Unified nomenclature of Ig VH genes in rainbow trout (Oncorhynchus mykiss): definition of eleven VH families.
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Animals↗
Biomedical subjects
L Pilström
Publications and source records attributed to L Pilström.
A second immunoglobulin light chain isotype in the rainbow trout.
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 Ckappa and Clambda 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.
Amino Acid Sequence↗
Lymphocyte expression in transgenic trout by mouse immunoglobulin promoter/enhancer.
Two groups of transgenic rainbow trout (Oncorhynchus mykiss, Walbaum) have been produced and compared. One group harbored the reporter gene of chloramphenicol acetyltransferase (CAT) associated with mouse immunoglobulin (Ig) promoter/enhancer (pUCL-CAT-E). The other group carried the same reporter gene under the control of the cytomegalovirus promoter/enhancer (pCMV-CAT). Slot blot analysis of DNA from blood cells and other tissues from pUCL-CAT-E fish showed variation of copy number between the major tissues but not between red and white blood cells. Southern blot analysis indicated that multiple copies organized in concatemers were incorporated into the genome. The pCMV-CAT fish had a pronounced expression of CAT in both white and red blood cells. In contrast, activity of CAT was found in the white blood cells of all pUCL-CAT-E fish but not in their red blood cells. Expression in white blood cells was found preferentially in sIg+ cells, indicating that B cells are the major expressors. High expression was also found in spleen and kidney, but the activity found in thymocytes was equal to the background level. Analysis of some major tissues showed high white blood cell expression associated with low tissue expression, except that liver (known to contain lymphoid tissue in fish) was higher. Thus the regulatory elements of the Ig gene from mouse induce a tissue-specific expression in fish.
Animals↗
A cluster type organization of the loci of the immunoglobulin light chain in Atlantic cod (Gadus morhua L.) and rainbow trout (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 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 kappa nor lambda. 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 kappa and lambda 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.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.
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Amino Acid Sequence↗
Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.
Rabbit antibodies to Atlantic cod (Gadus morhua L.) immunoglobulin were affinity purified and used to screen cDNA libraries from spleen and head kidney mRNA. cDNA clones for both the secretory and membrane-bound heavy (H) chain were isolated, the nucleotide and deduced amino acid sequences of which are reported here. Comparisons of the cod secretory H chain amino acid sequence show 24%, 27%, 30% identity to the mu chain of Mus, Xenopus and Ictalurus, respectively. The highest degree of identity was observed in the CH4 domain. The cDNA encoding the transmembrane form shows a novel splicing pattern where the TM1 exon is spliced directly onto the CH3 domain and not to the CH4 domain as in other animal groups. Southern blot analyses with VH and C probes on genomic DNA from cod erythrocytes indicate that there is a unique C gene but several V genes in the cod immunoglobulin H chain locus.
Amino Acid Sequence↗
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.
Amino Acids↗
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.
Animals↗
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.
Animals↗
Studies on allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. III. Comparative investigations by skin prick tests and nasal provocation tests.
The allergenic activities of allergen (GEN) and allergoid (GOID) preparations from partially purified timothy pollen extract were investigated in quantitative skin prick tests and nasal provocation tests. In skin prick test GOID yielded significantly less activity than GEN. Approximately 90 times more protein of GOID than of GEN was necessary to elicit wheals of the same mean size as 1 mg/ml histamine control solution (1 HEP). In nasal provocation the patients seemed to react differently with respect to their nasal sensitivity to GOID. One group of patients showed equal sensitivity to GOID and GEN, whereas the second group showed higher thresholds to GOID. These differences in nasal sensitivity to GOID could be of practical value to predict the optimal dosage schedule for the treatment of patients with GOID.
Adolescent↗
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.
Adenocarcinoma↗
A simple and efficient method for objective discrimination between pseudostratified, metaplastic and dysplastic nasal epithelium.
