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E Akerblom

Publications and source records attributed to E Akerblom.

18 recordsLinked to original sources
PubMed2001

Inhibition of hepatitis C virus NS3 protease activity by product-based peptides is dependent on helicase domain.

Structure activity relationships (SARs) of product-based inhibitors of hepatitis C virus NS3 protease were evaluated using an in vitro assay system comprising the native bifunctional full-length NS3 (protease-helicase/NTPase). The results were compared to previously reported data derived from the corresponding NS3 protease domain assay. Shortening the length of the protease inhibitors from hexapeptides to tripeptides revealed that the decrease in potency was much less when determined in the assay system with the full-length NS3 protein. Disagreements in SARs at different positions (P5 P2) were also discovered. Taken together, the results suggest that the impact of the helicase domain upon protease inhibitor binding is substantial.

DNA-Binding Proteins↗
PubMed1993

T cell killing of human colon carcinomas by monoclonal-antibody-targeted superantigens.

The bacterial superantigen staphylococcal enterotoxin A (SEA) induces T cell activation as well as directing activated T cells to kill major-histocompatibility-complex-class-II-expressing tumours such as freshly prepared leukemia cells. We now report that conjugates of SEA and the colon-carcinoma-reactive mAb C215 mediate T-cell-dependent killing of freshly isolated cells obtained from surgical specimens of human colon carcinomas. Cytotoxicity was observed at nanomolar concentrations of conjugate while no or very low effects were seen with the mAb C215 or SEA alone. Tumour-infiltrating lymphocytes (TIL) did not exert any cytotoxicity against conjugate-treated tumour cells immediately after isolation. In vitro culture of TIL with interleukin-2 and SEA resulted in SEA-mAb-conjugate-dependent killing of freshly isolated tumour cells. This suggests that mAb-SEA conjugates may be of potential use to target T lymphocytes, including TIL, against colon carcinoma cells in vivo.

Antibodies, Monoclonal↗
PubMed1992

Chemical and physiological properties of polysucrose, a new marker of intestinal permeability to macromolecules.

To measure intestinal absorption of macromolecules we have developed a new technique employing synthetic polysucrose polymers as probe molecules. Polysucrose (PS) is water-soluble, nontoxic, resistant to intestinal enzymes, spherical and can be produced with a molecular weight distribution that relates to the size of many normal food proteins. Normally, a very small fraction of large molecules passes the exclusion barrier of the healthy intestine. Thus, quantification of resorbed macromolecules requires assays of high sensitivity. For detection of PS in various biological fluids, micro-ELISAs have been established. PS with a mean molecular weight of 14,700 daltons (PS 15,000) is rapidly excreted into the urine. Twenty-one healthy volunteers who orally ingested 1 g of this preparation showed a 12-hour urine excretion of 0.018% (interquartile range 0.014-0.022).

Adult↗
PubMed1991

Monoclonal antibody-targeted superantigens: a different class of anti-tumor agents.

The bacterial superantigen staphylococcal enterotoxin (SE) A (SEA) directs cytotoxic T lymphocytes (CTLs) expressing particular sequences of the T-cell receptor (TCR) beta chain to lyse tumor cells expressing major histocompatibility complex (MHC) class II molecules, which serve as receptors for SEs. We now report that chemical conjugates of SEA and the colon carcinoma-reactive monoclonal antibodies (mAbs) C215 or C242 mediate T cell-dependent destruction of colon carcinoma cells lacking MHC class II molecules. SEA was covalently linked to the mAbs C215 and C242 via a PEG-based hydrophilic spacer. The C215-SEA conjugate targeted CD4+ as well as CD8+ CTLs to lyse a panel of colon carcinoma cells lacking MHC class II molecules. T-cell recognition of mAb-SEA conjugates was SEA specific, since SEB-selective T-cell lines with potent cytotoxic activity towards Raji cells coated with SEB did not respond to the C215-SEA conjugate. Unconjugated SEA did not induce T-cell lysis of MHC class II- colon carcinoma cells but efficiently directed CTLs against MHC class II+ Raji cells and certain interferon-treated MHC class II+ colon carcinoma cells. These results suggest that SEA-mAb conjugates retain the SEA-related selectivity for certain TCR beta-chain variable region (V beta) sequences but, in contrast to unconjugated SEA, mediate the TCR interaction in a MHC class II-independent manner. The cytotoxic activity mediated by C215-SEA and C242-SEA conjugates was blocked by excess of C215 mAb and C242 mAb, respectively, showing that the specificity in the targeting of mAb-SEA conjugates is defined by the antigen reactivity of the mAb. These results demonstrate that bacterial superantigens may be successfully conjugated to mAb with preserved T cell-activating capacity. The circumvention of MHC class II binding of SEs by conjugation to mAb suggests that such conjugates may find general application as antitumor agents, taking advantage of the extreme T cell-activating potency of superantigens.

