies and cells reactive to the mycobacterial 65-kDa hsp were also found in mice immunized with DTP. Interestingly, whole-cell pertussis vaccine-induced anti-hsp antibodies cross-reacted with the Escherichia coli GroEL hsp, and at a some extent with the human 60-kDa hsp, belonging to the same hsp family. These data suggest that priming of the immune system to hsp is a common phenomenon occurring very early in life.

Animals↗

PubMed1993

Preparation and immunogenicity of an inactivated hepatitis A vaccine.

A hepatitis A vaccine was prepared by formaldehyde inactivation of purified hepatitis A virus (HAV) LSH/S strain grown on human diploid MRC-5 cells. The vaccine was devoid of residual infectivity in vitro and failed to induce in marmoset monkeys any pathological features or variations of haematological and clinical chemistry values. Infectious HAV particles were not detected in faeces and sera of the vaccinated primates by ELISA or after passages in MRC-5 cells. The immunogenicity of the vaccine was evaluated by injecting guinea-pigs with 0.8, 0.2 or 0.05 micrograms of HAV antigen adsorbed onto 0.5 and 1 mg of Al (OH)3 or 0.3 mg of AlPO4. The antibody response, measured by a competitive radioimmunoassay, was dose- and adjuvant-dependent. One injection of 0.2 micrograms of AlPO4-adsorbed HAV antigen induced seroconversion in 100% of animals and high levels of specific and neutralizing serum antibodies. A further increase of antibody titres was observed after the second and third inoculations. These results show that this vaccine formulation is safe and immunogenic in animal models, and suggest that it should be evaluated further by human clinical studies.

Adjuvants, Immunologic↗

PubMed1993

Absence of protective immunity against diphtheria in a large proportion of young adults.

The schedule of vaccination recommended worldwide for diphtheria, tetanus and other diseases, provides good immunity during childhood. However, little attention has been paid to effective immunity in adults. We have collected sera from 334 Italian Army recruits and tested them for the presence of protective immunity against diphtheria and tetanus. In vivo neutralization assays were performed on rabbits and mice and values below 1/100 IU ml-1 were considered negative. Of the recruits, 22.9% were negative for diphtheria, while only 5.3% had no protective immunity against tetanus. This finding shows that a large proportion of Italian young adults are susceptible to diphtheria, and this could be dangerous if they travel to sites where this disease is still endemic, or if they come into contact with people coming from such areas. A booster vaccination of young adults against diphtheria should become common practice to avoid this risk. To reduce the side effects which are often associated with diphtheria vaccination in adults, we have developed a vaccine which contains a highly purified, non-toxic mutant of diphtheria toxin. This vaccine is combined with tetanus toxoid and can be routinely used as a booster in adults.

Adolescent↗

PubMed1993

Studies of safety, infectivity and immunogenicity of a new temperature-sensitive (ts) 51-1 strain of Salmonella typhi as a new live oral typhoid fever vaccine candidate.

This report describes the results of a phase 1 study evaluating the safety, infectivity, and immunogenicity of a new live oral Salmonella typhi temperature-sensitive (ts) 51-1 typhoid fever vaccine in the human. Three normal male subjects aged 23-32 years received three oral doses of S. typhi ts 51-1, each dose containing 10(9) organisms. Prior to and following immunization each subject was carefully monitored by clinical and laboratory parameters over a 2 week period during which serial specimens of blood and stool were analysed for the presence of the organism. Blood specimens were also obtained for the determination of serum antibody and cell-mediated immune responses and stool filtrates were analysed for the development of coproantibody. The results of these studies indicate that: (1) the vaccine is well tolerated with no clinical or laboratory evidence of adverse reactions; (2) ts 51-1 was detected in only one stool specimen from one volunteer; the organism recovered displayed characteristics of the ts 51-1 vaccine strain; and (3) although no significant humoral or cell-mediated lymphocytotoxic immune responses were detected in the blood, coproantibody was detected in stool specimens from all of the three immunized subjects and IgA-armed ADCC activity was detected in two of three subjects. These studies indicate that S. typhi ts 51-1 may be a suitable strain for the development of an improved oral typhoid fever vaccine. Studies are in progress to determine optimal methods of vaccine delivery preparatory to large phase 2 studies of efficacy.

Administration, Oral↗

PubMed1993

Immunogenicity of an acellular pertussis vaccine composed of genetically inactivated pertussis toxin combined with filamentous hemagglutinin and pertactin in infants and children.

We studied the immunogenicity of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin, and a 69-kilodalton protein, pertactin, in 30 children aged 12 to 24 months and in 80 infants aged 2 to 4 months. A significant increase of the neutralizing titer and of the titers against pertussis toxin, filamentous hemagglutinin, and pertactin, as determined by enzyme-linked immunosorbent assay, was achieved after three doses of vaccine in all the children; a significant increase of these antibody titers was obtained in 100%, 96.1%, 93.5%, and 98.7% of the infants, respectively.

