Different Inbred Strains Of Mice Research Articles

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lactococcus lactis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 × LD 50) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (10–20 fold) more immunogenic than the alternative forms of the protein.

Large-Scale Genomic Comparison Using Two-Dimensional DNA Gels

Two-dimensional electrophoresis (2DE) of DNA fragments, in which separation occurs first by size and then by sequence variation, is a method enabling large-scale comparison of complex genomes. Combining 2DE with probing for various classes of repetitive genomic elements allows rapid and efficient comparison of thousands of fragments and millions of base-pairs of DNA distributed across most genomic regions. This approach is demonstrated here by analyzing the extent of genomic relatedness of different inbred strains of mice. Such strains are shown to differ from each other by approximately 0.2-1% of their nucleotides, above which level reproductive speciation occurs. The 2DE method of assessing the overall relationship between two genomes represents an appropriate tool for analyzing members of a single species, but is too sensitive for use in interspecies comparisons.

Neuroregulation of natural suppressor cell activity.

The neuronal control of murine natural suppressor (NS) cell activity was studied in mice following lesion of the arcuate nucleus of the hypothalamus performed by the postnatal treatment with monosodium glutamate (MSG). The suppressor activity was evaluated as the ability of spleen or bone marrow cells (suppressors) to suppress the mitogen-induced lymphocytes proliferation of allogeneic Balb/c spleen cells (indicators). The study was carried out on three different inbred strains of mice (C57BL/6, DBA/2 and CBA). The results demonstrated that, in all strains used, the treatment of mice (12 day-old) with MSG led to a marked reduction of natural suppressor cell activity. This reduction was observed in both spleen and bone marrow, the lymphoid organs in which the NS cell activity is usually present. A reduced NS activity was also present in the bone marrow of MSG-treated adult mice, suggesting a permanent alteration of NS cell activity. These results provide additional support on neuromodulation of immune system.

The susceptibility of inbred mice to the production of malignant thymoma by N-methyl-N-nitrosourea.

The susceptibility of inbred mice to the induction of malignant thymoma by N-methyl-N-nitrosourea (MNU) has been quantified and compared. Strain differences emerged and this was also apparent in congenic lines of C57BL/10 mice, differing at the H-2 locus. However, different strains of inbred mice, with identical H-2 haplotypes, also showed varying susceptibility to MNU, indicating that the role of the MHC was not a simple one, but apparently depended on other, undefined genes. Proficiency of repair of O6-methylguanine by thymic tissue was not responsible for these strain differences and the increased resistance to induction of thymoma by MNU in older mice was due to age-dependent changes within the thymus gland. Interestingly, the thymus of older mice, grafted with neonatal thymic tissue, still provided a suitable environment for the development of thymic tumours.

The Cellular Immune Response Against Yersinia enterocolitica in Different Inbred Strains of Mice: Evidence for an Important Role of T Lymphocytes

Resistance of mice against infection with Yersinia enterocolitica has been shown to be related neither to the Ity locus coding for resistance against infection with Salmonella typhimurium and other pathogens nor to the H-2 locus. From other mouse infection models, e.g., murine leishmaniasis, there is evidence that a different T cell-dependent regulation of the host immune response in various inbred strains of mice determines the susceptibility to the infectious agent. However, until recently, little was known about the cellular immune response against Y. enterocolitica. Thus, in a first approach we used the highly virulent Y. enterocolitica strain WA of serotype O:8 and different inbred strains of mice (C57 BL/6, Balb/c and athymic T cell-deficient C57 BL/6 nude mice) to investigate the cell-mediated immunity against parenteral infection. Comparison of the median lethal dose and of the net-bacterial growth in the spleens of infected mice indicated that Balb/c mice could be considered as Yersinia-susceptible whereas C57 BL/6 mice were relatively resistant. However, in contrast to normal C57 BL/6, athymic T cell-deficient C57 BL/6 nude mice have proved to be highly susceptible to Yersinia infection suggesting that T cells are required for the elimination of the pathogen. This conclusion was supported by histomorphological and immunohistological results indicating that T lymphocytes were present in Yersinia-induced tissue lesions. Moreover, the adoptive transfer of Yersinia-specific T cell lines and clones into naive animals mediated significant protection against the pathogen in both Yersinia-resistant C57 BL/6 and in Yersinia-susceptible Balb/c mice. These findings emphasize an important role of T lymphocytes in the host response against Y. enterocolitica infection.

