Lars Pilström

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.

A cluster type organization of the loci of the immunoglobulin light chain in Atlantic cod (Gadus morhua L.) and rainbowttrout (Oncorhynchus mykiss Walbaum) indicated by nucleotide sequences of cDNAs and hybridization analysis

Antibody screening and colony hybridization of cDNA libraries have been used to isolate clones of the immunoglobulin light (IgL) chain from Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss Walbaum). Sequence analysis shows dissimilarities in in the constant part of the molecule (Cl) within each species. Comparisons of the amino acid sequences of the constant parts of the IgL chains show a 55% identity between the two teleost species. When compared with other species the highest similarities are found to the constant domain of the IgL chain from mammals (30%-37%), but the teleost IgL chain can be classified neither as κ nor λ. The Vl domain in Atlantic cod and rainbow trout is also more similar to those of mammals than to those of other animal species, but no difference between κ and λ was noticed. Genomic Southern blots hybridized with fragments coding for the constant part of IgL gave several bands larger than 2 kilobases and a similar pattern was obtained with fragments coding for the variable part. These results show that the locus of the IgL chain has a multiple organization in teleost fish and that the locus has an organization similar to that of sharks. Several of the cDNA clones isolated from both the head kidney and the spleen represent nonrearranged or nonspliced mRNA, and northern blot analysis shows that such transcripts are present in both the head kidney and the spleen.

Immunoglobulin Isotypes: Structure, Function, and Genetics

Immunoglobulin (Ig) classes (in mammals, IgM, IgA, IgD, IgG, IgE) are defined by the isotypes of heavy (H) chains (µ, α, δ, γ, and e). Each isotype is in turn distinguished by unique structures in its constant region domains. These different structures confer distinctive functions on the Ig classes. When two or more Ig classes are very similar, as occurs with the four different types of IgG found in man and mouse, they are usually termed subclasses. Each isotype is encoded by a distinct gene and multiple heavy chain isoforms can be produced by alternative pathways of RNA processing, such as the secreted (slg) and membrane (mlg) forms of all H chains, or the full-length and truncated H chain isoforms of certain avian antibodies. Allelic variation in the constant (C) regions gives rise to allotypes. The different types of light (L) chains (in mammals, к and λ) are also typically referred to as isotypes. This system of classification of Igs was developed from studies of man and his immunological understudy, the mouse, and has proven useful not only in these two species, but also in other mammalian species. Although the classification of mammalian Ig classes and isotypes is quite clear, the situation with Igs from nonmammalian vertebrates is not. For example, is the shark molecule referred to as IgM really IgM? Should we call the predominant low molecular weight Ig in chickens IgG or IgY? This chapter discusses the ways in which these and similar questions have been approached.

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.

A SECOND IMMUNOGLOBULIN LIGHT CHAIN ISOTYPE IN THE RAINBOW TROUT

Abstract A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription – polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Cκ and Cλ isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

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.

Why is the antibody response of Atlantic cod so poor? The search for a genetic explanation

The immune system of the Atlantic cod (Gadus morhua L) is unusual in that it cannot produce a specific antibody response upon immunization. Despite this, the cod is not particularly susceptible to infectious disease in its normal environment. This review examines the potential genetic basis for the lack of a specific antibody response in the cod. The genetics of cod immunoglobulins are compared with those of other well-characterized teleost fish. A review of the evidence suggests that deficiencies in the number, structure, organization, diversity and expression of both Immunoglobulin (Ig) heavy and Ig light chain genes in cod cannot explain its unusual antibody response. It is concluded that a deficiency in cod major histocompatibility (MH) class II molecules is a prime suspect for the lack of a specific antibody response in cod, and that testing of this hypothesis must await future experimentation.

T-cell antigen receptors in Atlantic cod (Gadus morhua L.): structure, organisation and expression of TCR α and β genes☆

Abstract 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 TCRα and β chain full length cDNAs in Atlantic cod. The Vα and Vβ 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 Jα and Jβ 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 Cα and Cβ sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic regions. The Atlantic cod Cβ 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 Cβ 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 Cβ. Based on Southern blot analyses, the TCRα and β 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 TCRα and β chains in thymus, spleen and head kidney, expression of the TCRβ 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.

Activity of alcohol dehydrogenase and acetaldehyde dehydrogenases in the liver and placenta during the development of the rat.

The ontogenetic development of the enzymes alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenases (ALDH I and II) was followed in rats. ADH could be detected just before birth and increased gradually to reach 82% of adult values at 47 days. ALDH I and II were present from day 15 of gestation, increased rapidly at birth, and reached 80-90% adult values at 47 days. The ratio between ALDH and ADH activities decreased gradually during ontogenesis. The relative subcellular distribution of all enzymes was identical before birth, 7 days after birth and in adults. The placental activities of ADH and ALDH I and II were studied at 15 and 20 days of pregnancy. ADH could not be detected in placentas. Low activities of ALDH I and II were present in placentas studied at 15 days of gestation, and still lower activities were found in placenta at 20 days.

CHARACTERIZATION OF MHC CLASS I AND BETA 2-MICROGLOBULIN SEQUENCES IN ATLANTIC COD REVEALS AN UNUSUALLY HIGH NUMBER OF EXPRESSED CLASS I GENES

Abstract Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and β2-microglobulin (b2m) genes in Atlantic cod (Gadus morhua L.). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic α1-, α2-, and α3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the α2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an α3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an α1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b2m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean β2m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b2m locus in Atlantic cod.