Morphologic features may sometimes be interpreted differently by different pathologists. By introducing objective methods in the histological evaluation the subjectivity of the judgement is reduced and a higher degree of consistency achieved. In the present study three linear parameters of nasal epithelial basal cells: the largest transversal nuclear diameter, the sum of the longitudinal and transversal nucleolar axes and the basal cell width, i.e. the width of the attachment face to the basement membrane, were classified by means of three-class rulers. From this classification weighted indices were made which were then given equal weight and transformed, so that when added up their sum, the joint index (Q) to the largest possible extent was consistent with pseudostratified/stratified cuboidal, stratified squamous and dysplastic epithelium when Q less than or equal to 1, 1 less than Q less than or equal to 2 and Q greater than 2 respectively. Tested prospectively we found 91% agreement between the morphometric and histological classification. The method presented is extremely simple and rapid to perform, and appears to have diagnostic potential in distinguishing various types of epithelial alterations of the nasal mucosa. The method may also be useful in approaching similar problems in other epithelia.
Cell Nucleolus↗
Papillary carcinomas of the thyroid have pore-deficient nuclei.
A significant reduction in nuclear pore density observed in papillary carcinomas compared to normal thyroid, adenomas and follicular carcinomas probably disturbs the nucleo-cytoplasmic transfer of ribonucleoproteins. This may contribute to the slow growth and indolent biological behavior of these tumors where even the presence of regional lymph node metastases does not worsen the prognosis. The signs of nuclear hyperactivity may result from an attempt to compensate for the nuclear membrane defect.
Adenocarcinoma↗
Studies on vinblastine-induced autophagocytosis in mouse liver. III. A quantitative study.
The microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection. The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.
Animals↗
Studies of allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. I. Physicochemical characteristics and binding to allergen-specific human IgE.
Physicochemical and in vitro allergenic properties of two different preparations of timothy (Phleum pratense) pollen were compared. The refined allergen extract, timothy B, contained proteins of molecular weights from 10,000 to 45,000 daltons. The allergoid preparation, which was prepared by mild treatment of the partially purified extract with formaldehyde, possessed only a trace of activity in the RAST inhibition assay. The formaldehyde treatment also resulted in a shift of the net charge of proteins to the more acidic site, as demonstrated by isoelectric focusing. Furthermore, it was shown that the activities of naturally occurring enzymes of native allergen extracts were reduced considerably. Only traces of acid phosphatase activity could be demonstrated in timothy B allergoid.
Acid Phosphatase↗
Studies on allergen and allergoid preparations from purified timothy (Phleum pratense) pollen extracts. II. Anaphylaxis studies in rats and histamine release from human leukocytes.
In vivo and in vitro allergenic activities of allergen and allergoid preparations from partially purified timothy pollen extract were measured in three different systems. In heterologous PCA titration, the allergen preparation was found to be 32 times more allergenic than the allergoid preparation. By intravenous chaled that treatment of allergen preparations with formaldehyde led to a preparation with the properties of an allergoid. In addition, it was found that the carbohydrate fraction of the refined allergen preparation of timothy a reduction in blood pressure. Thus, the allergen preparation was at least 50 times more potent in inducing systemic anaphylaxis in sensitized rats than the allergoid preparation. In the histamine release assay of grass-sensitive human leukocytes, approximately 1,000 times more allergoid protein than allergen protein was required to achieve 30% histamine release. Although to different degrees, in all three test systems in could be demonstrated that treatment of allergen preparations with formaldehyde led to a preparation with the properties of an allergoid. In addition, it was found that the carbohydrate fraction of the refined allergen preparation of timothy pollen decreased the blood pressure even in nonsensitized animals. The effect of this carbohydrate fraction on blood pressure was immediate but short-lived and immunologically nonspecific.
Allergens↗
Skeletal muscle lactate dehydrogenase isozymes and fibre composition in viral, mycoplasma and bacterial infections in young and old men.
The activities of LD isozymes were studied by thermoinactivation in skeletal muscle biopsies from young and old patients suffering from viral, mycoplasma or bacterial infections and from corresponding controls. The activities of LD2-5 decreased 35-45% and that of LD1 17-29% in the muscle of the patients compared to that of the controls. There were no differences between age groups. In the controls good correlations were demonstrated between the relative activities of the LD isozymes and the muscle fibre composition, but such correlations could not be found in the patients. This indicates that the decrease of the activity of muscle LD in these infections is not associated with alterations in any specific muscle fibre type but rather with an effect of a general reduction of the LD activity.
Adolescent↗