Modification of the immune reaction by antigen-immunosuppressive-agent conjugates. II. Immunogenicity of antigen-immunosuppressive-agent conjugates]

The maintenance of specific immunogenicity of carrier proteins is a necessary condition for the successful use of antigen-immunosuppressive agent-conjugates (AIC) for an antigen specific suppression of the immune response. The experimental results indicate that, in spite of the binding of 6-mercaptopurine (6-MP) and toluyl (T) to bovine gamma globulin (BGG) and human serum albumin (HSA), the carrier specific immunogenicity is not significantly altered. The intradermal application of 6-MP-BGG and T-BGG emulsified with complete Freund's adjuvant in guinea pigs results, in all cases, in a well detectable anti BGG hemagglutination and precipitation titer. This kind of immunization leads also to a formation of anti 6-mercaptopurine and anti new antigen determinants (NAD's) antibodies.     

Modification of the immune reaction by antigen-immunosuppressive-agent conjugates. IV. Studies on the specific suppression of humoral immune response in guinea pigs by antigen-immunosuppressive-agent conjugates]

Bovine gamma globulin (BGG) antigens were modified by the binding of 6-mercaptopurine and toluyl residues, and their influence on the humoral immune response in guinea pigs was investigated. The antigen-immunosuppressive agent-conjugates (AIC) were different, depending on the method used for their preparation and the number of coupled residues per one molecule of BGG. Conjugates denoted as MPI-n-BGG were prepared by special chemical binding of corresponding thioisocyanates. MPII-n-BGG were synthetized by acetylation, and MPIII-n-BGG conjugates, by reductive alkylation. Pretreatment of guinea pigs with MPIII-19-BGG, MPII-16-BGG resulted in a stimulatory effect on the subsequent humoral immune response induced by BGG application. A significant suppressive influence was detectable if the animals had been pretreated with MPII-6-BGG and MPI-26-BGG. MPI-13-BGG and MPI-36-BGG had no effect on the later induced anti-BGG antibody formation. The immune response against a second antigen (human serum albumin) was not influenced by this kind of pretreatment of the animals. Therefore it seems justified to conclude that both stimulatory and suppressive effects seen here were antigen specific and that both the method for chemical modification and the number of coupled 6-MP residues are very important for their effectivity.     

Modification of immune reactions by antigen-immunosuppressive agent conjugates. V. Studies on antigen-specific activity of 6-mercaptopurine bovine gamma globulin conjugates on the humoral immune response in rabbits]

Rabbits treated daily and for seven consecutive days with 6-mercaptopurine bovine gamma globulin conjugates (MPI--n-BGG; 'I'--characterizes the king of chemical binding and 'n' the number of coupled MP-residues per one mole BGG) show an altered immunological reactivity. A following intradermal immunization with BGG alum adsorbate results in a suppressed anti BGG antibody production on the third day after antigen application (antibody titer 1:320, antibody titer of control animals--pretreated with BGG and uncoupled 6-MP equals 1:5120). Already three days later the antibody titers of the test groups show a significant increase and are two dilution stages higher than the titers of the controls. A suppressive effect on the third day is induced by MPI--13-BGG, MPI--26-BGG, MPI--36-BGG and MPI--46-BGG; the later adjuvant effect can only be seen in the MPI--26-BGG, MPI--36-BGG and MPI--46-BGG but not in the MPI--13-BGG pretreated animal group. While the short time suppression was antigen specific--the humoral immune response against a second unrelated antigen was not reduced--the adjuvans effect was not antigen specific. A pretreatment with the substances mentioned above results in an increased anti BGG and anti HSA serum antibody level. Comparing investigations on the unspecific immunosuppression in rabbits by 6-mercaptopurine shows that application of 10 mg 6-MP/kg/day for seven days at first leads to a suppression but later on to an enhancement of antibody production. Application of 10 mg 6-MP/kg/day for 10 days results in a long lasting suppression without enhancement effect. As a reason for these differences the different catabolism of immunosuppressive agent and antigen is discussed. For the phenomena following antigen specific immunosuppression, similar mechanisms can be responsible.     

Receptor-mediated oligodeoxynucleotides delivery by estradiol and folic acid polylysine conjugates

The lack of efficient and specific delivery to target cells still limits the potential application of antisense oligodeoxynucleotides as therapeutic agents in cancer disease. We have covalently linked a polylysine chain (10,000–20,000 mW) to compounds as folic acid, retinoic acid, transferrin, insulin and estradiol, to deliver c-myb antisense oligonucleotide into tumor cells. Using these complexes as carriers for the oligodeoxynucleotides can be achieved an increase in their uptake into target cells through a natural endocytosis pathway.     

