Complement activation is required for IgM-mediated enhancement of the antibody response.

The ability of IgM antibodies to specifically enhance the thymus-dependent humoral immune response to particulate antigens is well documented. We have used two approaches to test whether complement factors play a role in this process. First, mice were depleted of C3 by treatment with cobra venom factor (CVF) and then immunized with SRBC with or without IgM-anti-SRBC. CVF treatment severely impaired the capacity of IgM to induce an enhanced anti-SRBC response. Moreover, it was shown that IgM can potentiate the response in C5-deficient AKR mice, thus demonstrating that the complement factors acting before C5 are the crucial ones. A second test compared the enhancing properties of two monoclonal IgM-anti-TNP antibodies where, because of a point mutation in the mu chain constant region, one of the antibodies is impaired in its capacity to activate complement. We show that the mutant antibody lacks the enhancing properties of the wild-type IgM. Activation of C3 by IgM antibodies as well as localization of antigen in the spleen seem to be necessary steps in the IgM-mediated enhancement of antibody responses. Our data offer an explanation to the immunosuppression described in CVF-treated animals as well as the low humoral immune responses in certain hereditary complement deficiencies. It is suggested that IgM indeed has an important physiological function in enhancing antibody responses to foreign substances.

Characterisation of rainbow trout cDNAs encoding a secreted and membrane-bound Ig heavy chain and the genomic intron upstream of the first constant exon.

Two different rainbow trout cDNA sequences encoding a heavy chain secreted Ig (Hs) and a part of a membrane-bound heavy chain Ig (Hm) are reported. The sequences were most similar to those encoding the Ig heavy chains (IgH) of other teleost fish. As in the Hm of the other teleost fish the rainbow trout Hm results from the splicing of the 3' end of the third constant exon (CH3) to the sequence encoding the membrane-bound domain. Analysis of a rainbow trout IgH genomic clone revealed that a joining heavy chain (JH) segment, different to the one observed in the cDNA, is located 825 bp 5' of the CH1 exon. The sequence also contains possible enhancer-like and octamer-like motifs.

Primary and secondary IgG are equally efficient immunosuppressors in relation to antigen binding capacity.

Secondary, hyperimmune IgG antibodies can suppress the humoral immune response against the relevant antigen. Whether IgG antibodies derived from a primary antigen response also have this capacity is not clear, although the role of primary IgG is of great interest in a physiological situation. In this study we compared the in vivo immunosuppressive potential of primary and secondary IgG anti-SRBC (sheep erythrocytes) on the primary anti-SRBC PFC response in CBA/Ca mice. Both primary and secondary IgG antibodies are potent immunosuppressors causing more than 99% specific suppression. Preparations of primary and secondary IgG antibodies which, measured by an ELISA method, were shown to bind to SRBC to the same extent, also had very similar immunosuppressive potency. This emphasizes the strong correlation between the antigen binding and the immunosuppressive capacities of IgG antibodies.

Isolation and partial characterization of immunoglobulin from cod (Gadus morhua L.).

Serum immunoglobulins (CS-Ig) from cod (Gadus morhua L.) were prepared by precipitation with ammonium sulphate and molecular sieving. The molecular weight estimated from molecular sieving and electrophoresis was 851 kD for the whole molecule and 81 and 27.5 kD for the two subunits. This suggests a tetrameric structure of the molecule. The isoelectric point of CS-Ig was determined to approximately pH 5.0. The amino acid composition and absorbancy at 280 nm are very similar to published data of IgM from other fish species as well as from several mammals. CS-Ig has a natural binding capacity to a number of antigens used for immunization. Assays of antibody activity of fractionated cod serum indicates that CS-Ig does not exist as a monomeric molecule. As the characteristics of CS-Ig are very similar to those found in other fish species, we believe that CS-Ig is an IgM-like molecule.

T-cell antigen receptors in Atlantic cod (Gadus morhua l.): structure, organisation and expression of TCR alpha and beta genes.

