Methylcellulose - a versatile printing material that enables biofabrication of tissue equivalents with high shape fidelity.

With the aid of biofabrication, cells can be spatially arranged in three dimensions, which offers the opportunity to guide tissue maturation in a better way compared to traditional tissue engineering approaches. A prominent technique allowing biofabrication of tissue equivalents is extrusion-based 3D (bio)printing, also called 3D (bio)plotting or robocasting, which comprises cells embedded in the biomaterial (bioink) during the fabrication process. First bioprinting studies introduced bioinks allowing either good cell viability or good shape fidelity. Concepts enabling printing of cell-laden constructs with high shape fidelity were developed only rarely. Recent studies showed the great potential of the polysaccharide methylcellulose (mc) as supportive biomaterial that can be utilized in various ways to enable biofabrication and especially extrusion-based bioprinting of bioinks. This minireview highlights the multiple applications of mc for biofabrication: it was successfully used as sacrificial ink to enable 3D shaping of cell sheets or biomaterial inks as well as as internal stabilizing component of various bioinks. Moreover, a brief overview about first bioprinted functional tissue equivalents is given, which have been fabricated by using mc. Based on these studies, future research should consider mc as an auxiliary material for bioinks and biofabricated constructs with high shape fidelity.

The immune response of carp against dextran. An analysis of the idiotype heterogeneity.

The expression of a "species-specific" idiotype with specificity for alpha(1-6) dextran in carp is described. All tested carp anti-dextran antisera react with a rabbit anti-Id (carp anti-dex) serum. The rabbit anti-Id serum can be partially inhibited by the specific ligand, dextran T70 or isomaltodecaose but not or only weak with other, unrelated carbohydrates. These data confirm our statement, that a limited germline V-gene repertoire may code for antibodies in fish.

Anti-dextran antibodies of carp detected by the enzyme-linked immunosorbent assay (ELISA).

Carp produce anti-alpha (1-6) dextran antibodies after immunization with a vaccine of Leuconostoc mesenteroides B512. These antibodies can be determined by passive haemagglutination, quantitative precipitation or by ELISA. In our hands the ELISA has proved to be 100 times more sensitive than the passive haemagglutination test. We could not found any correlation between the anti-dextran activity estimated with the passive haemagglutination on the one and the ELISA on the other hand.

Immune response of carps (Cyprinus carpio L.) against dextran.

In carps no antibodies could be evoked by immunization with soluble high molecular weight dextran using doses of 1-5000 mg dextran per kg body weight. On the other hand, an immune response against dextran could be elicited by immunization with a vaccine of Leuconostoc mesenteroides B512 as well as a dextran-BSA-conjugate. These carp anti-dextran antibodies showed precipitating and agglutinating activities with specificity against alpha linked dextran determined by the almost complete inhibition of the dextran-anti-dextran reaction by isomaltodecaose.

[Specificity of carp antihuman myeloma protein sera depending on the holding temperature]. / Untersuchungen zur Spezifität von Karpfen-anti-Humanmyelomprotein-Seren in Abhängigkeit von der Hälterungstemperatur.

While in carps kept in warm water (28 degrees C) and immunized with a human myeloma protein a challenge leads to an increase, the antibody titres in carps kept in cold water (14 degrees C) are changed only unsignificantly. Antibody titres of carps immunized once respectively three times and kept in cold water are not markedly different. In testing the specificity of the antisera taken from each of the three groups no differences could be recognized. With carp anti-IgGZei sera using the passive hemagglutination inhibition test a paraprotein characterizing determinant (s) is just as well demonstrable as by using a rabbit antiserum.

[A further confirmation of the high idiotypic specificity of carp anti-human myeloma protein sera]. / Eine weitere Bestätigung für die hohe idiotypische Spezifität von Karpfen-anti-Humanmyelomprotein-Seren.

Previous investigations about the specificity of carp anti-idiotypic sera could be confirmed by comparison with corresponding antisera of guinea pigs. 3 carp antihuman myeloma-protein sera rendered as idiotypic specific after an appropriate absorption were tested in the passive hemagglutination inhibition test. They showed no difference to the parallel tested guinea pig antisera. It can be assumed that between fish antibodies and antibodies of mammals no functional difference exists in respect of antigen binding. On the other hand mammals exhibit a higher extent of structural antibody heterogeneity.

[Tolerance induction and production of anti-idiotypic antisera in carp]. / Toleranzinduktion und Herstellung anti-idiotypischer Antiseren bei Karpfen.

Tolerance in carps can be induced by intracardial injection of 10-30 mg of ultracentrifuged human gamma globulin (HGGu), with strong individual variation being observable. Out of 21 carps pretreated with 10 mg HGGu, 10 animals showed 14 days after i.p. test immunization with 1 mg HGGu a log2 anti-HGG titre of 1.55 +/- 0.60 (control group - log2 7.33 +/- 1.47), while 11 animals exhibited a log2 titre of 7.45 +/- 2.75. Low doses of HGGu (0.01-1.0 mg) led to stimulation of immune response. If 1-2 mg of a human IgG myeloma protein are applied i.p. after intracardial injection of 10 mg HGGu either simultaneously or after a longer break, then a high-titre anti-idiotypical antiserum with low activity against HGG can be obtained in single cases. The experiments have shown wide individual variation in tolerance induction which may be due also to seasonal factors.

Detection of a monoclonal human myeloma protein of IgM and IgG class by carp anti-idiotypic sera.

In the Patient St. with a Morbus Waldenström macroglobulinemia a double paraproteinemia could be detected. Besides the IgM myeloma protein an IgG myeloma protein was identified during the clinical course. A strong cross reactivity between the IgM and the IgG myeloma proteins was shown using anti-idiotypic antisera. This is an indirect indication for a common precursor cell clone of the IgM- and IgG-myeloma protein producing cells. The anti-idiotypic antisera were made in carp. The high specificity of these antisera could be confirmed by inhibition assays. The double paraproteinemia has been proved to be convenient model for testing the idiotypic specificity of anti-Id antisera of carp.

