Ultraviolet radiation--induced chromosomal abnormalities in fetal fibroblasts from New Zealand black mice.

Fibroblasts from New Zealand Black mouse fetuses manifest increased frequency of chromosomal breaks and interchanges after exposure to ultraviolet radiation when compared with cells from BABL/c fetuses. This chromosomal instability is similar to what has been reported in cells from patients with xeroderma pigmentosum and may be related to the chromosomally abnormal clones and malignancy previously reported in adult New Zealand Black mice.

Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid prevents proteinuria and prolongs survival in NZB x NZW F1 mice.

Prostaglandins and related compounds are active mediators of inflammation, but data concerning their role in the pathogenesis of the glomerulonephritis of New Zealand Black x New Zealand White (NZB x NZW) F1 mice are conflicting. Dietary eicosapentaenoic acid (EPA, C20:5), a fatty acid analogue of arachidonic acid (C20:4), has been shown to impair platelet aggregation in humans, apparently through inhibition of the synthesis of prostaglandins and thromboxanes from arachidonic acid. We report here the effects of a diet high in EPA on the development of renal disease and survival in female NZB x NZW F1 mice. Animals from 4--5 wk of age were fed diets containing 25% lipid, supplied either as beef tallow or menhaden oil, with fatty acid analysis of less than 0.05 and 14.4% EPA, respectively. In the first experiment, by 13.5 mo of age, mice on the beef tallow diet had all (9/9) developed proteinuria and the majority (6/9) had died, with renal histologic examination revealing severe glomerulonephritis. In contrast, none of 10 menhaden oil-fed animals had developed proteinuria, and all were alive at this time (P less than 0.005 for both proteinuria and survival). In a second experiment using 50 mice in each dietary group, 56% of the beef tallow group vs. none of the menhaden oil group had developed proteinuria at 9 mo of age (P less than 0.005). Native DNA binding at 6 mo of age was 23.9 +/- 14.7 vs. 10.1 +/- 9.7% in the beef and menhaden oil groups, respectively (P less than 0.01). Weights were similar in all groups, and there was no evidence of essential fatty acid deficiency in any group. These results demonstrate that a diet high in EPA protects NZB x NZW F1 mice from the development of glomerulonephritis.

The NZB mouse as a model for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and the only leukemia for which a possible genetic component has been described. Analysis of this genetic component has been hindered by the fact that disease onset normally occurs after age 50. We report here the aged NZB mouse as an animal model for CLL. NZB mice have a genetically regulated, age-dependent onset of clonal, aneuploid cells which are IgM+ and Ly1+ (CD5+ B-cells). Peripheral blood smears from old NZB mice show an increase in circulating lymphocytes and "smudged" or ruptured cells, often seen in human CLL. Electron microscopic examination of these cells shows them to be mature lymphocytes. Light microscopy of the spleen shows infiltration of small lymphocytes and is consistent with CLL pathology. These long-lived, CLL-like cells can be serially passaged into recipient animals. This continued passage occasionally results in the development of a large cell lymphoma detectable in the spleen, lymph nodes, and liver. The histology of this lymphoma is quite distinct from that of the CLL-like cells, but the phenotype is that of an aneuploid CD5+, IgM+ cells. This apparently represents a continued transformation of the CLL-like clone similar to the development of Richter's syndrome in human CLL. Therefore, the NZB mouse can be a valuable tool for the determination of the genetic basis of CLL ontogeny and the conversion of CLL into Richter's syndrome.

Increased vascular permeability of Brucella abortus bacilli in the thymus of NZB/W F1 mice.

Various amounts of the bacterium, Brucella abortus (BA) were injected intravenously into autoimmune NZB/W F1 mice and non-autoimmune BDF1 mice and then the localization of BA in the thymus was traced using an immunohistochemical method at 30 min and 3 h after injection. The results showed that a greater amount of BA became consistently localized in the thymic parenchyma in a free form or in a phagocytized form in NZB/W F1 mice in comparison with BDF1 mice, indicating a marked increase of vascular permeability in the thymus of NZB/W F1 mice. The extravascular leakage of BA was clearly dose-dependent. The significance of invasion of bacterial antigens from the general circulation into the thymic parenchyma is discussed in relation to autoimmune states.