Antibodies, Monoclonal↗
PubMed1990

An immunoassay for histamine based on monoclonal antibodies.

Monoclonal antibodies with high specificity for histamine as well as for 1-methylhistamine were obtained after immunization of mice with a conjugate where the histamine was coupled via its ring 1-nitrogen to dog serum albumin. An immunoassay was developed for the quantitation of histamine release from basophils and 1-methylhistamine release from mast cells after provocation. The test method is based on competitive inhibition between histamine and a labelled histamine conjugate for the antigen binding sites of the antibodies. The separation step is performed by the addition of solid phase bound anti-mouse subclass specific antibodies. The sensitivity of the assay is 2 micrograms/l for histamine and 0.1 micrograms/l for 1-methylhistamine. No cross-reactivity was obtained with other metabolites of histamine or with histidine. Serotonin and dopamine were detectable, but only in doses (mg/l) well above the normal concentration found in the circulation. The immunoassay has been evaluated for its capacity to measure histamine release in vitro. A good correlation with the conventional fluorometric assay was obtained when histamine released from allergen stimulated leucocytes from allergic patients was tested. Urinary samples from patients undergoing hyposensitization showed a mean excretion of 1-methylhistamine at a level of 131 mumol MeHi/mol creatinine. The release of histamine and 1-methylhistamine in vivo was examined in plasma samples taken during a bronchial provocation test. A significant elevation above the basal analyte level occurred ten minutes after provocation.

Animals↗
PubMed1988

Induction of basophilic differentiation in the human basophilic cell line KU812.

The potential for differentiation of the human basophilic leukaemia cell line KU812 was examined by means of a panel of physiologic and non-physiologic substances used as inducers. The phenotypic characteristics of non-induced KU812 cells included an immature morphology with scanty cytoplasmic granulation, expression of a low amount of high affinity, but no low affinity receptors (CD 23) for IgE, and a capacity for low-rate histamine synthesis. The differentiation process was characterized by a rapid (24 h) increase in histamine production a slower morphological maturation with the development of Alcian blue stainable granula demonstrable after 72 h. Concomitant with the phenotypic alterations, cell growth was inhibited. Differentiation in KU812 cells was inducible by Ara-C and to some extent by sodium butyrate, but not by dimethyl sulphoxide, retinoic acid, or gamma-interferon. Conditioned medium (CM) from cultured peripheral blood cells from atopic individuals and 18 out of 22 analysed glioma cell lines induced differentiation of the KU812 cells, whereas supernatant from only 1 out of 21 other cell lines, including carcinoma, melanoma, sarcoma, leukaemia, and normal fibroblasts had this activity. CM from the T-leukaemic cell line, Mo, also induced KU812 differentiation. A primary fractionation of the active substance from this cell line by reversed phase chromatography eluted the active substance at a concentration of 42-44% acetonitrile. Our present study has shown that the KU812 may serve as an appropriate model to study differentiation of basophils. In addition, its fast and specific response to biological factors makes it suitable as a biological assay for determination of active factor produced by atopic individuals.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1986

Stimulation of maturation of large immature histamine-containing basophilic cells from human peripheral blood, cord blood and bone marrow.

