CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1.

Identification and isolation of hematopoietic stem cells (HSCs) in mice is most commonly based on the expression of surface molecules Kit and Sca-1 and the absence of markers of mature lineages. However, Sca-1 is absent or weakly expressed in hematopoietic progenitors in many strains, including nonobese diabetic (NOD), BALB/c, C3H, and CBA mice. In addition, both Kit and Sca-1 levels are modulated following bone marrow injury. In these cases, other markers and dye exclusion methods have been employed to identify HSCs, yet there is no antibody-based stain that enables identification of HSCs and early progenitors when Kit and Sca-1 are inadequate. CD201 is a marker that is highly restricted to HSCs and progenitors, and CD27 is expressed at moderate-to-high levels on HSCs. We show here that combining CD201 and CD27 enables highly efficient isolation of long-term HSCs in NOD mice as well as in other strains, including SJL, FVB, AKR, BALB/c, C3H, and CBA. We also find that HSCs appear to maintain expression of CD201 and CD27 after hematopoietic injury when Kit expression is downregulated. These results suggest a widely applicable yet simple alternative for HSC isolation in settings where Kit and Sca-1 expression are insufficient.

Reduced sulfate plasma concentrations in the BTBR T+tf/J mouse model of autism.

Clinical studies have shown that children diagnosed with autism show abnormal sulfate chemistry, which is critical for cellular and metabolic processes. To determine if the inbred BTBR T+tf/J mouse shows autism-relevant aberrations in sulfate chemistry, the present study examined plasma sulfate concentrations in BTBR T+tf/J, inbred C57BL/6J, and outbred CD-1 mice. Results showed that the BTBR T+tf/J mouse exhibits significantly lower plasma sulfate concentrations in comparison to both C57BL/6J and CD-1 mice. These results suggest that the BTBR mouse shows autism-relevant abnormalities in sulfate chemistry and may serve additional utility in examining the role of sulfate and sulfate-dependent systems in relation to autism-relevant behavioral aberrations.

Unique inbred strain MSM/Ms established from the Japanese wild mouse.

Most laboratory mice belong to a species of house mouse, Mus musculus. So far, at least three subspecies groups have been recognized; domesticus subspecies group (DOM) distributed in western Europe, musculus subspecies group (MUS) distributed in eastern Europe and northeast Asia, and castaneus subspecies group (CAS) found in southwest and southeast Asia including southern China. These subspecies are estimated to have branched off roughly one million years ago. Genetic comparison between subspecies' groups and common inbred strains (CIS) have revealed that the genetic background of CIS is derived mainly from DOM. This shows the importance of non-DOM wild mice as valuable genetic resources. We started to establish our unique strain, MSM/Ms, from MUS in Japan in 1978. In the beginning, we kept wild mice trapped in Mishima in large plastic buckets. In 1979, breeding by sister-brother mating started. The MSM/Ms inbred strain was established in 1986 and 21 years later it reached F(100). During breeding, no significant fluctuations in litter size and sex ratios have been observed. Extensive genetic analyses of chromosome C-banding pattern, biochemical markers and microsatellite DNA (MIT) markers of this strain have demonstrated the characteristics of MUS. A phylogenetic tree constructed from MIT markers has confirmed the MUS nature of MSM strain. Taken together with its genetic remoteness from CIS, MSM appears to maintain many valuable alleles for investigation of biological functions and diseases. Some of these alleles have avoided selection during breeding as either fancy mice or laboratory mice. The MSM-specific genetic traits discovered to date are discussed.

Cytokine profile in collagen-induced arthritis: differences between syngeneic and allogeneic pregnancy.

