A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides

During the last decade, episodes of sepsis have increased and Escherichia coli has remained the most frequent clinical isolate. Lipopolysaccharides (LPS; endotoxin) are the major toxic and antigenic components of gram-negative bacteria and qualify as targets for therapeutic interventions. Molecules that neutralize the toxic effects of LPS are actively investigated. In this paper, we describe a murine monoclonal antibody (MAb; WN1 222-5), broadly cross-reactive and cross-protective for smooth (S)-form and rough (R)-form LPS. As shown in enzyme-linked immunosorbent assay and the passive hemolysis assay, WN1 222-5 binds to the five known E. coli core chemotypes, to Salmonella core, and to S-form LPS having these core structures. In immunoblots, it is shown to react with both the nonsubstituted core LPS and with LPS carrying O-side chains, indicating the exposure of the epitope in both S-form and R-form LPS. This MAb of the immunoglobulin G2a class is not lipid A reactive but binds to E. coli J5, an RcP+ mutant which carries an inner core structure common to many members of the family Enterobacteriaceae. Phosphate groups present in the inner core contribute to the epitope but are not essential for the binding of WN1 222-5 to complete core LPS. Cross-reactivity for clinical bacterial isolates is broad. WN1 222-5 binds to all E. coli clinical isolates tested so far (79 blood isolates, 80 urinary isolates, and 21 fecal isolates) and to some Citrobacter, Enterobacter, and Klebsiella isolates. This pattern of reactivity indicates that its binding epitope is widespread among members of the Enterobacteriaceae. WN1 222-5 exhibits biologically relevant activities. In vitro, it inhibits the Limulus amoebocyte lysate assay activity of S-form and R-form LPS in a dose-dependent manner and it neutralizes the LPS-induced release of clinically relevant monokines (interleukin 6 and tumor necrosis factor). In vivo, WN1 222-5 blocks endotoxin-induced pyrogenicity in rabbits and lethality in galactosamine-sensitized mice. The discovery of WN1 222-5 settles the long-lasting controversy over the existence of anti-core LPS MAbs with both cross-reactive and cross-protective activity, opening new possibilities for the immunotherapy of sepsis caused by gram-negative bacteria.     

High-performance liquid chromatographic separation of carminic acid, alpha- and beta-bixin, and alpha- and beta-norbixin, and the determination of carminic acid in foods

During a study of natural food colours, a simple and reliable high-performance liquid chromatography system was developed for use with cochineal and annato. An isocratic mobile phase, consisting of methanol and 6% aqueous acetic acid, resolved bixin and norbixin, while a gradient system was used to separate carminic acid and the annato compounds. The carminic acid contents of cochineal extract, carmine and carmine hydrosoluble were determined using an isocratic mobile phase (40:60, v/v). The detection limit for carminic acid in the various products was approximately 100 ng/g. Carminic acid was determined quantitatively in fruit beverages, yogurt and candies. It was demonstrated that, because of decomposition, carminic acid was not suitable for use in candies when manufacturing temperatures above 100 degrees C were required. Most membrane filters are not suitable for use with cochineal solutions, but a cellulose membrane filter did not adsorb carminic acid and was used successfully to remove impurities from water-based cochineal products and food extracts containing carminic acid.     

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.     

A new epitope presenting system displays a HIV-1 V3 loop sequence and induces neutralizing antibodies

The principal neutralizing domain, IGPGRAF sequence, from the V3-loop of HIV-1 was inserted in two positions on the surface of the protein that makes up the capside shell of the insect Flock House Virus. The hybrid proteins were expressed in insect cells via recombinant baculoviruses. Three different hybrids were used as immunogens: two with a single copy of the insert in different positions of the carrier protein and a third with two copies of the insert at the same positions as before. All hybrid proteins induced strong and broad specific immune response in guinea pigs against different V3-loop sequences. However, only one of the hybrid proteins was able to induce a strong neutralizing response against MN and IIIB HIV-1 isolates. Our results demonstrate that a very short peptide sequence of HIV-1 can constitute a valuable immunogen able to induce a neutralizing response if presented to the immune system in the context of the FHV capsomer structure.     

Deletions in one domain of the Friend virus-encoded membrane glycoprotein overcome host range restrictions for erythroleukemia

Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.     

Managing side effects of interferon-beta in patients with relapsing-remitting multiple sclerosis

In separate clinical trials, two preparations of recombinant interferon (IFN)-beta, IFN beta-1a and IFN beta-1b, reduced exacerbation rates in relapsing-remitting multiple sclerosis (RR-MS). Further, IFN beta-1a slows the progression of disability in patients with RR-MS. Although they are effective in the treatment of MS, use of these drugs is associated with both class-specific and agent-specific side effects. Class-specific side effects include fever, chills, myalgias, arthralgias, and other flulike symptoms beginning 2 to 6 hours after injection and resolving within 24 hours of injection. Transient worsening of preexisting MS symptoms also occurs infrequently. Agent-specific side effects include injection-site reactions with IFN beta-1b. Simple management strategies can be used to minimize these reactions, including patient education; tailoring the dose and time of administration of IFN-beta; and prescribing appropriate combinations of acetaminophen, non-steroidal anti-inflammatory drugs, and steroids. Although side effects tend to diminish with treatment, successful management allows long-term administration of these drugs to achieve a reduction in disease activity and commensurate improvement in outcomes.     

The effect of antimicrobial agents on the induction of tumour necrosis factor by alveolar macrophages in vitro in response to endotoxin

1. In the present study, we evaluated the role of repeated administration on conditioning place preference (CPP) induced by fencamfamine (FCF) in male rats. 2. Repeated FCF (3.5 mg/kg) or saline once or daily for ten consecutive days enhanced sniffing duration and decreased locomotion and rearing duration. 3. At the 3.5 mg/kg dose, FCF produced a significant place-preference effect. 4. Repeated exposures to FCF intensified its reinforcing properties. 5. These results suggest that repeated FCF administration sensitizes its rewarding effects, as with other addictive substances.