Soy isoflavones, lactobacilli, Magnolia bark extract, vitamin D3 and calcium. Controlled clinical study in menopause.

AIM: Affective and behavioural disorders possibly concomitant to the vasomotor menopausal symptoms worsen quality of life. A rational formulation containing soy isoflavones (60 mg), lactobacilli (500 millions spores), calcium (141 mg) and vitamin D3 (5 microg) was added of Magnolia bark extract (60 mg) and magnesium (50 mg) (Estromineral serena, ES). The Magnolia extract active principles interact with GABA system and exhibit a sedative central action. Magnesium intervenes in enzymatic reactions of the energetic metabolism and protects the bone integrity. Aim of this controlled study was to compare the clinical activity and safety of ES versus calcium+vitamin D3 (Ca+D) in menopause. METHODS: A controlled, randomised, multicentre study was carried out in symptomatic menopausal women with sleep or mood alterations. Women received 1 tablet/day of ES or Ca+D for 24 weeks. Symptoms during the treatment and final judgements on efficacy and acceptability were evaluated. RESULTS: Eighty-nine women (44 ES and 45 Ca+D, mean age 53.8 years, in menopause since 56.6 months) participated to the study. Flushing, nocturnal sweating, palpitations, insomnia, asthenia, anxiety, mood depression, irritability, vaginal dryness, dyspareunia, and libido loss, significantly decreased in severity and frequency during ES versus Ca+D treatment even since the fourth week. Woman wellbeing (good/very good 66.7% vs 20%) judgement on efficacy (72.7% vs 17.1%) and acceptability (93.9% vs 31.4%) were significantly better for ES. CONCLUSIONS: The controlled study showed the efficacy of Magnolia extract and magnesium on psycho-affective and sleep disturbances in menopause, in addition to the effects of isoflavones on vasomotor symptoms. A global natural approach to menopause with ES evidenced its therapeutic usefulness and safety.

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.

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.

Cellular pertussis vaccine containing a Bordetella pertussis strain that produces a nontoxic pertussis toxin molecule.

Bordetella pertussis 165-9K/129G, which produces a nontoxic form of pertussis toxin (PT), was used to prepare a whole-cell diphtheria-tetanus-pertussis (DTP) vaccine. The in vivo potency and the serological response induced by this vaccine were comparable to those of the conventional DTP vaccine which contains active PT. The toxic activities induced by PT such as leukocytosis, histamine sensitivity, and potentiation of anaphylactic reactions, which are present in the conventional DTP vaccine, were absent in the new vaccine. These results suggest that the introduction of a whole-cell vaccine containing B. pertussis 165-9K/129G would induce the same immunity as the conventional vaccine and would avoid the administration of a harmful toxin to children.

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.

Properties of the B oligomer of pertussis toxin.

The B oligomer of pertussis toxin was purified from culture supernatants of Bordetella pertussis strains which do not secrete the S1 subunit. The purified B oligomer is devoid of toxicity for CHO cells and other in vivo properties of the toxin, such as leukocytosis and histamine sensitization, but it retains the abilities to agglutinate erythrocytes and to induce the proliferation of T lymphocytes. The B oligomer is also able to induce protective immunity in mice but is less potent than molecules containing the S1 subunit also.

Phase I clinical trial of an acellular pertussis vaccine composed of genetically detoxified pertussis toxin combined with FHA and 69 kDa.

An acellular pertussis vaccine composed of genetically detoxified pertussis toxin (PT-9K/129G), filamentous haemagglutinin (FHA) and pertactin (69 kDa protein) was evaluated in adult volunteers, in double blind, versus placebo. No fever was reported in either group. Mild local reactions were reported after injection of both vaccine and placebo. After the first dose a marked increase in antibodies to PT, FHA and 69 kDa protein was seen in vaccinated subjects with the exception of one who responded well to PT and FHA but did not show a humoral response to the 69 kDa protein. All vaccinees acquired cellular immunity against the three antigens. No significant variation was observed in the humoral or cellular responses after the second dose.

Properties of pertussis toxin mutant PT-9K/129G after formaldehyde treatment.

Formaldehyde treatment is a method routinely used to detoxify diphtheria, tetanus, and pertussis toxins as well as other molecules suitable for vaccine production. To investigate whether chemical detoxification alters the immunological properties of vaccine components, we have treated the pertussis toxin mutant PT-9K/129G with formaldehyde and tested the properties of the resulting molecules. Very low concentrations of formaldehyde stabilize the molecule without affecting the physicochemical and immunological parameters. Increasing doses of formaldehyde abolish the mitogenic and hemagglutinating activities of PT-9K/129G. At the same time, the molecule loses the ability to be recognized by a monoclonal antibody specific for a major protective epitope on the S1 subunit of pertussis toxin and its affinity for anti-pertussis toxin polyclonal antibodies is also reduced. In marked contrast, the ability of PT-9K/129G to be recognized by human T-cell clones is not affected by Formalin treatment. In vivo, the formaldehyde-treated molecules induce amounts of specific antibodies comparable with those of untreated molecules but significantly lower levels of toxin-neutralizing antibodies. Furthermore, the formaldehyde-treated molecules also show a reduced protective activity in the intracerebral challenge assay.

Differential activity of interleukin 1 alpha and interleukin 1 beta in the stimulation of the immune response in vivo.

