Use of 2-Hydroxypropyl-β-cyclodextrin as a Solubilizing and Stabilizing Excipient for Protein Drugs

A chemically modified, amorphous -cyclodextrin, namely, 2-hydroxypropyl--cyclodextrin (HPCD), was examined as a solubilizing and stabilizing agent for protein drugs. The aqueous solubility of ovine growth hormone at pH 7.4 was increased through the use of HPCD. This effect was manifested by higher UV transparency at 600 nm. Interleukin-2 (IL-2) is rendered insoluble upon lyophilization in the absence of stabilizers. Use of aqueous HPCD provides a clear solution, as indicated by fluorometric light scattering, and inhibits aggregate formation, as shown by ultracentrifugation and Western blot analyses. In addition, there were no major conformational changes of IL-2 in HPCD formulation as indicated by fourth-derivative ultraviolet spectroscopy. Finally, IL-2 retained 100% of its biopotency when prepared in HPCD solutions. Aggregation of insulin was also suppressed by HPCD. These data, as well as the i.v. safety of HPCD and its well-characterized chemical composition, suggest that this starch derivative may be a potentially useful excipient for protein drugs intended for parenteral use.     

Differential expression of CD44(S) and variant isoforms v3, v10 in three-dimensional cultures of mouse melanoma cell lines

Multi-cellular spheroids (MCS) generated from tumor cells serve as excellent in vitro models for understanding the mechanisms of tumor progression and micro-metastasis. We have compared the expression of molecular markers with reference to their growth as conventional adherent monolayers (2-D) and anchorage independent cultures (3-D) using two mouse melanoma cell lines, B16F10 and Clone M3. The two cell lines differed in their ability to form spheroids with respect to their aggregation potential, with B16F10 forming large clusters compared to Clone M3. A panel of molecular markers comprising cell adhesion molecules, cyclin dependent kinase inhibitors and members of the cadherin–catenin complex were analyzed by flow cytometry in 2-D and 3-D cultures. There was a distinct difference in the patterns of expression of CD44(S) and variant isoforms v3,v10 in spheroids compared to cells grown as monolayers in both cell lines. Also, there was an increase in cells positive for CDK inhibitor p27 in 3-D cultures from the B16F10 cell line. The expression of alpha and gamma catenin was down regulated in spheroids. As these molecules are implicated in the regulation of cell proliferation, alterations in the expression of these molecules in 3-D cultures compared to their 2-D counterparts suggests the importance of spheroids as experimental model for tumorigenesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1

BACKGROUND: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. OBJECTIVE: To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. METHODS: Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. RESULTS: The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. CONCLUSIONS: The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.     

Phage-displayed Bet mim 1, a mimotope of the major birch pollen allergen Bet v 1, induces B cell responses to the natural antigen using bystander T cell help

BACKGROUND AND OBJECTIVE: In previous studies we have generated mimotopes of Bet v 1, the major birch pollen allergen, by biopannings of phage-display random peptide libraries. In the present study, we analysed the humoral and cellular immune response to Bet v 1-mimotopes. METHODS: The mimotope CFPYCYPSESA, designated Bet mim 1, was used for intraperitoneal immunizations of BALB/c mice in phage-displayed form. For examination of the humoral immune response, enzyme-linked immunosorbent assay (ELISA) experiments were applied. Stimulation capacities were investigated in cultured mouse splenocytes and in humoral Bet v 1-specific T cell clones. RESULTS: We demonstrated that the Bet mim 1-induced murine antibody response against Bet v 1 was predominated by the IgG1 isotype. In these mice only the phage-displayed mimotopes, but neither the allergen nor the synthetic Bet mim 1-mimotopes were able to stimulate proliferation of cultured splenocytes. Using Bet v 1-specific T cell clones of allergic patients, phage-displayed and synthetic mimotopes were unable to stimulate T cell proliferation. Moreover, tolerance induction to Bet v 1 in mice by intranasal administration of Bet mim 1-phages or Bet mim 1-peptide failed. CONCLUSION: Taking these results together, our data indicate that Bet mim 1 mimics a Bet v 1-epitope on the B cell but not on the T cell level. We suggest that the phage itself is responsible for the recruitment of T cells providing bystander help in the formation of a mimotope-specific humoral response.     