Adenine salvage enzymes and intracellular nucleotide triphosphate content in Physarum flavicomum amoebae during growth and development

The specific activity of adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) and adenosine phosphorylase (EC 2.4.2.-), two enzymes involved in the utilization of exogenous adenine, was measured in extracts of myxamoebae-swarm cells of Physarum flavicomum undergoing growth, microcyst formation (control), and during adenine inhibition of encystment. Both enzymes showed a higher specific activity in adenine-inhibited cells (AIC) compared to normal control (NC) or growing cells (GC). These experiments revealed that the specific activity of APRT was 7.1-, 5.3-, and 1.7-fold higher than that of adenosine phosphorylase in AIC, GC, and NC, respectively. This suggests a predominant role for the enzyme APRT in the salvage of adenine in this organism. The major route for the utilization of adenine thus seems to be by its direct conversion to AMP rather than via its riboside adenosine. HPLC analysis of the ribonucleotide triphosphates in cell extracts of GC, NC, and AIC revealed a 2.6- and a 3.3-fold increase in the ATP and GTP content, respectively, in the AIC compared with the NC cells. The ATP content in the GC was higher by a factor of 2.2 compared with the NC cells, while the GTP content in the GC was only 0.6 times that in the NC cells. UTP levels in AIC and GC were 1.3- and 1.4-fold higher than in the NC cells. In contrast, the CTP level in AIC was lower than in NC cells and was not detectable in the growing cells.     

Structure of mouse interleukin 3 (IL-3) binding protein (AIC2A). Amino acid residues critical for IL-3 binding.

Despite the high degree of sequence homology between two mouse proteins AIC2A and AIC2B (91% at the amino acid level), only the AIC2A protein binds interleukin 3 (IL-3). Soluble AIC2A protein bound IL-3 with affinity similar to the membrane-bound AIC2A protein, indicating that binding of IL-3 to AIC2A was mediated by the external domain alone. The extracellular domain of the AIC2A protein has two repeats of the common motif shared by members of the cytokine receptor family. Neither one of these repeats alone bound IL-3. Hybrids of AIC2A and AIC2B revealed that the first domain of the cytokine receptor motif could be replaced with the AIC2B sequence without an affinity change, suggesting the importance of the second domain. By changing individual amino acid residues of AIC2A in the second domain which differ from those of AIC2B, we identified several amino acid residues critical for IL-3 binding. All these residues are located at the putative hinge region within the second domain.     

Variable selection with incomplete covariate data

SUMMARY: Application of classical model selection methods such as Akaike's information criterion (AIC) becomes problematic when observations are missing. In this article we propose some variations on the AIC, which are applicable to missing covariate problems. The method is directly based on the expectation maximization (EM) algorithm and is readily available for EM-based estimation methods, without much additional computational efforts. The missing data AIC criteria are formally derived and shown to work in a simulation study and by application to data on diabetic retinopathy.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates.

gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs.     

Synthetic developments towards PNA-peptide conjugates

Since the discovery of peptide nucleic acids (PNAs) as DNA mimics in the early 1990s, a tremendous effort has been directed to their application as antisense and antigene probes. With the aim of further enhancing their properties, PNAs have been conjugated to a variety of effector molecules. Among these, small peptide fragments, often derived from functional proteins, are able to convey their specific properties to the conjugate.     

AIC649 Induces a Bi-Phasic Treatment Response in the Woodchuck Model of Chronic Hepatitis B

AIC649 has been shown to directly address the antigen presenting cell arm of the host immune defense leading to a regulated cytokine release and activation of T cell responses. In the present study we analyzed the antiviral efficacy of AIC649 as well as its potential to induce functional cure in animal models for chronic hepatitis B. Hepatitis B virus transgenic mice and chronically woodchuck hepatitis virus (WHV) infected woodchucks were treated with AIC649, respectively. In the mouse system AIC649 decreased the hepatitis B virus titer as effective as the “gold standard”, Tenofovir. Interestingly, AIC649-treated chronically WHV infected woodchucks displayed a bi-phasic pattern of response: The marker for functional cure—hepatitis surface antigen—first increased but subsequently decreased even after cessation of treatment to significantly reduced levels. We hypothesize that the observed bi-phasic response pattern to AIC649 treatment reflects a physiologically “concerted”, reconstituted immune response against WHV and therefore may indicate a potential for inducing functional cure in HBV-infected patients.     

Ferrocene-avidin conjugates for bioelectrochemical applications.

