Prediction of the intramembranous tissue formation during perisprosthetic healing with uncertainties. Part 2. Global clinical healing due to combination of random sources.

This work proposes to examine the variability of the bone tissue healing process in the early period after the implantation surgery. The first part took into account the effect of variability of individual biochemical factors on the solid phase fraction, which is an indicator of the quality of the primary fixation and condition of its long-term behaviour. The next issue, addressed in this second part, is the effect of cumulative sources of uncertainties on the same problem of a canine implant. This paper is concerned with the ability to increase the number of random parameters to assess the coupled influence of those variabilities on the tissue healing. To avoid an excessive increase in the complexity of the numerical modelling and further, to maintain efficiency in computational cost, a collocation-based polynomial chaos expansion approach is implemented. A progressive set of simulations with an increasing number of sources of uncertainty is performed. This information is helpful for future implant design and decision process for the implantation surgical act.

Introduction of the maleimide function onto resin-bound peptides: a simple, high-yield process useful for discriminating among several lysines.

Incorporation of epsilon-Adpoc-lysine as a residue in solid phase peptide synthesis allows selective deprotection of this residue on the resin-bound peptide relative to other acid labile groups such as Boc. Premature resin cleavage is avoided. A maleimide group, a useful thiol-capture reagent, was readily introduced by reacting the liberated amino function with an acylating agent containing the maleimide functionality. Acidic cleavage from the resin, with an appropriate scavenging system, afforded peptides that are derivatized with a maleimide functionality on a specific lysine. This is advantageous for producing peptide-carrier conjugates of defined specificity, useful as immunogens, by maleimide-thiol coupling. The derivatization and resin removal chemistries appear to proceed in excellent yield with respect to the maleimide group. The structures were confirmed by tandem mass spectrometry.

Haemophilus influenzae type b conjugate vaccines.

In summary, all of the Hib conjugate vaccines are highly immunogenic and efficacious in children older than 12-15 months of age, and HbOC, PRP-OMPC, and PRP-T are highly immunogenic and demonstrated to be efficacious in infants as young as 2 months old. HbOC, PRP-OMPC, and PRP-T have been licensed in numerous countries for infants and are recommended for infant immunization. However, perhaps the greatest tribute one can pay to all four Hib vaccines described in this review is to note the dramatic decrease in the incidence of Hib disease that has occurred since their introduction. In fact, according to the Morbidity and Mortality Weekly Report (March 4, 1994), the incidence of Hib disease in children less than 5 years old has declined by 95% from 41 cases per 100,000 in 1987 to 2 cases per 100,000 in 1993, timing that coincides with the availability and use of the Hib conjugate vaccines (Anderson, 1994). As universal administration is achieved and the apparent vaccine-induced reduction in carriage of Hib by the population continues, Hib vaccines may follow the lead of past vaccines (such as smallpox, measles, mumps, rubella, and polio) toward eradication of disease or at least a high degree of medical control, thereby virtually eliminating the mortality and insidious morbidity associated with invasive Hib diseases.

Immunogenicity of synthetic HIV-1 gp120 V3-loop peptide-conjugate immunogens.

A successful prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine must elicit an immune response that will prevent establishment of the persistent viral infection. The only response shown to be effective in this regard is virus-neutralizing antibody directed against the viral gp120 hypervariable V3-loop region. Conjugate immunogens, containing cyclic peptides representing the V3 determinant covalently bound to a carrier protein, were capable of eliciting virus-neutralizing antibodies. The consistency of the response was related to peptide size. The smaller cyclic peptides, expressing relatively conserved sequences from the V3-loop apex, were poor inducers of neutralizing activity. In contrast, the largest cyclic peptides mediated neutralizing responses that were similar to those observed and previously reported for intact gp120 immunogens. A cyclic synthetic peptide expressing most of the prototypic HIV-1 MN variant V3 determinant warrants further study as a potentially effective vaccine immunogen.

Cyclic V3-loop-related HIV-1 conjugate vaccines. Synthesis, conformation and immunological properties.

Branched undecapeptides with sequences related to the virus glycoprotein V3 domain sequences of the MN and IIIB variants of HIV-1 were synthesized and cyclized with a peptide (amide) closure to cyclic decapeptides. Two-dimensional NMR studies allowed protons for the MN variant-related cycle (L-697,250) to be assigned. Molecular modelling with distance geometry methods permitted a conformation to be identified which showed good agreement with ROESY and 2D NMR study data. A molecular dynamics simulation showed that the highly conserved loop tip sequence (Gly-Pro-Gly-Arg) was in a conventional beta-turn less than 50% of the time. For evaluation of immunogenicity and antibody characterization studies, covalent carrier conjugates were prepared. 3-Maleimidopropionylation of the Nle amino group of the cyclic peptides gave an electrophilic tether which captured a thiol group from a thiolated carrier protein, OMPC (outer membrane protein complex of Neisseria meningitidis). Through the use of a novel co-conjugation procedure, soluble immunogen-carrier molecules were prepared which had suitable physical properties for use as a vaccine. These V3-loop-based vaccines could elicit neutralizing antibody, but not consistently in all animals. Characterization of sera showed that responses were broadly virus neutralizing.