Synthesis of α-fetoprotein,albumin, and transferrin by continuous mouse hepatoma cells

The ability of continuous cultures of MGXXIIa mouse hepatoma cells to synthesize -fetoprotein, albumin, and transferrin was studied by an immunoautoradiographic method. Albumin and transferrin were found in the growth medium of hepatoma cells in the 5th year of culture (55th month), concentrated with polyethylene glycol; no -fetoprotein could be detected. Only transferrin was found in the growth medium of hepatoma cells in the 8th year of culture (92nd month). Two clonal cultures obtained in the 8th year of culture of hepatoma cells also showed ability to synthesize transferrin. Continuous hepatoma cells preserved their malignancy. In Lyphogel-concentrated sera of mice with tumors formed after inoculation of hepatoma cells in the 5th year of culture, -fetoprotein was found by the microprecipitation test in agar. No -fetoprotein was found in the sera of mice with tumors formed after inoculation of hepatoma cells in the 8th year of culture.Laboratory of the Molecular Basis of Immunogenesis, Institute of Experimental Biology, Academy of Sciences of the Armenian SSR, Erevan. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 76–78, July, 1979.     

Detection of specific IgE antibodies in the sera of patients allergic to birch pollen using recombinant allergens Bet v 1, Bet v 2, Bet v 4: evaluation of different IgE reactivity profiles

BACKGROUND: Birch pollen is a significant cause of immediate hypersensitivity among susceptible subjects in temperate climates, affecting 5-54% of the population in western Europe. We examined the specific serum IgE antibodies towards recombinant allergens Bet v 1, Bet v 2 and Bet v 4 in birch-sensitive patients from the province of Cuneo, north-west Italy. METHODS: Sera were obtained from 372 patients with symptomatic birch pollen-induced allergic rhinitis and/or asthma. A subgroup of these patients suffered from oral allergy syndrome after eating apple. Their sera were evaluated for specific IgE against natural birch pollen and apple extract, as well as Bet v 1, Bet v 2 and Bet v 4 using Pharmacia CAP system (Pharmacia, Uppsala, Sweden). RESULTS: Of 372 patients 215 (57.80%) had serum-specific IgE towards Bet v 1. A total of 166 sera (44.62%) contained serum-specific IgE to Bet v 2, while Bet v 4 IgE reactivity was documented in 35 subjects (9.41%). Moreover, 146 (39.25%) patients were monosensitized to Bet v 1; 96 (25.81%) patients were monosensitized to Bet v 2; only four sera (1.08%) contained specific IgE towards Bet v 4. Thirty-nine sera (11.02%) did not contain specific IgE to these individual birch pollen allergens. Of course, all 372 sera (100%) had specific IgE against natural birch pollen extract, of which 162 (43.55%) contained specific IgE to apple extract (75.35% of Bet v 1 positive sera). CONCLUSION: In this study we observed that three birch pollen recombinant allergens alone, could sufficiently identify 90% of birch pollen-sensitive patients. Therefore, for a more precise IgE profile of patients allergic to birch, further purified birch pollen allergens (i.e. Bet v 6, Bet v 7, Bet v 8) will be required.     

Characteristics of human embryonic hepatocytes cultivated in vitro

A simple and reproducible method of monolayer cultivation of hepatocytes from 7–12-week human fetuses is suggested. Such cultures are capable of limited growth, they can be maintained without subculaute for 4–6 weeks, and they preserve certain specific characteristics: synthesis of such characteristic serum proteins as albumin and -fetoprotein, the presence of a high glycogen content and high monoamine oxidase activity, and the characteristic reaction to high concentrations of glucocorticoid. These properties can be used in experimental genetics as markers or for selection purposes.Laboratory of Human Cytogenetics, Institute of Medical Genetics, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 5, pp. 123–125, May, 1975.     

Role of lipid peroxidation andα-tocopherol in conductivity of artificial membranes made from liver phospholipids of rats with burns

The state of membrane permeability for potassium and calcium ions was studied in bilayer phospholipid membranes (BPM) from the liver of rats with burns. A sharp increase in the conductivity of BPM was found under these circumstances. This process was accompanied by an increase in lipid peroxidation. Administration of-tocopherol (1 mg/kg body weight) restored these indices to normal. Model experiments with methyl oleate and cumyl hydroperoxide confirm the peroxide mechanism of injury to membrane formations.Department of Biochemistry, Erevan Medical Institute. Department of Biophysics, Central Research Laboratory, Erevan Postgraduate Medical Institute, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 5, pp. 422–425, May, 1979.