Age-dependent enhancement of diazepam sensitivity is accelerated in New Zealand Black mice.

Separate age groups of C57BL/6 and autoimmune New Zealand Black (NZB) mice were compared for diazepam-induced ataxia and barbiturate-induced loss of righting reflex. Between 1 and 3 months of age, both strains showed a similar age-related decrease in ED50 for diazepam-induced ataxia. However, between 3 and 12 months the decrease in ED50 was markedly greater in NZB mice. In contrast, age-related increases in the durations of loss of righting reflex following hexobarbital or barbital were similar in both strains. The results suggest that NZB mice show relatively accelerated age-related increases in sensitivity to benzodiazepine, but not to barbiturates.

Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice.

In various types of autoimmune disease, an increased frequency of spontaneous chromosome breaks has been reported. Plasma from such patients induces chromosome breaks in normal cells. Exposure of plasma to superoxide radicals increases the breakage activity, and addition of superoxide dismutase protects against it. The New Zealand black mouse is an animal model of autoimmune disease which displays the breakage phenomenon. To test for the possibility that the breakage is related to deficient protection against superoxide radicals, the activities of superoxide dismutase isoenzymes were determined in tissues and blood from New Zealand black mice and compared with the activities of normal BALB/c mice. No differences between the strains were revealed in tissue EC-superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase activity. The erythrocyte superoxide dismutase activities were also equal. The plasma EC-superoxide dismutase activity was 35% lower in the New Zealand black mice than in the BALB/c mice. Between euthymic BALB/c mice and nude mice, previously reported to be deficient in tissue superoxide dismutase activity, no difference could be demonstrated.

[Trial selection of 2 sub-strains of NZB mice based on the level of chromosome aberrations]. / Tentatives de sélection de deux sous-lignées de Souris NZB sur la base du taux de remaniements chromosomiques

Increased chromosomal breakage is observed in NZB mice. Breeding experiments with mice selected according to breakage frequencies provide evidence that the proportion of animals with high and low breakage figures in the first and second generation progeny depends on the phenotype of the parents.

[Occurrence of the heterozygosity at Es-1 locus in a sub-strain of the NZB mice].

In the course of inspection of the biochemical marker genes in inbred strains of mice maintained in our laboratory, a female mouse of the NZB strain was found to be heterozygous for the Es-1 locus. Namely, it was Es-1a/Es-1b type. After this finding, many heterozygous mice were found among her sisters and the descendants. However, these heterozygotes (Es-1a/Es-1b) showed no heterozygosity for other 11 characters, i.e., the 6 biochemical markers (Hbb, Trf, Es-2, Id-1, Mod-1, Gpd-1) and the 5 coat colour markers (A, B, C, D, AND S) were idential as those previously described. It was, moreover, observed that they possessed the immunological characteristics typical of the NZB mice. Therefore, it could be concluded that the heterozygosity had been originated from a single mutation at the Es-1 locus, i.e., from Es-1a to Es-1b or vice versa. With regard to the alleles at the Es-1 locus, an investigation was carried out in two sub-strains of the NZB mice having different breeding history and the followings were clarified. One substrain imported from Karolinska Institute, Sweden, had been fixed with the Es-1a allele and the other imported from England was found to be Es-1b/Es-1b type. The NZB mice which displayed the heterozygosity had been derieved from the Karolinska sub-strain. Importance of biochemical marker genes for inspection of proper maintenance of inbred strains has been discussed.

Intravenous glucose tolerance tests in the New Zealand strains of mice.

Intravenous glucose tolerance tests (I.V.G.T.T.) were carried out in fasting NZO, NZB and NZB/W mice. NZO and NZB mice exhibited an impaired rate of glucose decay, while NZB/W mice cleared glucose very rapidly. The possibility that autoimmune processes are responsible for the glucose intolerance observed in NZO and NZB mice is discussed.

Calories versus protein in onset of renal disease in NZB x NZW mice.