OBJECTIVE: To identify the differences in cytokine profile between allogeneic and syngeneic pregnancy in mice with collagen-induced arthritis (CIA). METHODS: Mice (strain B10.RIII) were injected with bovine collagen. Females were mated with males of the same strain (syngeneic pregnancy) or with males of strain B10. Q (allogeneic pregnancy). Concentrations of cytokines were measured during pregnancy and after delivery, and the onset and evolution of arthritis was followed in all female animals throughout the study period. RESULTS: In female mice that developed CIA, cytokine concentrations were lower in allogeneic pregnancies than syngeneic pregnancies. When paired cytokine concentrations were compared in each animal during and after pregnancy, MCP-1 was lower during gestation than after delivery in both groups of pregnant mice, IL-6 was lower during gestation than after delivery only in allogeneic pregnancies, and IL-10 was lower during gestation than after delivery in allogeneic pregnancies, whereas in syngeneic pregnancies IL-10 was higher during gestation than after delivery. CONCLUSIONS: Allogeneic pregnancy was associated with less arthritis because of lower concentrations of proinflammatory cytokines (IL-6 and others), not because of an increase in the concentration of antiinflammatory cytokines (IL-10).

Relationships of dietary fat, body composition, and bone mineral density in inbred mouse strain panels.

Laboratory inbred mouse strains show a broad range of variation in phenotypes, such as body composition, bone mineral density (BMD), plasma leptin, and insulin-like growth factor I (IGF-I), and thus provide a basis for the study of associations among them. We analyzed these phenotypes in male and female mice from 43 inbred strains fed on a high-fat (30% caloric content) diet and from 30 inbred strains fed on a low-fat (6%) diet. Structural equation modeling of these data reveals that the relationship of body fat content and areal BMD is altered by dietary factors and genotypes. Sex has no net effect on areal BMD, but after accounting for body mass difference females have higher areal BMD. Leptin is affected by relative fat mass and has no net effect on areal BMD. IGF-I has a direct effect on areal BMD.

Quantitative trait locus analysis of serum insulin, triglyceride, total cholesterol and phospholipid levels in the (SM/J x A/J)F2 mice.

Quantitative trait locus (QTL) analysis of serum insulin, triglyceride, total cholesterol and phospholipid levels at 10 weeks of age was performed in 321 F2 offspring from SM/J and A/J mice. Interval mapping revealed a total of 22 suggestive QTLs affecting the four traits: two insulin QTLs on Chromosomes (Chrs) 6 and 8; six triglyceride QTLs on Chrs 4, 8, 9, 11, 12 and 19; six total-cholesterol QTLs on Chrs 1, 3, 4, 14, 17 and 19; and eight phospholipid QTLs on Chrs 2, 3, 4, 6, 8, 10 and 19. Gender influenced the expression of eight of the suggestive QTLs. The total-cholesterol QTLs on Chrs 4, 14 and 17, the triglyceride QTL on Chr 9 and the phospholipid QTL on Chr 4 were specific to females. The phospholipid QTLs on Chrs 2 and 6 and the insulin QTL on Chr 8 were specific to males. In addition, common QTLs involved in the regulation of some of the traits were identified. The female-specific QTL on Chr 4 appeared to be involved in the regulation of total cholesterol and phospholipid levels. The QTL on Chr 8 affected insulin and phospholipid levels, whereas the Chr 19 QTL was common to the three lipid parameters.

Quantitative trait loci regulating relative lymphocyte proportions in mouse peripheral blood.