The development of FcR epsilon-bearing histamine-containing basophilic cells was studied in cultures of peripheral blood leucocytes and bone marrow leucocytes from normal individuals. The determination of basophilic cells was performed blindly. Before cultivation there were fairly similar numbers of basophilic cells in the samples from the three different sources (1.6 +/- 1.6 n = 13; 1.4 +/- 2.0 n = 21; 4.3 +/- 4.7 n = 8, respectively). During cultivation spontaneously appearing large blast-like basophilic cells were seen in good correspondence with a formation of histamine in the cultures (Spearman rank correlation coefficient = 0.9135, n = 13, P less than 0.001). This was more accentuated with bone marrow cells than with peripheral blood and cord blood cells. Conditioned medium (CM) was prepared from cells isolated from various tissues and stimulated by different means, e.g. peripheral blood from atopic individuals stimulated with allergen and unstimulated tonsil cells. Addition of the CM resulted in increased development of histamine-containing basophilic cells. Optimum stimulation was achieved with 10% CM. The basophilic stimulation by CM, as assessed as indices vs. unstimulated cultures, was more accentuated in cultures of peripheral blood cells than of bone marrow and cord blood cells (2.8 +/- 1.2; 1.8 +/- 0.5; 2.0 +/- 0.4, respectively). In contrast, the histamine formation was particularly evident in stimulated cultures of bone marrow cells, where more than four-fold increases of histamine were found. In bone marrow cells the histamine levels per basophilic cell also increased, whereas this was not the case in cord blood cells. A pronounced development of basophilic cells was achieved when using leukoagglutinin, provided the mitogen in the CM was eliminated. The formation of basophilic cells was prevented with mitomycin c and cycloheximide. In conclusion, the system described may provide important information on the development of histamine-containing basophilic cells at various maturation stages from different compartments, and mechanisms in a developing atopic disease.

Basophils↗
PubMed1984

Use of capillary isotachophoresis to measure the degree of chemical substitution of dog serum albumin.

Analytical capillary isotachophoresis may provide a means for the rapid determination of the degree of substitution of proteins. Dog albumin conjugates with trinitrophenyl (TNP), fluorescein isothiocyanate (FITC) or monomethoxy polyethylene glycol (mPEG) have been studied as models for protein modification. An increase in the degree of covalent substitution of dog albumin with TNP, FITC or mPEG could be monitored rapidly using the UV signal or the conductivity signal as a measure of the increased protein zone width. These findings are encouraging with regard to the possible use of capillary isotachophoresis for screening the extent of protein modification.

Animals↗
PubMed1984

Polyethylene glycol reactive antibodies in man: titer distribution in allergic patients treated with monomethoxy polyethylene glycol modified allergens or placebo, and in healthy blood donors.

Antibodies to polyethylene glycol (PEG) were analyzed in patients with various allergies and in healthy blood donors employing passive hemagglutination. In untreated allergic patients and in healthy blood donors, naturally occurring anti-PEG antibody titers between 32 and 512 were seen in 3.3 and 0.2%, respectively. During hyposensitization with monomethoxy polyethylene glycol modified ragweed extract and honey bee venom, respectively, the patients showed an anti-PEG antibody response. Titers of 32-512 were found in 50% of the patients directly after the first treatment course. After 2 years of treatment the percentage of patients with such titers declined to 28.5%. Mercaptoethanol treatment of sera indicated that the anti-PEG antibodies predominantly were of the IgM isotype. The weak IgM response found in treated patients is considered to be of no clinical significance.

Allergens↗
PubMed1983

Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins.