By using short degenerate primers complementing conserved T-cell antigen receptor (TCR) variable and constant region segments for PCR, we were able to isolate putative TCRalpha and beta chain full length cDNAs in Atlantic cod. The Valpha and Vbeta domains have the canonical features of known teleost and mammalian TCR V domains, including conserved residues in the beginning of FR2 and at the end of FR3. The Jalpha and Jbeta region possess the conserved Phe-Gly-X-Gly motif found in nearly all TCR and immunoglobulin light chain J regions. Similar to other vertebrates, the Atlantic cod Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cbeta sequence lacks a cysteine in its connecting peptide region, but other motifs proposed to be important for dimerisation and cell surface expression are observed. Four different cod Cbeta sequences were identified, two of which share 3' untranslated regions different from one of the other two sequences, suggesting the existence of isotypic gene variants of Cbeta. Based on Southern blot analyses, the TCRalpha and beta gene loci appear to be arranged in translocon organisation (as opposed to multicluster) with multiple V gene segments, some (D) and J gene segments and a single or few C gene segments. Northern blot analyses show expression of the TCRalpha and beta chains in thymus, spleen and head kidney, expression of the TCRbeta chain was also detected in the ovary. Interestingly, no expression was detected in intestine even though the existence of T-cells in intestine has been proposed in other teleost species.

Variability of the immunoglobulin light chain in the Siberian sturgeon, Acipenser baeri.

All sturgeon VL segments isolated in this study belong to a single family, VLI, which can be divided into two subfamilies. Of the 79 cDNA clones isolated, 76 belong to the larger subfamily, VLIa, and only 3 clones constitute the smaller subfamily, VLIb. To evaluate variability, the Shannon entropy was estimated for each individual amino acid position, and to facilitate comparisons of variability between species the mean entropy of the CDR regions was calculated. In such a comparison, the sturgeon was found to have CDR1 and CDR3 variability approaching those found in mouse and clawed frog, but showed very low variability for CDR2. Amino acid position 50 does however display variability in the range of mouse and clawed frog. It is further confirmed that the sturgeon has numerous J segments, but that the junctional diversity does not contribute greatly to the diversity of the light chain. Comparisons of cDNA clones and a genomic VL segment indicate that the VL undergoes changes, particularly in the CDR regions, in a manner that can be explained by somatic hypermutation and/or gene conversion.

Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica.

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (gamma chain), IgE (epsilon chain) and IgA (alpha chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (mu chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.

Immunoglobulin VH regions in Atlantic cod (Gadus morhua L.): their diversity and relationship to VH families from other species.

Variable regions (VH) of Atlantic cod cDNA clones have been isolated by the polymerase chain reaction (PCR) technique, using primers for the first constant domain of heavy chain (CH1) and lambda gt11. Based upon sequence analysis and comparisons these clones have been divided into three different VH families. The approximate number of members in each family was estimated by genomic Southern blot. Comparisons of complementarity determining regions (CDRs) and frameworks (FRs) show that FR2 and the last part of FR3 are the most conserved regions. The CDR3 is very heterogeneous and gives a major contribution to VH diversity. Possible relationships between VH sequences from 17 species are shown graphically. Different VH families are often more conserved between species than within any one species. Two genomic VH clones have been isolated and partially sequenced. The VH genes have an octamer and TATA motif in the 5' region, followed by an 18-amino-acid-long hydrophobic leader, and the mature VH coding region. The characteristics heptamer-nonamer recombination signals for VH to D joining are present 3' of the VH segment.

Ig light chain variability in DNP(494)-KLH immunised sea bass (Dicentrarchus labrax L.): evidence for intra-molecular induced suppression.

The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.

Light chain variable region diversity in Atlantic cod (Gadus morhua L.).

This study was undertaken to determine if a lack of V(L) domain variability could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod V(L) regions was studied in 33 cDNA and two genomic clones. The variability of the CDRs was estimated by the Shannon entropy method and compared with that in other species. It was found to be lowest in the little skate (Raja erinacea), higher in cod, and highest in Xenopus and mouse. While the variability of the CDRs is slightly lower in cod than in Xenopus and mouse, it is spread over broader areas of the amino acid sequence. The length of CDR1 and CDR3 in cod is equal to or exceeds that found in skate, Xenopus, chicken and mammals. Isoelectric points and hydrophobicity vary substantially among the studied Ig light chain domains. Thus, neither the length, nor the variability, nor the physicochemical properties (pI and hydrophobicity) of the L chain CDRs can explain the absence of antibody response to immunization in cod.

Ultrastructural morphometry of thyroid neoplasms.