Adhesins, Bacterial↗

PubMed1992

Effect of tuftsin and its retro-inverso analogue on the release of interferon (IFN-gamma) and tumor necrosis factor (TNF-alpha) by human leucocytes.

The aim of this work was to demonstrate whether natural tuftsin or a retro-inverso (r.i.) analogue may induce interferon (IFN) and tumor necrosis factor (TNF) in peripheral-blood-mononuclear-cells (PBMC). For this purpose tuftsin or its analogue were added at different molar concentrations to PBMC and the supernatants were tested for IFN and TNF activity. Both cytokines were released after 12 hours incubation with r.i. tuftsin at an optimum concentration of 10(-10) M. Under the same conditions no activity was observed in the presence of natural tuftsin. In comparison to natural tuftsin the stimulatory activity of this tuftsin analogue is likely to be due to its high stability.

Adult↗

PubMed1992

Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C.

A conjugate vaccine against meningococcus A and C was prepared using the non-toxic mutant of diphtheria toxin CRM 197 as a carrier protein. Capsular polysaccharides of Neisseria meningitidis group A and C were hydrolysed and the resulting oligosaccharides were then coupled to CRM 197 in order to obtain conjugates with a carbohydrate content of 25-30%. The final vaccine that contained 11 micrograms of each oligosaccharide and 88 micrograms of CRM 197 was used to immunize mice and rabbits. After the preclinical studies which showed that the vaccine was safe and immunogenic in animal models, a pilot phase 1 clinical trial, blind versus placebo, was performed on adult volunteers. The difference between the incidence of adverse reactions associated with vaccine and placebo administration was not statistically significant. All the volunteers who received the vaccine had a significant increase in antibodies to group A and C meningococcal capsular polysaccharides after the first dose.

Animals↗

PubMed1992

Progress towards the development of new vaccines against whooping cough.

Acellular vaccines against whooping cough are in the final stage of clinical testing and are likely to become available for mass immunization in the near future. Over a dozen vaccines of similar composition have been developed by vaccine companies and research laboratories; all of them contain a detoxified form of pertussis toxin (PT) that may be present alone or combined with one or more other non-toxic proteins, such as filamentous haemagglutinin (FHA), pertactin (69 kDa), and the agglutinogens (AGG). Most of the vaccines contain a PT that has been inactivated by chemical treatment, a process that reduces the immunogenicity of the molecule and may not completely eliminate the risk of reversion to toxicity. To avoid these problems, we have constructed by genetic manipulation a mutant of Bordetella pertussis that produces a non-toxic form of PT. This molecule (PT-9K/129G) contains two amino acid substitutions in the S1 subunit (Arg9-->Lys and Glu129-->Gly) which abolish the enzymatic activity of the S1 subunit and all the toxic properties of PT, without changing the immunological properties of the wild-type toxin. Following extensive preclinical studies, which have shown that PT-9K/129G is safe and more antigenic than the toxin treated with chemical agents, this molecule was tested for safety and immunogenicity in adult volunteers, 18-month-old children and 2-month-old infants. The molecule has been tested alone, combined with FHA and pertactin and also combined with diphtheria and tetanus toxoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Acellular pertussis vaccine composed of genetically inactivated pertussis toxin: safety and immunogenicity in 12- to 24- and 2- to 4-month-old children.

To determine whether a nontoxic derivative of pertussis toxin obtained by recombinant DNA technology, PT-9K/129G, is a good candidate for a new pertussis vaccine, we examined the safety and the immunogenicity in children of a vaccine containing 15 micrograms of PT-9K/129G protein and 0.5 mg of aluminum hydroxide per dose. Fifty-three children 12 to 24 months of age and 21 infants aged 2 to 4 months were injected with two and three doses, respectively. The vaccine did not induce significant local or systemic reactions and elicited an increase of antibody titer in more than 98% of the children. The geometric mean of the toxin-neutralizing titers increased after each dose and was 85 units in children given two doses and 196 units in those given three doses. Two children who had detectable antibody levels before the first immunization had a high response (greater than 320 units) to the first vaccine dose. The findings suggest that PT-9K/129G is a promising antigen to be included in the development of acellular pertussis vaccines.

Antibodies, Bacterial↗

PubMed1992

Development and clinical testing of an acellular pertussis vaccine containing genetically detoxified pertussis toxin.

In 1924 Ramon described the inactivation of diphtheria toxin by formaldehyde treatment. This method allowed the introduction of mass vaccination against diphtheria and tetanus and opened the way to the inactivation of viruses by chemical treatment. In this review we describe the use of genetic manipulations for the inactivation of pertussis toxin. The toxin inactivated by this new method is an antigen superior to those obtained by chemical treatment and has been used to develop a new vaccine against whooping cough.

Animals↗

PubMed1992

Recombinant acellular pertussis vaccine--from the laboratory to the clinic: improving the quality of the immune response.

Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.

Amino Acid Sequence↗