Relative proportions of peripheral blood (PB) B lymphocytes (B220%) as well as CD4 (CD4%) and CD8 (CD8%) T lymphocytes differ significantly among inbred mouse strains: B220% is high in C57BL/6J (B6) and C57BR/cdJ, intermediate in BALB/cByJ (BALB) and DBA/2J (D2), and low in NOD/LtJ (NOD) and SJL/J (SJL) mice, whereas CD4% and CD8% are high in NOD and SJL mice and low in the other 4 strains. By following segregating genetic markers linked to these traits in (B6 x D2) recombinant inbred (BXD RI) mice, the study defined 2 quantitative trait loci (QTLs) for the B220% phenotype: Pbbcp1 (peripheral blood B cell percentage 1, logarithm of odds [LOD] 4.1, P <.000 01) and Pbbcp2 (LOD 3.7, P <.000 04) on chromosome 1 (Chr 1) at about 63 cM and 48 cM; one suggestive locus for the CD4% phenotype (LOD 2.6, P <.000 57) on Chr 8 at about 73 cM; and one QTL for the CD8% phenotype: Pbctlp1 (peripheral blood cytotoxic T lymphocyte percentage 1, LOD 3.8, P <.000 02) on Chr 19 at about 12 cM. The study further segregated PB lymphocyte proportions in B6SJLF2 mice by using DNA markers adjacent to these mapped QTLs and found that the Pbbcp1 locus (LOD 5.6, P <.000 01) was also important in this mouse population. In both BXD RI and B6SJLF2 mice, QTLs regulating B-cell proportions showed no significant effect on T-cell proportions and vice versa. Thus, PB B- and T-lymphocyte proportions are regulated separately by different genetic elements.

Distribution of body weight, blood insulin and lipid levels in the SMXA recombinant inbred strains and the QTL analysis.

In the SMXA recombinant inbred (RI) strains, we measured body weight, blood insulin and lipid (triglyceride, total cholesterol and phospholipid) levels in each strain. In the five traits, mean values of substrains varied remarkably and showed a continuous spectrum of distribution, suggesting control by multiple genes at distinct loci for each trait. We also screened for quantitative trait loci (QTLs) involved in the five traits. Suggestive QTLs for body weight (Chromosomes 1 and 6), insulin (Chromosomes 1, 3, 10 and 17), triglyceride (Chromosomes 4 and 11) and phospholipid (Chromosome 18) levels were detected. The SMXA RI strains are unique tools for analyzing genetic factors that influence body weight, blood insulin and lipids levels.

Micronucleated erythrocyte frequency in peripheral blood of B6C3F(1) mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.

The mouse peripheral blood micronucleus (MN) test was performed on samples collected from 20 short-term, 67 subchronic, and 5 chronic toxicity and carcinogenicity studies conducted by the National Toxicology Program (NTP). Data are presented for studies not previously published. Aspects of protocol that distinguish this test from conventional short-term bone marrow MN tests are duration of exposure, and absence of repeat tests and concurrent positive controls. Furthermore, in contrast to short-term bone marrow MN tests where scoring is limited to polychromatic erythrocytes (PCE), longer term studies using peripheral blood may evaluate MN in both, or either, the normochromatic (NCE) or PCE populations. The incidence of MN-PCE provides an index of damage induced within 72 hr of sampling, whereas the incidence of MN in the NCE population at steady state provides an index of average damage during the 30-day period preceding sampling. The mouse peripheral blood MN test has been proposed as a useful adjunct to rodent toxicity tests and has been effectively incorporated as a routine part of overall toxicity testing by the NTP. Data derived from peripheral blood MN analyses of dosed animals provide a useful indication of the in vivo potential for induced genetic damage and supply an important piece of evidence to be considered in the overall assessment of toxicity and health risk of a particular chemical. Although results indicate that the test has low sensitivity for prediction of carcinogenicity, a convincingly positive result in this assay appears to be highly predictive of rodent carcinogenicity.

Distinct immunomodulation by autoimmunogenic xenobiotics in susceptible and resistant mice.

HgCl(2) and diphenylhydantoin (DPH) are prototype chemicals associated with diverse (auto)immune effects in genetically susceptible individuals. Both chemicals activate T cells, and the balance of Th1 versus Th2 activation may influence the clinical outcome of exposure. It is unknown which chemically created neoantigens are responsible for Th activation. We therefore investigated the effect of DPH and HgCl(2) on specific responses to TNP-ovalbumin, in mouse strains with varying sensitivity for the adverse effects. HgCl(2) was found to enhance Th2-driven antibody responses in susceptible B10.s, but protective type 1 responses in resistant B10.d2 mice. This was chemical-specific, as DPH enhanced type 2 responses in both strains. DBA/2 mice were relatively unresponsive to HgCl(2), whereas DPH stimulated type 1 responses in these mice. Interestingly, prior exposure to HgCl(2), but not DPH, facilitated IC deposition in B10.s mice only. Thus, we demonstrate that, depending on MHC-II and background genes, HgCl(2) and DPH preferentially adjuvate type 1 or type 2 responses. In case of HgCl(2), the type of response corresponds with susceptibility to antibody-mediated autoimmunity induced by this chemical. In addition, we demonstrate that, within one strain, different autoimmunogenic chemicals can enhance distinct responses to the same antigen.