Autoimmunity-prone (NZB x NZW)F1 (B/W) female mice are used as a model of human lupus erythematosus. When full-fed, these mice die of glomerulonephritis between 7 and 11 (average 9) months of age. When food intake is restricted to 60% of calories, the onset of this disease is delayed and the mice live greatly prolonged lives free of disease. Since high protein intake is commonly associated with acceleration of kidney damage in humans and experimental animals, the current experiments were designed to employ diets in which protein concentration was as high as possible. The observations demonstrate clearly that with this model of autoimmune disease, total calorie intake (from whatever source) exerts an overriding influence on life span. A higher calorie intake leads to early death and restricted-calorie intake leads to an increased life span. When B/W mice are full-fed, with respect to calories, feeding diets of greatly differing protein composition did not influence life span significantly. By contrast, calorie restriction of diets, even of very high protein content or of lower protein content, greatly prolonged life of B/W mice. Even with exceedingly high protein intake (greater than 83% of the calories) it is not protein per se but the total calorie intake that exerts the greatest influence that determines length of life in mice of this autoimmunity- and glomerulonephritis-prone strain.

DNA repair, H-2, and aging in NZB and CBA mice.

Current evidence suggests that a correlation exists between the capacity to perform excision repair of UV-induced DNA damage and maximum lifespan in different species. Preliminary evidence has also indicated differences of DNA repair capacities in lymphocytes of several strains of mice congenic at the H-2 locus. It is known that the H-2 system influences maximum lifespan potential in mice. In the present studies excision repair of UV-induced DNA damage, but not gamma-induced damage, was found to correlate the mean survival in the adult inbred mouse strains NZB and CBA, using PHA stimulated splenic lymphocytes. Furthermore, in (NZB X CBA)F2 hybrid with adult progeny the level of DNA repair of UV-induced damage corresponded to the H-2 allele (H-2d/2d from NZB or H-2b/2b from CBA) inherited from the parental strain. These studies suggest the possibility of a tricornered relationship between the main histocompatibility complex, one form of DNA repair, and lifespan within the species.

H-2-linked regulation of serum gp70 production in mice.

By using many congenic strains of C57BL/10 (B10) mice and NZB mice, we have demonstrated a genetic system that controls the production of serum gp70. Our system has been tentatively designated as Sgp-1 and is composed of three phenotypes, Sgp-1a, 1b, and 1c. This system appears to be closely linked to, but not in the H-2 region on chromosome 17. Sgp-1a, which is associated with H-2d, correlates with relatively large amounts of serum gp70 in B10 congenic lines. Sgp-1b, which appeared in most of the H-2 types tested, corresponds with low serum gp70 output in B10 congenic lines and F1 hybrid offspring of B10 and NZB crosses and with increased gp70 production after lipopolysaccharide (LPS) stimulation. Sgp-1c, which is associated with H-2s, also relates to small amounts of serum gp70 in B10 congenic lines and their F1 hybrids from NZB matings, but shows lack of serum gp70 responsiveness to LPS. This failure to accelerate serum gp70 production after injection of LPS is independent of other acute phase responses and polyclonal activation of B cells.

Studies of congenitally immunologically mutant New Zealand mice. IX. Age-related microenvironmental effects on autoantibody production in NZB and NZB.Xid mice studied by transplantation.

The mechanism of polyclonal expansion of B cells and subsequent autoantibody production in New Zealand mice remains a critical question. We have been studying the requirements for autoantibody production both in NZB mice as well as NZB mice congenic with the Xid gene of CBA/N mice. In this study, we have attempted to alter the immunologic phenotype of NZB.Xid mice by transfer of cells from young and old NZB mice. There was little difficulty in restoring normal levels of serum IgM, IgG3, splenic Lyb-5 cells, and response to DNP-Ficoll in young NZB.Xid mice that were injected with young NZB bone marrow cells. Although such animals had an almost immediate change in their immune profile to values characteristic of NZB mice, they required, much like unmanipulated NZB mice, a latency period of an additional 6 mo before autoantibodies were detected. In contrast, adult NZB.Xid mice, who likewise developed an immune profile similar to NZB after transfer of bone marrow cells from young NZB mice, began to express autoantibodies immediately without any latency period. NZB.Xid mice who were recipients of adult NZB bone marrow cells did not show sustained autoantibody production, reflecting the limited state of B cell precursors in adult NZB mice. Thus, the age of both donor cells and the age of recipient mice are critical factors for determining the latency period and the age at which autoantibodies will appear. Similarly we attempted to alter the production of autoantibodies in NZB mice that were irradiated and injected with bone marrow cells from NZB.Xid animals. NZB mice had a major amelioration of disease when they received cell transfers from young NZB.Xid mice. This amelioration, which included the acquisition of the immune profile of NZB.Xid animals, was not seen in adult NZB mice that were recipient of young NZB cells. We suggest that although Lyb-5 cells may be the effective mechanism for autoantibody production, there are other interacting influences that may selectively turn on or turn off autoantibodies and that are required and are responsible for the latency period.