Immunogenicity of conjugate vaccines consisting of pneumococcal capsular polysaccharide types 6B, 14, 19F, and 23F and a meningococcal outer membrane protein complex.

In an effort to prepare pneumococcal (Pn) capsular polysaccharide (Ps) vaccines that would be immunogenic in infants, covalent conjugates were prepared for Pn types 6B, 14, 19F, and 23F. Each Ps type was covalently bound to an outer membrane protein complex from Neisseria meningitidis serogroup B and evaluated for immunogenicity in mice and infant monkeys. The conjugates induced specific anti-Ps antibody responses in mice and in infant rhesus and African green monkeys; a conjugate of 6B and outer membrane protein complex was immunogenic at Ps doses as low as 20 ng. Although low levels of the Pn group-common cell wall polysaccharide were present in all type-specific Ps preparations, anti-cell wall polysaccharide responses induced by covalent conjugates were < 1% of the total anti-Ps response after two doses of vaccine. In contrast, the anti-cell wall polysaccharide response of a noncovalent conjugate represented 41% of the anti-Ps response after two doses. Relative T-cell dependence, a requirement for the human target population of infants less than 18 months old, was demonstrated for all four Pn Ps conjugates in an athymic mouse model. Therefore, these Pn Ps-outer membrane protein complex conjugate vaccines are excellent candidates for evaluation in human infants.

Acidic derivatives of homocysteine thiolactone: utility as anionic linkers.

Homocysteine thiolactone (2) derivatives in which the nitrogen is acylated with groups containing acidic functionalities have been synthesized. These include the succinyl (3), the carboxymethylglutaryl (4), the 3-phosphonopropionyl (7), and the 3-sulfopropionyl (8) derivatives. These thiolactones can be used to introduce a thiol functionality into proteins such as the outer membrane protein complex of Neisseria meningitidis (OMPC) allowing conjugation with electrophilic ligands. This chemistry is the same as with N-acetylhomocysteine thiolactone (1), but their pKa values are such that at pH 7 concomitant negative charge is introduced into the conjugate. Such negative charge should neutralize some excess positive charge introduced when arginine- and lysine-rich peptides are bonded as ligands. In the case of OMPC, introduction of such positive charge appears to effect irreversible precipitation. The system has been studied using the maleimidopropionyl and bromoacetyltriarginine (9 and 10) derivatives as models. In select instances anionic spacers reduce the degree of precipitation relative to N-acetyl-homocysteine thiolactone derivatives.

Immunogenicity of a new Haemophilus influenzae type b conjugate vaccine (meningococcal protein conjugate) (PedvaxHIB).

Haemophilus influenzae type b is responsible for an estimated 15,000 to 20,000 cases of meningitis per year in the United States, mainly in children 2 months to 5 years old. The mortality rate from meningitis due to H influenzae type b infections ranges from 5% to 10%. Despite antibiotic treatment, up to 35% of survivors have permanent neurologic sequelae. In addition to meningitis, H. influenzae type b is responsible for other invasive infections, including epiglottitis, septicemia, cellulitis, septic arthritis, osteomyelitis, pneumonia, pericarditis, and otitis media; approximately 30,000 cases H influenzae diseases occur annually in the United States. The diseases peak in incidence between 6 and 12 months of age, with almost one half of the cases occurring before 1 year of age. About 75% of disease caused by H influenzae type b occurs in children younger than 24 months old. The incidence of disease is higher in children of certain groups, including blacks, Hispanics, Eskimos and Native Americans, young children attending day-care facilities, patients with asplenia or antibody-deficiency syndromes, and children of lower socioeconomic status. There is considerable evidence that antibody to the capsular polysaccharide (polyribosylribitol-phosphate [PRP] of H influenzae type b is protective.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemistry on solid supports: defining events and titers by use of cleavable, assayable linking molecules.

The cleavable diamines cystamine, 5, and 1,6-diamino-3,4-dihydroxyhexane, 1, were bonded to solid supports and, with a simple, newly developed dinitrofluorobenzene-based assay, were used to define (a) titers of ligands and (b) chemistry distal to the support. Compound 1, which is cleavable with periodate, becomes a linking molecule which is stable to almost all conditions encountered in biochemistry and enjoys considerable hydrophilic character. Compound 5, which is cleavable with dithiothreitol, can be usefully applied to those systems which do not require reducing agents. These nucleophilic linking moieties were converted to cleavable electrophilic linkers by succinylation and p-nitrophenyl ester activation. The first preparation of a polysaccharide-linked support is described. The method also allows the chemical definition of ligands containing amino groups which are prepared by deblocking of protecting groups while on the support. The methodology should promote greater understanding of affinity chromatography materials and processes.

Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C.

Pseudomonas exotoxin A: toxoid preparation by photoaffinity inactivation.

A method for toxoid preparation has been developed in which toxins expressing enzymatic activity can be detoxified by photoaffinity labeling techniques. In the case of Pseudomonas aeruginosa exotoxin A, the method relies on the affinity of azido-substituted analogs of the substrate (NAD) for the proenzyme form of the toxin. Photolysis of the putative toxin-analog complex results in irreversible inactivation of the toxin without loss of antigenic character. It is proposed that this occurs by nitrene insertion into a chemical bond on the toxin molecule. This affinity photoinactivation process should be applicable to other ADP-ribosylating toxins.