The biological activities of human recombinant interleukin (IL) 1 alpha and IL 1 beta were compared in different biological systems. The two IL 1 forms were equally active in vitro in inducing proliferation of murine thymocytes and of the murine T helper clone D10.G4.1, and in triggering release of prostaglandin E2 from human skin fibroblasts. In vivo, IL 1 alpha and IL 1 beta were similarly pyrogenic both in rabbits and mice, and could equally increase the circulating levels of the acute phase protein serum amyloid A in mice. However, only IL 1 beta showed immunostimulatory activity in vivo, as it could enhance the number of specific antibody-producing cells in the spleen of mice immunized with either a T-dependent or a T-independent antigen. Although devoid of immunostimulatory activity, IL 1 alpha could efficiently compete immunostimulation induced by IL 1 beta, suggesting an effective interaction with the IL 1 receptor. Thus, IL 1 beta appears to have an important role in the positive regulation of immune responses, while IL 1 alpha may act as down-regulator of the IL 1 beta effect.

One-step immunoaffinity purification of bioactive human recombinant IL-1 beta with a monoclonal antibody directed to a well-exposed domain of the protein.

A monoclonal antibody (mAb) specific for human recombinant IL-1 beta (hu rIL-1 beta) was produced by immunizing BALB/c mice with hu rIL-1 beta purified with classical methods. This mAb recognizes an epitope within the highly hydrophylic fragment spanning amino acid 133-147. The affinity constant of this mAb towards IL-1 beta was determined by RIA. An affinity column was prepared by covalent binding of the mAb to Sepharose CL-4B. The column was capable of selectively binding hu rIL-1 beta produced in Escherichia coli directly from crude homogenates. The IL-1 beta protein yield was higher than 90% with a very good recovery of IL-1 beta biological activity. Moreover, the immunosorbent retained at least two thirds of its IL-1 beta-binding capacity after 20 cycles of purification.

A monoclonal antibody to the IL-1 beta peptide 163-171 blocks adjuvanticity but not pyrogenicity of IL-1 beta in vivo.

The synthetic fragment VQGEESNDK, corresponding to the amino acid sequence in position 163-171 of human IL-1 beta, possesses the immunostimulatory but not the pyrogenic activity of the mature IL-1 beta polypeptide in vivo. To assess the relevance of this domain of IL-1 beta for its biologic activities, a mAb was raised against the synthetic peptide 163-171. The mAb Vhp20 could effectively recognize human rIL-1 beta in RIA and immunoblotting. In vivo, the mAb Vhp20 was able to selectively inhibit the immunostimulatory activity of IL-1 beta, but it could not affect the fever-inducing capacity of IL-1 beta. It is proposed that functional domains could be identified in the human IL-1 beta protein and that the fragment in position 163-171 is of major importance for the adjuvant capacity of the entire molecule, but irrelevant to its pyrogenic activity.

Interleukin 1 and its synthetic peptides as adjuvants for poorly immunogenic vaccines.

The adjuvant activity of the peptide corresponding to the fragment 163-171 of human interleukin 1 beta (VQGEESNDK) has been evaluated on the immune response to both T-dependent and T-independent antigens. The hydrochloride derivative of the peptide showed an effect quantitatively comparable in molar terms to that of the entire protein. At variance with the entire IL 1 protein the peptide appeared devoid of inflammatory effects and therefore it may find clinical applications as adjuvant for poorly immunogenic vaccines or as immunorestorative agent. The activity of other fragments in proximity of the sequence 163-171 was also evaluated. The shorter fragment 165-171 appeared as active as the 163-171 peptide.

Influence of the peptide insolubilization method on detection of anti-peptide antibodies in ELISA. Evaluation of nonspecific interactions.

Different methods of peptide insolubilization in solid phase were compared in ELISA, to verify the influence of the peptide antigen presentation in the interaction with related antibodies. Our studies were performed using as model the peptide fragment 163-171 of human Interleukin 1 beta, and polyclonal or monoclonal anti-peptide antibodies. It was found that the peptide, N-terminally linked to a protein carrier before the adsorption on microtiter wells, interacted with specific polyclonal and monoclonal antibodies with high sensitivity and specificity. In contrast the recognition of similar random conjugates, prepared using a bivalent cross-linking reagent or the peptide covalently linked to poly-L-Lysine-pretreated wells, was hampered generally by very high levels of nonspecific binding. On the other hand, the free peptide adsorbed directly to the solid phase interacted with antibodies with very low sensitivity and specificity. Nonspecific interactions were found in particular between peptides and hyperimmune sera or nonrelated monoclonal antibodies. On the contrary pre-immune sera and normal mouse immunoglobulins never showed significant interactions with any of peptides. This nonspecificity was also overcome when N-terminally linked peptide-protein conjugates were used for the assay.

A short synthetic peptide fragment of human interleukin 1 with immunostimulatory but not inflammatory activity.

Short peptide fragments of human and murine interleukin 1 (IL 1) were synthesized on the basis of their predicted exposure on the surface of the molecule in an attempt to identify the minimal structure responsible for the immunostimulatory activity of IL 1. One of these peptides, a fragment of nine residues of human IL 1 beta (VQGEESNDK, fragment 163-171), showed high T cell activation capacity, as judged by its ability to stimulate murine thymocyte proliferation and to potently induce interleukin 2 production in spleen cells. On the other hand, the 163-171 peptide was devoid of prostaglandin-inducing capacity in vitro and pyrogenic activity in vivo, two inflammatory features peculiar to the entire hu IL 1 beta molecule. Thus we propose that this peptide may represent one of the portions of hu IL 1 beta responsible for its immunostimulatory capacity.