Coupling of ferrocene moieties to avidin via a flexible spacer molecule yields a conjugate which combines the unique biotin-binding properties of avidin with the reversible redox characteristics of ferrocenes. Synthesis of the conjugate has been optimised and the conjugates were characterised bio- and electrochemically. Covalent immobilisation of the conjugate on gold electrodes in a dense monolayer results in electrodes with a high binding capacity for biotinylated molecules as well as good electron transfer properties. The application potential of such electrodes for bioelectrochemical systems is demonstrated by electrochemical reduction of hydrogen peroxide under mild conditions catalysed by a bound biotin-microperoxidase MP11 conjugate.     

Targeting of HIV gp120 by oligonucleotide-photosensitizer conjugates. Light-induced damages

The effects of the lysosphingolipid, sphingosylphosphorylcholine (SPC), on the cardiac ryanodine receptor (RyR) were examined. The open probability of cardiac RyR incorporated in lipid bilayers was decreased by cytoplasmic, but not lumenal side application of micromolar concentrations of SPC. Modification of channel function was characterized by the appearance of a long-lived closed state in addition to the brief channel closings observed in the presence and absence of SPC. Open channel kinetics and ion conduction properties, however, were not altered by this compound. These results suggest that SPC, a putative second messenger derived from sphingomyelin, may regulate Ca(2+) release from the sarcoplasmic reticulum by modifying the gating kinetics of the RyR.     

Receptor inactivation by dye-neuropeptide conjugates. 3. Comparative binding of dye-neuropeptide conjugates to FMRFamide receptors of Helix aspersa and Loligo pealei

Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [¹²⁵I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [¹²⁵I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC₅₀ of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.     

The Effect of Branch Length Variation on the Selection of Models of Molecular Evolution

Models of sequence evolution play an important role in molecular evolutionary studies. The use of inappropriate models of evolution may bias the results of the analysis and lead to erroneous conclusions. Several procedures for selecting the best-fit model of evolution for the data at hand have been proposed, like the likelihood ratio test (LRT) and the Akaike (AIC) and Bayesian (BIC) information criteria. The relative performance of these model-selecting algorithms has not yet been studied under a range of different model trees. In this study, the influence of branch length variation upon model selection is characterized. This is done by simulating sequence alignments under a known model of nucleotide substitution, and recording how often this true model is recovered by different model-fitting strategies. Results of this study agree with previous simulations and suggest that model selection is reasonably accurate. However, different model selection methods showed distinct levels of accuracy. Some LRT approaches showed better performance than the AIC or BIC information criteria. Within the LRTs, model selection is affected by the complexity of the initial model selected for the comparisons, and only slightly by the order in which different parameters are added to the model. A specific hierarchy of LRTs, which starts from a simple model of evolution, performed overall better than other possible LRT hierarchies, or than the AIC or BIC. Received: 2 October 2000 / Accepted: 4 January 2001     

Total strain fields of the antero-inferior shoulder capsule under subluxation: a stereoradiogrammetric study

The antero-inferior capsule (AIC) is the primary restraint to antero-inferior glenohumeral dislocation. This study utilizes a biomechanical model to determine the total strain field of the AIC in a subluxed shoulder. Strains were calculated from two capsule states: a nominal strain state set by inflation and a strained state set by subluxation. Marker coordinates on the AIC were reconstructed from stereoradiographs and strain fields calculated. Peak strain on the glenoid side of the AIC was significantly greater than the humeral side and strain fields were highly variable. This study reports an accurate method for measuring planar strains in a three-dimensional membrane.     

Protein-polymer conjugates: synthetic approaches by controlled radical polymerizations and interesting applications

Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, anchoring to surfaces, and supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to prepare protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted.     

Preparation and evaluation of tumor-targeting peptide-oligonucleotide conjugates

Enormous progress has been made in the development of antisense oligodeoxynucleotides (ODNs) as therapeutic agents inhibiting gene expression. Unfortunately, the therapeutical application of ODNs is still held back because of the low cellular uptake and the lack of specific transport into particular cells. In this paper, we report a drug-targeting system using somatostatin receptors (SSTRs) which are overexpressed in various tumors. Phosphorothioate ODNs were covalently linked to Tyr(3)-octreotate, an analogue of somatostatin. The peptide was assembled by solid-phase synthesis, oxidized to form the cyclic disulfide, and subsequently derivatized with a N-terminal maleimido functionality. 5'-Thiol derivatized phosphorothioate-ODNs directed against the protooncogene bcl-2 were conjugated to this maleimido-modified peptide. Binding studies revealed that the conjugates retain specific binding with nanomolar affinities to SSTRs (IC(50)-values between 1.83 and 2.52 nM). Furthermore, melting studies with complementary DNA revealed that the terminal conjugation of the ODNs did not significantly affect their hybridization affinity.