Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned with Poly(ethylene glycol) Hydrogels

A simple method for controlling the spatial positioning of mammalian cells and bacteria on substrates using patterned poly(ethylene glycol) (PEG) hydrogel microstructures is described. These microstructures were fabricated using photolithography on silicon, glass or poly (dimethylsiloxane) (PDMS) surfaces modified with a 3-(trichlorosilyl) propyl methacrylate (TPM) monolayer. During the photogelation reaction, the resulting hydrogel microstructures were covalently bound to the substrate via the TPM monolayer and did not detached from the substrate upon hydration. For mammalian cell patterning, microwell arrays of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding hydrophobic TPM floors inside the microwells. Murine 3T3 fibroblasts and transformed hepatocytes were shown to selectively adhere to the TPM monolayer inside the microwells, maintaining their viability, while adherent cells were not present on the hydrogel walls. The number of cells inside one microwell could be controled by changing the lateral dimension of the microwells, thus allowing only a single cell per microwell if desired. In the case of 30×30 m microwells, as many as 400 microwells were fabricated in 1 mm². In addition, PEG hydrogel microstructures were also shown to effectively resist the adhesion of bacteria such as Escherichia coli.     

Interactions of γ-immunoglobulins with lipid mono- or bilayers and liposomes. Existence of two conformations of γ-immunoglobulins of different hydrophobicities

Interactions between rabbit-γ-immunoglobulins and model membranes (lipid monalayers, planar lipid bilayers, liposomes) have been investigated. No significant interaction was observed with immunoglobulins. However, immunoglobulins dialysed first vs aqueous buffer having pH 2 or 3 and then dialysed against pH 7 buffer presumably adopt a new conformation which allows their bindings to model membranes. This binding is hydrophobic and the immunoglobulin region interacting with the lipid acyl chains is probably located in the heavy chain, as suggested by labelling in this region by a photosensitive probe previously incorporated into the lipid hydrophobic core. Cleavage at the hinge region by papain or pepsin, or heating above 38°C, induces the loss of the hydrophobic conformation responsible for hydrophobic bindings. The binding capacity of immunoglobulins heated above 38°C is restored after momentary dialysis at pH 2. The possible existence of two Ig isomers is discussed in relation to the mechanism of γ-immunoglobulin passage through the endoplasmic membrane and fixation into the plasma membrane.     

A Preliminary Study of Immunotherapy with a Monomethoxy Polyethylene Glycol Modified Honey Bee Venom Preparation

Monomethoxy polyethylene glycol (mPEG) modified honey bee venom (HBV) immunotherapy (IT) has been studied in 14 patients allergic to honey bee venom. Doses could be increased more rapidly and higher doses were reached compared to regular venom immunotherapy. No general side effects were seen, although large local swellings were found somewhat more often than with regular HBV. Most patients could easily be switched from the modified to the unmodified venom. Eight patients experienced and tolerated field stings. Skin testing showed a decreased allergenicity of the mPEG-HBV. The mean HBV-specific IgE level was below pre-treatment level already after only 6 weeks of IT. The HBV-specific IgG response was very good.     

新型可降解聚合物聚乙二醇对苯二甲酸酯/聚对苯二甲酸丁二醇酯的细胞相容性研究

目的：研究新型可降解聚合物聚乙二醇对苯二甲酸酯／聚对苯二甲酸丁二醇酯(PEGT／PBT)的人脐静脉内皮细胞(HUVEC)相容性，对其在组织工程血管中的应用进行探讨。方法：对PEGT／PBT进行细胞毒性评价。观察并检测HUVEC在PEGB／PBT、Ⅰ型胶原改性(Col-)的PEGT／PBT、纤维连接蛋白改性(Fn-)的PEGT／PBT上的粘附和增殖，对细胞在粘附过程中的粘着斑蛋白进行免疫荧光染色观察。结果：PEGT／PBT细胞毒性不大于1级，能支持脐静脉内皮细胞的粘附和增殖。纤维连接蛋白和Ⅰ型胶原处理可促进HLIVEC在PEGT／PBT膜上的增殖，而且纤维连接蛋白可增加HUVEC在PEGT／PBT上20min、2h的粘附率，并促进细胞形成局部粘附结构。结论：新型可降解聚合物PEGT／PBT具有良好的血管细胞相容性，对其在组织工程血管中的应用值得进一步开发研究。     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