The FLS mouse: a new inbred strain with spontaneous fatty liver.

BACKGROUND AND PURPOSE: A new strain of mouse, named FLS (fatty liver Shionogi), which develops spontaneous fatty liver without obesity, was established by inbreeding. Morphologic, physiologic, and genetic characterization of the strain was done. METHODS: Characteristics of male FLS mice were compared with those of the sister strain, dd Shionogi (DS), which does not develop spontaneous fatty liver. A genetic cross experiment was performed by mating FLS with C3H/He/Shi mice. RESULTS: The hepatocytes of neonatal FLS mice contained fine lipid droplets throughout the lobules, and large lipid droplets appeared as mice aged. Liver triglyceride concentrations of FLS mice were fivefold higher than those of DS mice, but serum lipid concentrations and the lipoprotein profile did not indicate abnormalities. Higher plasma aspartate transaminase and alanine transaminase activities in FLS, compared with DS mice, suggested hepatocellular lesions. The genetic cross experiment suggested that the fatty liver formation is a complex polygenic trait. CONCLUSION: The FLS mice develop a progressive hepatic steatosis without obesity and diabetes. The FLS mouse might be a good model for investigating hepatic disorders accompanied by fatty liver unrelated to alcoholism or obesity.

A twenty strain survey and backcross localization of the erythrocytic GTP concentration determining locus Gtpc on mouse chromosome 9.

Erythrocyte nucleotide concentrations were surveyed among 20 inbred strains of mice in order to further assess the variability in GTP concentration. There was no significant difference in erythrocytic ATP concentration (Scheffé's test at P = 0.01), 678-1154 nmol/mL packed cells, among the strains surveyed. Two groups were distinguishable with respect to erythrocytic GTP concentration, 8 strains having high GTP, 215 +/- 44 nmole/mL packed cells, and 12 strains having low GTP, 34 +/- 12 nmole/mL packed cells. The erythrocytic GTP concentration determining trait Gtpc was previously shown to be linked to transferrin, Trf, on chromosome 9. Analysis of 232 [(B6 x WB) F1 x B6] backcross individuals for Gtpc and 8 microsatellite markers restricted the localization of Gtpc to a 5.6 +/- 2.1 cM region. The gene order and genetic distances in cM +/- SE are: (D9Mit14) 0.4 +/- 0.4 (D9Mit24) 1.7 +/- 0.8 (Gtpc, D9Mit51, D9Mit116, D9Mit212) 3.9 +/- 1.3 (D9Mit200) 3.0 +/- 1.1 (D9Mit20) 7.8 +/- 1.8 (D9Mit18). The GTP concentration determining trait appears to be a property of erythrocytes as no differences were observed for GTP/ATP ratios of brain, kidney, liver, and tongue from a low GTP strain, C3H/HeHa x Pgk-la and a high GTP strain, C57BL/6J.

Improved radioimmunoassay for measurement of mouse thyrotropin in serum: strain differences in thyrotropin concentration and thyrotroph sensitivity to thyroid hormone.