Epidermal compromise in American cutaneous leishmaniasis.

In American cutaneous leishmaniasis (ACL), Leishmania parasites enter the epidermis of the host via the bite of infected sandflies. Immune responses against the parasite vary from "effective" in localized (LCL) to a state of "selective anergy" in diffuse (DCL) cutaneous leishmaniasis, whereas the intermediate muco-cutaneous form (MCL) is characterized by an exacerbated cell-mediated immunity. We have shown that in LCL epidermis, Langerhans cells (LC) are increased, HLA-DR is universally expressed and intercellular adhesion molecule-1 (ICAM-1) immunoreactivity is distributed in patches. In addition, mRNA for IL-1 beta, IL-8, TNF alpha, TNF beta, and INF gamma may be detected in epidermal sheets by reverse transcriptase followed by polymerase chain reaction (RT-PCR). In contrast, DCL epidermis shows fewer LC than LCL epidermis, and expression of ICAM-1, HLA-DR, and IL-1 beta mRNA cannot be detected. MCL lesions show a mucosal epithelium lacking LC, but ICAM-1 is universally expressed. The clinical manifestations of ACL can be reproduced experimentally in different strains of inbred mice. In healthy mice, we have shown a positive correlation between LC and dendritic epidermal T cells (DETC) numbers. This correlation was not, however, observed in L. mexicana-infected mice, suggesting that infection alters the balance between the two cell types. In addition, agents that modulate LC and DETC cell densities change the development of experimental leishmaniasis. These results suggest that the epidermis is essential in determining the type of immune response that is developed against the Leishmania parasites.

Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor [formula omitted] region

We have analyzed the organization, structure, and function of the murine T-cell receptor Cα Cδ region. This region spans 94.6 kb of DNA and contains the Cα and Cδ genes, as well as the Vδ5, Jδ2, and 50 different Jα gene segments. Within this sequence we have identified 15 new Jα gene segments, 40 new 5′ RNA splice signals, and 40 new DNA rearrangement signals for the Jα gene segments. The murine Cα Cδ sequence contains an exceptionally high level of coding sequence with over 5.7% of the total sequence found in the exons. This is much more than that found in the β-globin locus and the HPRT locus. Using the sequence data obtained from the Cα Cδ region, we have designed simple assays to test for Jα gene segment transcription and to determine the level of polymorphism for simple repeat sequences among different inbred strains of mice using the polymerase chain reaction. Furthermore, comparisons of this 95 kb of sequence with the available sequence from homologous regions of other species have led to the identification of a highly conserved sequence that is present throughout vertebrates and in the mouse binds lymphocyte-specific nuclear proteins. Comparisons of a 10-kb region, which includes the Cα gene in human and mouse, average 66% sequence similarity. These studies support the contention that large-scale DNA sequencing projects of homologous regions of mouse and human will provide powerful new tools for studying the biology and evolution of loci such as the T-cell receptor and for identifying and posing new questions about the functions of conserved sequences.

Damage in B2m genes and DNA methylation of H-2 genes are involved in loss of expression of class I MHC products on the membrane of LR.4, a cell line derivative of the T-cell lymphoma L5178Y.

We have isolated an H-2 deficient cell line (LR.4) from the T-cell lymphoma L5178Y which grew without restrictions in the peritoneal cavity of different inbred strains of mice. The use of polyclonal anti-H-2 antiserum and complement indicated that LR.4 cells did not express class I determinants on the cell membrane. Southern blots of genomic DNA of LR.4 cells showed that B2m genes were severely damaged and that class I H-2 genes were extensively methylated. Consequently, LR.4 cells failed to transcribe mRNAs for both B2m and class I H-2 genes. On the other hand, specific immunity to LR.4 was demonstrated in C57BL/6J mice since, in subsequent challenges with either LR.4 or EL4.4, LR.4 did not grow, whereas EL4.4 grew and killed the mice. In C57BL/6J mice, rejection of LR.4 was accompanied by the production of cytotoxic antibodies. The immune response induced in C57BL/6J mice was determined by non-H-2 antigenic determinants in LR.4 cells.

