Antibody studies in mice of outer membrane antigens for use in an improved meningococcal B and C vaccine

Abstract Since 1988, N. meningitidis , B:4:P1.15, ET-5 complex, has been responsible for an epidemic of meningococcal disease in Greater São Paulo, Brazil. Despite current trials to develop an effective vaccine against group B meningococci, children less than 2 years old have not been protected. It has been suggested that iron-regulated proteins (IRPs) should be considered as potential antigens for meningococcal vaccines. The vaccines under study consisted of outer-membrane vesicles depleted of lipooligosaccharide from three serogroup B strains and one serogroup C strain, IRPs, meningococcal group C polysaccharide and aluminum hydroxide. Four different protein and C polysaccharide concentrations were studied. The ELISA and bactericidal results showed a higher antibody response when 2 injections of 2.0 μg doses were administered. Despite higher IgG reactivity against antigen preparations containing IRPs seen in ELISA, the bactericidal activity was not increased if the target strain was grown in iron-restricted medium. The influence of addition of alkaline-detoxified lipooligosaccharide (dLOS) on immunogenicity of the vaccine was also investigated, and the dLOS provided for a more functionally specific antibody response.     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

rG-CSF reduces endotoxemia and improves survival during E.coli pneumonia

Freeman, Bradley D., Zenaide Quezado, Fabrice Zeni, CharlesNatanson, Robert L. Danner, Steven Banks, Marcello Quezado, YvonneFitz, John Bacher, and Peter Q. Eichacker. rG-CSF reduces endotoxemia and improves survival during E. coli pneumonia. J. Appl.Physiol. 83(5): 1467-1475, 1997.We investigatedthe effects of recombinant granulocyte colony-stimulating factor(rG-CSF) during canine bacterial pneumonia. Beagles with chronictracheostomies received daily subcutaneous rG-CSF (5 µg/kg body wt)or placebo for 14 days, beginning 9 days before intrabronchialinoculation with E. coli. Animalsreceived antibiotics and fluid support; a subset received humidifiedoxygen (fractional inspired O₂0.40). Compared with controls, rG-CSF increased circulating neutrophil counts (57.4 vs. 11.0 × 10³/mm³,day 1 after infection;P = 0.0001), decreased plasmaendotoxin (7.5 vs. 1.1 EU/ml at 8 h; P < 0.01) and serum tumor necrosis factor- (3,402 vs.729 pg/ml at 2 h; P = 0.01) levels,and prolonged survival (relative risk of death = 0.45, 95% confidenceinterval 0.21-0.97; P = 0.038).Also, rG-CSF attenuated sepsis-associated myocardial dysfunction(P < 0.001). rG-CSF had no effect onpulmonary function or on blood and lung bacteria counts (allP = not significant). Other animalschallenged with endotoxin (4 mg/kg iv) after similar treatment withrG-CSF had lower serum endotoxin levels (7.62 vs. 5.81 log EU/ml at 6 h; P < 0.01) and less cardiovasculardysfunction (P < 0.05 to < 0.002)but similar tumor necrosis factor- levels (P = not significant) compared withcontrols. Thus prophylactic rG-CSF sufficient to increase circulatingneutrophils during bacterial pneumonia may improve cardiovascularfunction and survival by mechanisms that in part enhance the clearanceof bacterial toxins but do not improve lung function.

     

Production of neosolaniol byFusarium tumidum

Extracts from autoclaved maize culture ofFusarium tumidum strain R-5823 were toxic towardsArtemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by¹H,¹³C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains ofF. tumidum andGibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin byF. tumidum.     

Influence of mature and immature myeloid cells on lymphocyte reactivity in chronic myelocytic leukemia

Summary Earlier studies have shown anergy in chronic myeloid leukemia (CML), and it is known that myeloid cells influence lymphocyte responses. Therefore, lymphocytes from CML patients who had received no cytostatics for 2 weeks were stimulated in 89 tests with PHA and ConA. In 39 control tests, normal lymphocytes were used.Lymphocytes from CML patients were significantly less (p<0.05) markedly stimulated than normal ones. Lymphocytes from CML patients with more than 10×10⁹ white blood cells (WBC) per liter blood were inhibited to a greater degree than those from patients with a normal WBC count.When normal lymphocytes were stimulated with PHA in the presence of mononuclear cells from the blood of CML patients (mostly leukemic myelocytes), their response was significantly (p<0.05) inhibited. Inhibition with leukemic myelocytes was significantly (p<0.05) greater than that with mature granulocytes from CML patients. The latter did not seem to have an inhibitory effect.We suggest that patients with manifest CML are anergic to some extent because leukemic myelocytes have a suppressor effect.Visiting scientist and Anna Villa Rusconi Fellow, on secondment from Institute of Medical Pathology, University of Ferrara, Italy     

New pulsed field gradient NMR experiments for the detection of bound water in proteins

Summary New H₂O-selective homonuclear and heteronuclear 2D NMR experiments have been designed for the observation of protein hydration (PHOGSY, Protein Hydration Observed by Gradient Spectroscop Y). These experiments utilize selective excitation of the H₂O resonance and pulsed field gradients for coherence selection and efficient H₂O suppression. The method allows for a rapid and sensitive detection of H₂O molecules in labelled and unlabelled proteins. In addition it opens a way to measure the residence time of water bound to proteins. Its application to uniformly ¹⁵N-labelled FKBP-12 (FK-506 binding protein) is demonstrated.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.