We report an improved heterologous radioimmunoassay (RIA) for the measurement of thyrotropin (TSH) in mouse serum. The assay components are: antirat thyrotropin (rTSH) serum from the National Hormone and Pituitary Program, a commercial [125I]-labeled rTSH and mouse thyrotropin (mTSH) serum standards produced by dilution of a serum pool from hypothyroid mice with high TSH with a serum pool from mice treated with excess levothyroxine (LT4) (mTSH-0). Sensitivity was increased by reducing the amount of antibody and tracer and by taking advantage of the disequilibrium technique. Accuracy was greatly improved by the preparation of mouse serum TSH standards. TSH in serial dilutions of individual mice with high TSH of different etiologies paralleled the mTSH standard curve but not that of rTSH or a crude mouse TSH/luteinizing hormone (LH) reference preparation. The high-mTSH-serum standard contained 20 mU TSH per milliliter, measured in a bioassay utilizing a cell line stably transfected with human TSH receptor cDNA, and a relative TSH concentration of 40 ng/mL. The sensitivity of the RIA is 0.01 to 0.02 ng/mL, depending on the quality of the tracer and the preparation of mTSH-0 serum. The intra-assay and interassay coefficients of variations were, respectively: 16% and 27% at 0.04 ng/mL; 6.3% and 8.2% at 0.4 ng/mL; 5.4% and 9.8% at 1.7 ng/mL; 10% and 24% at 4.0 ng/mL. The mean TSH concentration in serum of 60-80-day-old male mice was four-fold higher than that in females of the same age. The assay was able to distinguish differences in serum TSH concentrations in five different strains of mice. Baseline serum TSH concentrations (mean +/- SD) of 70-day-old male mice were: 0.143 +/- 0.065 ng/mL in the CD-1 strain; 0.229 +/- 0.042 ng/mL in C57BL/6 mice; 0.084 +/- 0.017 ng/mL in SWR/J mice; 0.133 +/- 0.057 ng/mL in NOD SCID mice, and 0.266 +/- 0.122 ng/mL in FVB mice. Mean serum thyroxine (T4) concentrations were also significantly different among the mouse strains but did not correlate with the serum TSH level. Administration of levotriiodothyronine (LT3) suppressed the serum TSH to a greater degree in mice with higher baseline TSH values. Suppression of the thyroidal radioiodide uptake with LT3 correlated with that of serum TSH.

Unexpected association between reproductive longevity and blood magnesium levels in a new model of selected mouse strains.

Two recently described mouse strains, with high (MGH) and low (MGL) blood magnesium (Mg) levels were obtained by selection over 19 generations. Both strains exhibit strong differences for characteristics generally known to be related to blood Mg levels, such as increased stress sensitivity and stress-induced aggressivity in MGL mice. In contrast, while experimental Mg deficiency due to low oral Mg intake has been shown to shorten life span and lower reproductive ability, reproductive longevity was longer in the MGL than in the MGH strain. Interestingly, the life spans of the two strains are very similar. Although this character could have been fixed in the strains by chance, with no relationship to the blood Mg level, the possibility of a causal link with the selection cannot be ruled out and is discussed. Regardless of the mechanisms at stake, the MGH and MGL strains appear to constitute a new model for the study of the relationships between reproductive longevity and blood Mg levels.

Influence of a psychogenic and a neurogenic stressor on several indices of immune functioning in different strains of mice.

It is demonstrated that cell proliferation in response to mitogens, natural killer cell (NK) activity, and macrophage functioning of mice may be influenced by either a neurogenic stressor (footshock) or a psychogenic stressor (exposing the mouse to a predator, namely a rat). The nature and magnitude of the immune changes, however, varied across three strains of mice (BALB/cByJ, C57BL/6ByJ, and CD-1), differing in reactivity to stressors and also as a function of the type of stressor employed. While footshock reduced mitogen-stimulated B-cell proliferation in BALB/cByJ mice, it had the opposite effect in the CD-1 strain. Exposure to the predator, however, had little effect in any of the strains. Macrophage activity and NK cytotoxicity were reduced in response to both stressors in a strain-dependent fashion. Plasma corticosterone in response to footshock was greater in BALB/cByJ than in C57BL/6ByJ mice; however, the strain difference was not evident in response to the psychogenic stressor. It is suggested that analyses of stressor effects on immune functioning need to consider the specific strain/species employed, the particular immune parameters being examined, and the nature of the stressor employed.

Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.

Atopic dermatitis (AD) usually develops in patients with an individual or family history of allergic diseases, and is characterized by chronic relapsing inflammation seen especially in childhood, association with IgE hyperproduction and precipitation by environmental factors. However, the exact etiology of AD has been unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is required. We found that skin lesions, which were clinically and histologically very similar to human AD, spontaneously appeared on the face, neck, ears and dorsal skin of inbred NC/Nga mice when they were raised in non-sterile (conventional) circumstances, but not under specific pathogen-free conditions. Plasma levels of total IgE in conventional NC/Nga mice were markedly elevated from 8 weeks of age, correlating with clinical skin severity of dermatitis. Immunohistochemical examination of the skin lesion showed increased numbers of mast cells and CD4+ T cells containing IL-4 necessary for IgE synthesis. Thus, NC/Nga mice suffered from dermatitis very similar to human AD with IgE hyperproduction, which may be triggered by some environmental factor(s).

[The level of sex steroids in the blood of inbred-strain mice during a normal pregnancy and after zygote retransplantation]. / Uroven' polovykh steroidov v krovi myshei inbrednykh linii vo vremia normal'noi beremennosti i posle retransplantatsii zigot.

The effect of genotypes on the blood content of progesterone and estradiol was studied in female CBA, C57BL and BALB mice. We also studied progesterone content at the early stages of pregnancy after the effect of stress factors, such as narcosis and surgery, involved in the retransplantation of the embryo from the donor to the recipient. The levels of sex hormones increased during pregnancy with two peaks on the 6-8th and 14-18th days. The pattern of changes in the level of both hormones depends on the female genotype: at some stages, reliable interstrain changes in the blood content of both progesterone and estradiol were found. At the same time, narcosis and bilateral laparotomy did not affect the blood level of progesterone within 12 h after mating, despite marked activation of the adrenocortical system. The data obtained should be taken into account when selecting mother-fetus pairs for retransplantation of embryos in experimental and farm animals.

Development of congenic strains of mice carrying amyloidogenic apolipoprotein A-II (Apoa2c). Apoa2c reduces the plasma level and the size of high density lipoprotein.

A congenic strain of mice with amyloidogenic apolipoprotein A-II (Apoa2c) on the genetic background of the amyloidosis-resistant SAM-R/1 strain was produced by 12 generations of backcrossing. Genome mapping using endogenous murine leukemia proviral markers was done in the congenic strain, termed R1.P1-Apoa2c. We confirmed that only a small region surrounding the apoA-II gene on chromosome 1 was transferred from the genome of the donor SAM-P/1 strain. The level and particle size of plasma high density lipoprotein were decreased in R1.P1-Apoa2c mice compared to those in the progenitor SAM-R/1 mice. The function of apoA-II can be studied using this strain of mice.

Elevated gene expression of argininosuccinate synthetase in peripheral lymphocytes from systemic lupus erythematosus (SLE) patients.

Argininosuccinate synthetase (ASS) is a rate-limiting enzyme of urea cycle and functions primarily in the liver, whereas ASS activity is hardly detected in normal lymphocytes. In this study, we examined the level of ASS gene expression in peripheral blood lymphocytes (PBL) from human SLE patients by amplification of reverse-transcribed mRNA using the polymerase chain reaction. We have demonstrated that (a) approximately 40% of SLE patients exhibited 2.5 to 5 times higher expression of ASS gene in PBL than those of healthy PBL and (b) the elevation of ASS gene expression of PBL significantly correlates with the active pathogenesis of SLE patients according to the criteria of Japanese Ministry of Health and Welfare (p < 0.001 by student's two-tailed t-test). Thus, it is suggested that ASS gene expression is a promising marker of hyperactivated lymphocytes uniquely generated in patients with systemic autoimmune disease.