Antibody responses to ganglio-series gangliosides in different strains of inbred mice

We studied antibody responses after immunization with ganglio-series gangliosides against 10 strains of inbred mice, including Balb/c, C57BL/6, A/J, C3H/HeN, C3H/HeJ, CBA/N, AKR/N, NZB/N, DBA/2 and nu/nu Balb/c. Twelve gangliosides having NeuAc as their sialic acid moiety (GM4, GM3, GM2, GM1, GD3, O-Ac-GD3, GD2, GD1a, GD1b, GT1a, GT1b and GQ1b), four gangliosides having NeuGc (GM3, GM2, GM1 and GD3) and four asialo-gangliosides (GA4, GA3, GA2 and GA1) were injected intravenously adsorbed to Salmonella minnesota. The antibody titers of the mice sera were determined by an enzyme-linked immunosorbent assay and an immune adherence assay. Antibody responses were found to depend not only on the ganglioside used as an immunogen but also on the mouse strain. Gangliosides having a trisaccharide sequence (NeuAcα2 → 8NeuAcα2 → 3Gal-) such as GD3, GD2, GDlb, GT1a and GQ1b, in particular O-Ac-GD3, induced high-titer antibody responses, whereas those having a disaccharide sequence (NeuAcα2 → 3Gal-) such as GM4, GM3, GM2, GM1, GDla and GTl1 induced low-liter antibody responses. On the other hand, gangliosides with NeuGc developed minimum titers. In contrast, asialo-gangliosides induced much higher responses than the corresponding gangliosides. The differences in ceramide portions of these gangliosides did not appear to be involved in inducing antibody responses. Mice could be divided into three groups according to the magnitude of their antibody responses: Group 1, those that produce the highest antibody responses (C3H/HeN and A/J); Group 2, those that demonstrate moderate antibody titers (Balb/c, C57BL/6, DBA/2 and nu/nu Balb/c); and Group 3, those that make minimum responses (AKR/N, C3H/HeJ, CBA/N and NZB/N). The pattern of reactivity to the various gangliosides was similar in all the strains tested.

Motor activity and the mesotelencephalic dopamine function. II. Multivariate analysis of genetically segregating generations

Previous experiments on genetically different inbred strains of mice demonstrated parallel variations between the activity of regional brain tyrosine hydroxylase (TH) and locomotor behavior. Based on these associations, it was hypothesized that genetic variations in mesotelencephalic TH activity, an index of dopamine neurotransmitter function, would correlate positively with exploratory and locomotor behavior. In order to test this hypothesis, open-field motor behaviors and mesencephalic and striatal TH activities were analyzed by multivariate statistical methods in genetically segregating (C57BL/6ByJ X BALB/cJ)F2 and (C57BL/6ByJ X CXBI/ByJ)F2 generations. Factor analysis, based on correlation matrices of variables with significant genetic dominance or additive effects, demonstrated that locomotor activity and frequency of occurrence of various motor patterns were not correlated with mesencephalic and striatal TH activity. These results indicate that the assumption of a positive phenotypic correlation between spontaneous motor activity and mesotelencephalic TH activity does not hold in genetically segregating populations. Strategies and problems in revealing the behavioral consequences of genetically based variations in the mesotelencephalic DA system are briefly discussed.

Immunobiology of experimental leishmaniasis.

Self-cure versus uncontrolled disease progression in experimental murine cutaneous leishmaniasis depends upon a delicate interplay among various activated cells of the host's immune system. Susceptibility or resistance to infection with Leishmania major is correlated with the ability of different inbred strains of mice to produce the characteristic spectra of lymphokines upon infection. Appropriate experimental interventions now allow the modulation of these responses, providing the possibility to render genetically susceptible mice resistant to infection and, vice versa, to cause genotypically "healer" strains to express a "non-healer" phenotype. These experimental manipulations have proven to be powerful tools in the dissection of the underlying immune mechanisms and cellular parameters responsible for susceptibility and resistance, and will perhaps allow the identification of molecules of parasite origin that induce deleterious immune responses to infection with Leishmania, and thus to exclude them from future vaccines. More importantly, rational immune intervention could permit the diversion of established host-damaging immune responses to host-protective immunization.