Functional absence of a B cell subpopulation in ageing New Zealand mice.

As has been found for spleen cells from ageing NZB x NZW (B/W) mice, ageing NZB mice were also found to make no antibody when stimulated in vitro with the polyclonal B cell activators (PBA) LPS and PPD. This immune defect was not due to the action of suppressor cells, since old NZB and B/W spleen cells did not suppress the PBA response of young spleen cells. Spleen cells from aged NZB mice were not able to generate antibody-forming cells when stimulated with the thymus-independent antigen, TNP-LPS, but were able to produce antibody in response to another thymus-independent antigen, TNP-AECM-Ficoll, thereby implying that there is a selective functional deletion of a B cell subpopulation in ageing New Zealand mice. The failure of B/W and NZB spleen cells to generate antibody in response to PBA is interpreted as a consequence of a continuing in vivo polyclonal B cell activation accompanying the development of autoimmune disease, leading to a scarcity of B cells available for activation by PBA and by some thymus-independent antigens in vitro.

Sex-dependent systemic lupus erythematosus-like syndrome in (NZB X SJL)F1 mice.

(NZB X SJL)F1 (NS) mice were previously shown to develop sex-dependent thymic abnormalities in the course of aging. The possible occurrence in these mice of various autoimmune manifestations characteristic of a systemic lupus erythematosus (SLE)-like syndrome is investigated. Female NS mice died faster and exhibited antinuclear (AN), anti-ds-DNA antibodies and circulating immune complexes earlier in life and in greater amounts than male NS mice. At 12 months of age immunoglobulin deposits were detected in the renal glomeruli and at the dermo-epidermal junction of the skin. These deposits were more frequent and more intense in females than in males. In addition, proteinuria was found to rise with aging in females but not in males. These data demonstrate that NS mice suffer from SLE symptoms which, like the thymic abnormalities, are influenced by sex-related factors. The study of castrated males and females and of androgen-treated females suggests that androgens exert an inhibitory effect on these SLE symptoms. Preliminary genetic analysis further indicates a probable polygenic control of these SLE symptoms in NS females.

Marked species and age-dependent differences in cell proliferation and neurogenesis in the hippocampus of wild-living rodents.

Variations in the extent of adult neurogenesis and natural and experimental factors controlling it have been described in laboratory animals. The wide range of variation seen even within a species, the mouse, raises the question as to which rates of neurogenesis can be expected in natural populations. Answering this question is important to evaluate the functional significance of adult neurogenesis under natural conditions and to define the factors controlling it. To address this issue, we investigated four species of wild-living rodents and outbred laboratory mice using markers for proliferating cells, Ki-67, and developing neurons, doublecortin and NeuroD. We found about four times as many Ki-67-positive cells per mm3 granule cell layer in two wood mouse species (Muridae; Apodemus spp.) than in bank and pine voles (Arvicolidae; Clethrionomys glareolus and Microtus subterraneus). Laboratory mice show proliferation rates between wood mice and voles. Markers for developing neurons, NeuroD and doublecortin, reflect the findings of proliferation activity. Hippocampal cell proliferation decreases dramatically with age in wild-living species. The onset of the downregulation varies among species. It occurs late in the life span of the yellow-necked wood mouse. In aged animals, the number of proliferating cells per mm3 granule cell layer is reduced to 19% of the adult value. Downregulation occurs early in pine voles, in which cell proliferation in adult animals is reduced to 33% of juvenile values. Proliferation and age-dependent changes along the deep border of the alveus and angular bundle follow those of the dentate gyrus. We conclude that cell proliferation and neurogenesis in the dentate gyrus vary significantly among wild-living rodents, and that they are downregulated with age, but at species-specific time points.