Prevention of collagen-induced arthritis (CIA) by treatment with polyethylene glycol-conjugated type II collagen; distinct tolerogenic property of the conjugated collagen from the native one

Administration of a soluble protein into animals prior to challenge immunization induces immunological tolerance which is specific for the protein. In addition, chemical modification of proteins with polyethylene glycol (PEG) has been reported to convert the immunogenic proteins to become tolerogenic. However, differences in tolerogenic properties between PEG-modified proteins and the native counterparts have never been analysed. The ability of PEG-conjugated type II collagen (PEG-CII) to attenuate CIA, an animal model for rheumatoid arthritis, was compared with the native unconjugated CII. Groups of DBA/1 J mice were treated weekly with i.p. injections with PEG-CII, native CII, or vehicle alone for 3 weeks, before they were challenged with CII in adjuvants. The induction of tolerance was confirmed in both PEG-CII- and CII-pretreated mice when suppression of lymph node T cell proliferation in response to CII was noted. The degrees of suppression of T cell proliferation were comparable between the two pretreated groups. However, induction of arthritis and production of IgG anti-CII antibody were more markedly suppressed in PEG-CII-pretreated mice than in native CII-pretreated mice, although the severity of arthritis and antibody levels in the latter group were also lower than in control mice. IgG2a and IgG2b antibody levels were equally suppressed in the two pretreated groups, whereas the IgG1 level was significantly lower in the PEG-CII-pretreated group than in the native CII-pretreated group. The results provide the first evidence that attachment of PEG to CII renders the protein more tolerogenic.     

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.     

Poly(ethylene glycol)-containing Hydrogels for Oral Protein Delivery Applications

Novel pH-sensitive hydrogels were developed as suitable candidates for carriers in bioMEMS devices as well as for oral delivery of therapeutic peptides and proteins due to their ability to respond to environmental pH change. Macromonomers containing various PEG molecular weights were synthesized and used to prepare P(MAA-g-EG) hydrogels were by photopolymerization. P(MAA-g-EG) hydrogels showed a drastic change of the equilibrium swelling ratio between pH 2.2 and 7.0. At pH 7.0, hydrogels with PEGMA2000 exhibited higher swelling ratio than hydrogels with PEGMA1000. For both hydrogels with PEGMA1000 and PEGMA2000, the swelling mechanism became more relaxation-controled as the environmental pH changed from 2.2 to 7.0 due to the ionization of the functional groups in polymer networks at high pH. In vitro release studies of insulin were conducted. P(MAA-g-EG) hydrogels exhibited drastic increase of insulin release as the pH of the medium was changed from acidic to basic. Insulin release from P(MAA-g-EG) hydrogels with PEGMA2000 was slower than from hydrogels with PEGMA1000 at both low and high pH. These results were used to design and improve protein release behavior from these carriers.     

Inhibition of experimental metastasis and cell adhesion of murine melanoma cells by chondroitin sulfate-derivatized lipid, a neoproteoglycan with anti-cell adhesion activity

Chondroitin sulfate dipalmitoylphosphatidylethanolamine (CS-PE), when immobilized onto substratum, inhibited the adhesion of B16F10 mouse melanoma cells to fibronectin-coated dishes (anti-adhesion activity). CS-PE showed the most potent anti-adhesion activity for the melanoma cells among various GAG-PEs. CS-PE also inhibited the adhesion of B16F10 cells to Matrigel and the invasion of the cells into Matrigel. In the in vivo system of experimental metastasis, administration of B16F10 cells with CS-PE into C57BL/6 mice significantly inhibited lung metastasis. The inhibition degree of CS or hyaluronic acid-PE was lower than CS-PE. CS-PE administered intravenously into mice before the injection of B16F10 cells also inhibited metastasis. Pretreatment of B16F10 cells with CS-PE caused some but a lower degree of inhibition. When CS-PE was injected intravenously into mice, more binding in the lung was found than when CS was injected. CS-PE but not CS inhibited the retention in the lung of fluorochrome-labeled B16F10 cells when injected intravenously into mice. Since there was no significant effect of CS-PE on the viability and growth of B16F10 cells, the results suggest that CS-PE immobilized onto the subendothelial matrix may prevent melanoma cells from adhering to the subendothelial substrata of lung capillaries and inhibit subsequent invasion processes of metastasis.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.