Antibodies to polyethylene glycol (PEG) were raised in rabbits by immunization with monomethoxy polyethylene glycol modified ovalbumin (OA), bovine superoxide dismutase (SOD), and ragweed pollen extract (Rag), given in Freund's complete adjuvant (FCA). Immunogenicity depended on the nature of the protein and the degree of modification. With modified OA, in the presence of FCA, the majority of animals showed an anti-PEG response. With modified SOD and Rag only a small proportion of animals responded. In the absence of FCA, modified OA, given s.c., did not elicit any anti-PEG antibody response in rabbits and only a weak response in mice. PEG of MW 10,000 and 100,000 given in FCA was found nonimmunogenic in rabbits, and PEG of MW 5.9 X 10(6), given s.c. to mice, showed no or very poor immunogenic properties. Gel diffusion, heterologous passive anaphylaxis and passive hemagglutination were used to demonstrate anti-PEG antibodies raised to PEG-modified proteins. Specificity was confirmed by hapten inhibition of precipitation, inhibition of passive hemagglutination and cross-reactivity tests. PEG of MW greater than or equal to 4,000 produced specific precipitates, smaller molecules acted as monovalent haptens. From hapten inhibition of precipitation by PEG of MW 300 it appears that the antigenic determinant of PEG may be a sequence of 6-7 -CH2CH2O-units. Anti-PEG antibodies can be used analytically. By gel diffusion, Peg was detected in minimal concentrations of 0.1-1 microgram/ml. The clinical relevance of these findings with regard to therapy with PEG-modified enzymes and allergens in humans remains to be established.

Animals↗
PubMed1983

Antibody responses to honey-bee venom and monomethoxy-polyethylene glycol-modified honey-bee venom in mice.

Antibody responses of the IgE isotype were raised in mice with honey-bee venom (HBV) administered in alum or by daily injections without adjuvant. The sensitized mice were treated with single injections of HBV modified with monomethoxy-polyethylene glycol. By such treatment with modified but not with natural HBV, suppression of the IgE antibody responses was achieved. The IgG antibody responses, in contrast, were unchanged or enhanced.

Adjuvants, Immunologic↗
PubMed1981

Suppression of reaginic antibodies with modified allergens. IV. Induction of suppressor T cells by conjugates of polyethylene glycol (PEG) and monomethoxy PEG with ovalbumin.

Administration of multiple injections of conjugates of ovalbumin (OA) and polyethylene glycol (PEG) or its monomethoxy derivative (mPEG) into mice which had been sensitized with 2,4-dinitrophenylated OA (DNP3-OA) abrogated both the anti-OA and anti-DNP IgE responses, in spite of additional injections of the sensitizing dose of DNP3-OA in the presence of A1(OH)3. Treatment of mice with OA-PEG in A1(OH)3 stimulated preferentially helper T cells, whereas injection of mice with OA-PEG in the absence of adjuvant elicited predominantly suppressor T cells. The unresponsive state of mice which had been treated 21 days earlier with OA-PEG could not be broken by the transfer of normal spleen cells and an additional sensitizing dose of DNP3-OA. Transfer of spleen cells from tolerized animals to normal mice dampened the capacity of the latter to mount both anti-DNP and anti-OA IgE responses; however, the suppressive effect of these cells was eliminated by treatment of the normal recipients with cyclophosphamide, which is a procedure known to inactive suppressor T cells, and hence it may be concluded that this effect was not due to the carryover of the tolerogen with the transferred cells. All these results provide strong support for the conclusion that the suppressor cells induced by the treatment of mice with OA-PEG and OA-mPEG conjugates belonged to a T cell subpopulation, and that the B cells of these mice were devoid of suppressive activity.

Allergens↗
PubMed1981

Suppression of reaginic antibodies with modified allergens. III. Preparation of tolerogenic conjugates of common allergens with monomethoxypolyethylene glycols of different molecular weights by the mixed anhydride method.

Our previous findings that antigens, such as ovalbumin (OA) and the extract of ragweed pollen (RAG), could be rendered nonantigenic, nonallergenic and tolerogenic by conjugation with polyethylene glycol (PEG) have been extended in the present study to the synthesis of conjugates of a variety of antigens with monofunctional monomethoxy-PEGs (mPEGS) of different molecular weights by the use of the mixed anhydride method. Thus, mPEGs with molecular weights of 2,000, 5,000, 10,000 and 20,000 were coupled to proteins such as dog serum albumin (DA), bovine pancreatic ribonuclease, OA and the constituents of pollen, helminth and bacterial allergens (RAG, Timothy grass pollen, Ascaris suum and Micropolyspora faeni). All these mPEG conjugates depressed markedly the ongoing IgE antibody formation in sensitized animals, in spite of additional injections of the sensitizing dose of the appropriate antigen. Moreover, the allergenicity of the proteins was either totally abolished or markedly reduced after coupling to mPEGs. Conjugates of DA and OA of varying degree of substitution (i.e. number of mPEG molecules attached per protein molecule) were prepared with mPEGs of different molecular weights and their immunological properties were assessed. It appears that, for a series of tolerogenic conjugates of the same antigen, there exists some inverse relationship between the degree of substitution and the molecular weight of mPEG, i.e. a high level of tolerogenicity with a concomitant reduction or total loss of allergenicity was achieved with a lower degree of substitution utilizing mPEGs of increasing molecular weights. On the basis of these results, it is concluded that a variety of allergens may be converted by conjugation with mPEGs to tolerogenic products with a potential for use in the therapy of patients allergic to a wide spectrum of common allergens.