Genetic variability of the murine creatine kinase B gene locus and related pseudogenes in different inbred strains of mice

The role of genetic variation in isoenzyme gene families is often poorly appreciated. We report here on the determination of DNA sequences and typing of genetic variability in four creatine kinase B (CKB) gene loci in different inbred strains of mice. The unique functional murine CKB gene was found to be nearly identical to the previously characterised rat and human sequences in both size and exon-intron structure. In this gene, approximately 0.5% allelic nucleotide positions as well as the lengths of simple A-rich and [TG] n repetitive elements located at the 5′ and 3′ sides of the transcribed segment, differed between inbred strains of mice. Preliminary experiments suggest that this sequence divergence is of importance for design of gene targeting strategies involving homologous DNA recombination. The three additional CKB-like gene loci in mice all had the characteristics of processed pseudogenes. By Southern blot analysis we could demonstrate that both the type and number of pseudogenes differed between inbred strains. Analysis of the CKB gene sequences enabled us to speculate about the evolutionary history of this highly polymorphic subfamily of genes.

Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein.

The immunogenicity of a chimeric T/B cell peptide corresponding to antigenically characterized epitopes of the Chlamydia trachomatis major outer membrane protein (MOMP) was studied in mice to further define its potential use in the development of a subunit vaccine in preventing blinding trachoma in humans. The chimeric peptide, designated A8-VDI, corresponds to a conserved MOMP T helper (Th) cell epitope(s) (A8, residues 106-130) and serovar A VDI (residues 66-80), which contains the serovar-specific neutralizing epitope 71VAGLEK76. Mice immunized with peptide A8-VDI produced high-titered polyclonal IgG antibodies which recognized the VAGLEK-neutralizing epitope. Peptide A8-VDI primed A/J mice to produce high-titered serum-neutralizing antibodies in response to a secondary immunization with intact chlamydial elementary bodies (EBs). Peptide A8-VDI, but not peptide VDI alone, was immunogenic in six different inbred strains of mice disparate at H-2, indicating that the Th cell epitope(s) contained in the A8 portion of the chimera was recognized in the context of multiple major histocompatibility complex (MHC) haplotypes. An unexpected finding of this work was that different inbred strains of mice immunized with the chimeric peptide produced antibodies of differing fine specificities to the VDI portion of the chimera. Some mouse strains produced anti-VDI antibodies that did not recognize the VAGLEK-neutralizing epitope. The ability of mice to respond to the VAGLEK-neutralizing site was not dependent on MHC haplotype since mouse strains of the same H-2 haplotype produced anti-VDI antibodies of differing fine specificity.

Immunologic and genetic characterization of S180, a cell line of murine origin capable of growing in different inbred strains of mice

Some of the immunologic and genetic properties of the cell line S180 have been examined. These cells grew without restrictions in the peritoneal cavity of different inbred strains of mice and invariably killed the animals. With Northern blots it was demonstrated that S180 cells contained class I mRNAs but failed to transcribe B2m genes. However, under experimental conditions, a protective humoral immune response mediated by cytotoxic antibodies and complement against S180 cells was obtained through non-H-2 antigens in C57BL/6J mice.

Mouse microsatellites from a flow-sorted 4:6 Robertsonian chromosome.

Twenty microsatellites were generated from a previously characterized lambda gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.

Egg production of Clonorchis sinensis in different strains of inbred mice.

In order to compare the intraspecific variation in host-parasite relationship of Clonorchis sinensis, six strains of inbred mice, ICR, DDY, GPC, BALB/c, nude and DS, were infected orally with 20 metacercariae of C. sinensis. The biologic incubation period of C. sinensis was the shortest in DDY mice, 21.2 days in average, followed by GPC 21.4, BALB/c and DS 23.2, ICR and nude 23.4 days, respectively. The fertile period of the fluke was also the longest in the DDY strain, 164 days on average, followed by GPC 132, BALB/c 97, nude 37, DS 32 and ICR 28 days. The egg-laying capacity of the fluke in DDY and GPC was relatively high and stable compared with the other four strains of mice. It was found that there are intraspecific variations in biologic incubation period, fertile period, and fecundity of C. sinensis. The DDY mouse is likely to be the most suitable experimental animal among the six strains of the mice tested.

PCR-analyzed microsatellites of the mouse genome--additional polymorphisms among ten inbred mouse strains.