[Comparative studies of the specificity of idiotypic guinea pig and carp antisera]. / Vergleichende Untersuchungen zur Spezifität von idiotypischen Meerschweinchen- und Karpfen-Antiseren.

The specificity of guinea-pig- and carp-anti-idiotypic antisera were compared by using the radioimmunoassay and the passive hemagglutination inhibition test. Guinea-pigs and carps were immunized with the IgA/k mouse myeloma protein S117 which has binding activity to the hapten N-acetylglucosamine. the resulting antisera were idiotypically specified by absorption and the activity was determined by the binding of the radiolabeled idiotype S117. The binding curves of guinea pig and carp anti-idiotypic antisera are very similar. The idiotype-anti-idiotype reaction could be partially inhibited by the specific hapten N-acetylglucosamine but not by N-acetylgalactosamine. Both antisera, guinea pig and carp, respectively, are different in respect to the immunoglobulin class of anti-idiotypic antibodies which react with the idiotype S117. In the case of the guinea pig antisera the anti-idiotypic antibodies are mainly of the IgG-type whereas the anti-idiotypic antibodies of carp are exclusively of IgM-type. This important difference is discussed.

Anti-idiotypic antibodies of IgM-type produced in carp (Cyprinus carpio L.).

Carp synthesize highly specific anti-idiotypic antibodies of the IgM class. Ant-idiotypic antibodies could be elicited in these animals by a human IgM myeloma protein and were detected in a passive hemagglutination assay. The agglutination was completely inhibited by approximately 0.15 microgram homologous antigen, whereas a 100 000-fold excess of a heterologous IgM myeloma protein of the same L chain type did not produce any inhibition. A possible subgroup specificity of carp anti-idiotypic antisera can be excluded.

[Studies of the antibody heterogeneity in carp (Cyprinus carpio L.). I. Electrophoretic and isoelectric spectra of anti-DNP-antibodies]. / Untersuchungen zur Antikörperheterogenität beim Karpfen (Cyprinus carpio L.). I. Elektrophoretische und isoelektrische Spektren von Anti-DNP-Antikörpern.

Carp IgM as well as carp anti-DNP-antibodies migrate electrophoretically very well as a diffuse band into polyacrylamide gel of large pore size. The isoelectric spectra of the carp anti-DNP-antibodies are heterogeneous and show bands in the pI-range of 4.0 to 6.4. The activity of focused anti-DNP-antibodies could be demonstrated in the pI range between 5.4 to 6.4 even in high antibody dilutions. The investigated structural heterogeneity of the anti-DNP-antibodies of carp is a further proof for the phylogenetically early onset of a large antibody heterogeneity of lower vertebrates.

[Structural and immunochemical studies of carp (Cyprinus carpio L.) immunoglobulin. V. Tryptic fragments of carp (Cyprinus carpio L.) immunoglobulin M]. / Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.). V. Tryptische Fragmente des Immunglobulins M des Karpfens (Cyprinus carpio L.).

Carp IgM, isolated from normal serum is more sensitive to trypsinization compared to a human myeloma protein IgMGo. Under the same conditions (treatment with trypsin at 56 degrees C for 30 min) carp IgM was degraded to small, mostly dialysable peptides to a larger extent than IgMGo. In both cases the fragmentation resulted in immunoelectrophoretically pure Fab mu and Fc mu fragments. The Fab mu fragments of human IgM (yield: 20% of used IgM material) had a molecular weight of 54,000, the Fc mu fragments (yield: 30%) were a heterogenous mixture as far as molecular sizes concerned with values of about 300,000. For the corresponding fragments of carp IgM we could analyze a molecular weight of about 43,000 for Fab mu (yield: 8%) and for Fc mu (yield 10%) three fractions of 160,000, 130,000 and 90,000. The reductive subunits of Fc mu fragments showed different molecular weights: 39,000 for IgMGo and 45,000 for carp IgM. The anti-fragment antisera prepared in rabbits were monospecific as demonstrated by immunodiffusion.

[Structural and immunochemical studies on carp (Cyprinus carpio L.) immunoglobulins. IV. In vitro reassociation of carp immunoglobulin]. / Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.) IV. In vitro Reassoziation von Karpfen-Immunglobulin.

Mildly reduced high molecular immunoglobulin and antibody of carp with tetrameric structure, carbohydrate content of 6 to 7% and absence of J-chain can reassociate to native molecules. The disulphide bonds between subunits and polypeptide chains are sensitive and can be completly splitted by treatment with only 1 mM DTE. One half of the immunoglobulins retained their high molecular structure as expression of strong non-covalent bonds between subunits of the tetrameric molecule. The other half of immunoglobulins dissociate into HL-halfmolecules. We suppose that carps possess 2 "typs of immunoglobulins" which differ in the tendency to aggregate to high molecular immunoglobulins.

[Structural and immunochemical studies of the immunoglobulins of carp (Cyprinus carpio L.) II. Analysis of the subunits]. / Strukturelle und immunchemische Untersuchungen am Immunglobulin des Karpfens (Cyprinus carpio L.) II. Analyse der Untereinheiten

The 14 S immunoglobulin of the carp (Cyprinus carpio L.) was split into subunits with 0,01 M dithioerythritol. These 5,7 S subunits have a molecular weight of 104 000 and a hexose and hexosamine content of 6.2%. It is likely that the subunits represent HL-half-molecules and not H2L2-monomeres. The values for the molecular of H- and L-chains were 77 000 and 24 000, respectively.