Albumins↗
PubMedDate not supplied

Targeting of superantigens.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.

Antibodies, Monoclonal↗
PubMedDate not supplied

Preparation and characterization of conjugates of monoclonal antibodies and staphylococcal enterotoxin A using a new hydrophilic cross-linker.

Conjugates between monoclonal antibodies recognizing human cancer cells and the superantigen staphylococcal enterotoxin A (mAb-SEA) represent a potential novel approach to tumor therapy. Such mAb-SEA conjugates direct T-cells to lyse colon carcinoma cells in vitro. The synthesis of mAb-SEA conjugates which were prepared by introducing thiol groups on SEA and iodoacetyl or maleimide groups on mAb forming a stable thioether linkage between SEA and mAb is described. A hydrophilic spacer, composed of repeated ethylene oxide units, was constructed to increase the distance between SEA and mAb, preserving biological activity of both proteins. The degree of modification of mAb with SEA was determined with SDS-PAGE. Variables influencing the composition of the conjugates and their effect on the tumor-cell cytotoxicity were studied and optimal conditions for the synthesis were established. Functionally active mAb-SEA conjugates were prepared from a panel of different mAb and T-cell-dependent cytotoxicity against several human cancer types including colon, ovarial, breast, and renal cancer was obtained. This suggests that mAb-SEA conjugates may be of value in the treatment of human neoplastic disease.

Antibodies, Monoclonal↗
PubMedDate not supplied

B-Domain deleted recombinant coagulation factor VIII modified with monomethoxy polyethylene glycol.

Recombinant coagulation factor VIII (r-VIII SQ) was chemically modified with monomethoxy poly(ethylene glycol) (mPEG). Three mPEG derivatives were used for coupling to the r-VIII SQ lysines, a mixed anhydride of monomethoxy poly(ethylene glycol) succinic acid (mPEG-SAH), monomethoxy poly(ethylene glycol) succinimidyl succinate (mPEG-SS), and monomethoxy poly(ethylene glycol) tresylate (mPEG-TRES). A consequence of the modification with all derivatives was a substantial reduction in coagulant activity, even at very low degrees of modification. A method was developed with the purpose of avoiding conjugation at certain important biological sites on the factor VIII and thereby producing conjugates with better retained activity. This was achieved by immobilizing the protein onto a solid matrix during the modification reaction. Characterization of conjugates by SDS-PAGE, western blots, interaction with von Willebrand factor (vWf), and thrombin activation/inactivation analyses was undertaken. The SDS-PAGE and western blots revealed coupling heterogeneity regarding degree of modification. The amount of factor VIII able to bind to vWf decreased with the conjugation. Thrombin activated the modified factor VIII to essentially the same extent as the reference preparation of r-VIII SQ. Inactivation of the modified factor VIII was, however, slower than inactivation of the unmodified protein. Finally, an in vitro study was performed to evaluate the influence of the mPEG modification on the protein stability in extract of porcine tissue. Despite that conjugates with low degrees of modification were included in the study, the coagulant activity was preserved to a significantly higher extent in all incubation mixtures containing conjugates compared to that with unmodified protein.

Animals↗
PubMedDate not supplied

An improved procedure for N- to C-directed (Inverse) solid-phase peptide synthesis.

A method for solid-phase peptide synthesis in the N- to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA(1)-AA(2)-AA(3)) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N, N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA(2) in the coupling of AA(3) was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA(1) and AA(2). Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.

Combinatorial Chemistry Techniques↗