Eighty sequences from the mouse genome database containing microsatellites (simple sequence repeats) have been analyzed for size variation among ten different inbred strains of mice; 62/80 (77.5%) showed polymorphism of at least three alleles. We have been able to detect all the polymorphisms by agarose gel electrophoresis, often running the gels for up to 3 h. Between individual pairs of mouse strains to be used in chromosomal mapping studies in our laboratory, 35-60% polymorphism occurred. There are potentially enough microsatellites within the mouse and human genome to have a marker at every 1-cM distance. This simple approach will, therefore, continue to be useful in genome mapping studies, leading eventually to high-resolution maps of both the mouse and human genomes; this should allow for physical mapping and cloning of specific genes.

Expression of the Calcium-Binding Proteins MRP8 and MRP14 by Early Infiltrating Cells in Experimental Contact Dermatitis

The immune response to an allergen is not only dependent on the inflammatory stimulus, but also on the genetic disposition of the individual. Important effector cells in the immune response are myelomonocytic cells in their various differentiation stages. We recently described the expression of MRP8 and MRP14, two calcium-binding proteins of the S-100 family, by these cells during inflammatory activation. Here, we investigated whether their expression in murine contact dermatitis is dependent on the stimulus by which dermatitis is elicited, and if it is related to the genetic constitution of different inbred strains of mice. Therefore we performed immunohistochemical studies on the distribution of MRP8- and MRP14-positive cells during experimentally induced allergic (ACD) and irritant contact dermatitis (ICD). Both forms of dermatitis were elicited in BALB/c and C57B1/6 mice. BALB/c mice were found to react with a more intense inflammatory response in both ACD and ICD (high responders) than C57Bl/6 mice (low responders). The expression of MRP8 and MRP14 in both forms of dermatitis correlated with the early influx of macrophages and with the cell density of the infiltrate. Also the percentage of MRP8- and MRP14-positive cells in the infiltrate during ACD or ICD was higher in the more intense inflammatory reaction of BALB/c mice compared to C57Bl/6 mice. We conclude that MRP8 and MRP14 define a differentiation stage of inflammatory macrophages and that their expression correlates with the activity of inflammatory processes.

A purified parasite antigen (p30) mediates CD8+ T cell immunity against fatal Toxoplasma gondii infection in mice.

Induction of protective immunity against acute and chronic toxoplasmosis can be achieved using p30, the major membrane and excreted/secreted protein of Toxoplasma gondii. This protein, when administered to outbred mice in the presence of the saponin Quil A, is able to induce almost 100% protection against acute infection without evidence of intracerebral cyst development. Adoptive transfer of immune splenocytes from immunized inbred A/J mice conferred a significant level (p less than 0.001) of protection against subsequent challenge. Phenotypic analysis in outbred as well as two different strains of inbred mice (A/J and C57BL/6) demonstrated that CD8+ T cells are selectively stimulated by this immunization protocol. T cell depletion studies using specific mAb directed at either CD3+ or CD8+ T cell phenotype, followed by adoptive transfer, failed to confer protective immunity, whereas CD4+ depletion had no effect. These cytotoxic CD8+ T cells produced high titers of both IFN-gamma and IL-2. Moreover, these CD8+ T cells were directly parasiticidal against radiolabeled extracellular T. gondii, further supporting the critical immune function of these p30 Ag-specific CD8+ T cells in host immunity against T. gondii infection.

Comparisons between patterns of convulsions induced by two β-carbolines in 10 inbred strains of mice

The β-carbolines, methyl-β-carboline-3-carboxylate (β-CCM) and 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) are known to have pharmacological properties opposite to those of agonistic benzodiazepines. Convulsions induced by these drugs lead to differential patterns, such as clonus, myoclonic or tonic seizures. In 10 different inbred strains of mice we investigated whether the responsiveness to the two drugs was the same, irrespective of the pattern of convulsions. We found the same ranking in the responsiveness of the strains to both drugs in the case of myoclonic seizures. No such correlation could be found for clonus or tonic seizures. Our conclusion is that the same genetic factors determine myoclonic seizures, whereas a plurality of mechanisms underly the other patterns. Thus, myoclonic seizures seem to be the most appropriate index for evaluating the convulsant action of β-carbolines in genetic experiments.