This paper presents the first comprehensive morphometry analysis of normal thyroid, adenomas, and follicular and papillary carcinomas. The mean nuclear volume and the mean nuclear surface increased, while the volume densities of rough endoplasmic reticulum and dense bodies decreased from normal thyroid through adenomas and follicular carcinomas to papillary carcinomas. The different amounts of cytoplasmic organelles probably are related to endocrine function rather than to malignant potential. Papillary carcinomas with a predominantly follicular growth pattern are related more closely to follicular carcinomas than to papillary carcinomas dominated by papillae. This probably indicates a more active endocrine function rather than a different degree of malignancy. Papillary carcinomas, where follicular structures dominate, are therefore expected to respond to radioiodine treatment more favorably than those mainly forming papillae. Papillary carcinomas with abundant ground glass nuclei do not seem to have a lower volume density of heterochromatin than tumors lacking this nuclear feature. In the individual problem case with a follicular tumor, electron microscopy and morphometry cannot even distinguish between an adenoma and a well-differentiated follicular carcinoma.

TGF-beta3 exists in bony fish.

A partial nucleotide sequence of transforming growth factor-beta3 (TGF-beta3) has been isolated from the Siberian sturgeon (Acipenser baeri), rainbow trout (Oncorhynchus mykiss) and European eel (Anguilla anguilla), confirming a ubiquitous presence in the ray-finned (Actinopterygian) bony fish. The bony fish TGF-beta3 is highly conserved, with some 83-84% nucleotide identity (coding region) and 90-95% predicted amino acid identity to known homeotherm TGF-beta3's. Far lower homologies are apparent with other known TGF-beta isoforms in fish (e.g. 64-66% and 81-82% amino acid identity to trout TGF-beta 1/5 and carp TGF-beta2 respectively). Phylogenetic tree analysis showed that the fish TGF-beta3's clustered with the known homeotherm TGF-beta3's. The relatively tight clustering of TGF-beta1, TGF-beta2 and TGF-beta3 was in contrast to the TGF-beta5's, which are clearly a more heterogenous group.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) I. The effects of environmental temperature.

The effects of environmental temperature on certain humoral immune parameters in Atlantic cod (Gadus morhua L.) were studied. Serum samples were collected from captive cod, of wild origin, kept at different temperatures for 12 months. It was found that immunoglobulin and natural antibody levels increased with increasing temperature whereas the total serum protein concentration, anti-protease activity, iron concentration, unsaturated and total iron binding capacity decreased with increasing temperature. Haemolytic activity and percentage iron saturation also tended to decrease with increasing temperature although this was not statistically significant.

Humoral immune parameters in Atlantic cod (Gadus morhua L.) II. The effects of size and gender under different environmental conditions.

The effects of size and gender on several humoral immune parameters in cod were examined under different environmental conditions. Serum samples were collected from wild cod of different sizes. Two samplings were undertaken: In the spring in relatively cold waters off the north west coast of Iceland and in the fall in relatively warm waters off the west coast of Iceland. Most of the parameters increased with increasing cod size, except the haemolytic activity which decreased. Higher serum protein levels were seen in cod sampled in the fall than in the spring. In cod sampled in the spring there was an apparent difference between specimens < 75 cm in length and the larger specimens with respect to haemolytic activity and iron concentration. None of the parameters were influenced by the gender of the cod.

Evolution of adaptive immunity.

Antigen receptors and major histocompatibility molecules, key elements required for adaptive immunity, are first seen in jawed fish. So, how did they evolve and have they changed?

Purification of birch pollen allergen extract by gel filtration. I. Chemical and immunological characterization of the fraction.

Birch pollen extract was fractionated by gel filtration on a Sephadex G-75 column. The physicochemical characterization of fractions included the determination of protein, carbohydrate, molecular weight and pI. The immunological properties of the fractions were measured by skin prick testing of allergenic individuals and by indirect RAST. The cross-reactivity of the fractions was investigated by heterologous PCA in rats after producing specific reaginic sera in mice. The allergenically most active fractions were composed mainly of proteins with a molecular weight range of 10,000--50,000 daltons. These active fractions represented only 3% of the total protein and carbohydrate content of the crude extract. These results indicate that it is possible to purify birch pollen allergen extract from the bulk of nonallergic contaminants, mainly low molecular weight carbohydrates, by a single step gel filtration procedure.