Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20–30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176–185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-γ response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.     

CXCR3基因敲除小鼠慢性乙型肝炎病毒复制模型的建立

目的建立CXCR3基因敲除小鼠慢性乙型肝炎病毒复制模型。方法繁育CXCR3基因敲除小鼠,并抽提组织DNA进行聚合酶链反应及凝胶电泳鉴定基因型。CXCR3基因敲除小鼠纯合子9只与野生型C57BL/6小鼠9只同时高压水注射pAAV/HBV1.2质粒,按既定时间点采血检测HBsAg、HBeAg、HBV DNA,以及取肝组织行免疫组织化学检测HBcAg表达。结果本实验室繁殖的CXCR3基因敲除小鼠均为纯合子基因型。在pAAV/HBV1.2质粒转染后第1天,CXCR3基因敲除小鼠与野生型C57BL/6小鼠血清HBsAg水平分别为1134.69±244.42和1759.63±881.20(P=0.096);第4天分别为5305.29±1395.06和7493.29±658.63(P=0.003);第15天分别为1615.04±1187.16和1536.19±1046.02(P=0.905);第40天为229.45±79.27和228.19±295.02(P=0.996);第1天血清HBeAg水平分别为6.65±1.50和20.61±4.03(P=0.000);第4天为6.33±1.61和9.79±2.31(P=0.007);第15天为3.52±1.97和2.85±0.74(P=0.425);第40天为1.28±0.06和1.90±1.01(P=0.431);第10天血清HBVDNA水平分别为4.38±0.22 lgcopies/ml和6.56±0.16lgcopies/ml(P=0.008);第15天为4.41±0.88lgcopies/ml和5.69±0.04lgcopies/m(l P=0.177);第25天为4.48±0.04lgcopies/ml和6.44±0.16lgcopies/ml(P=0.004);第40天为3.66±0.45lgcopies/ml和5.20±0.28lgcopies/ml(P=0.055);两组血清HBV DNA持续存在,在转染后第40天仍为阳性。肝组织HBcAg持续阳性,在转染后第4天、15天和40天均呈高表达,但CXCR3基因敲除小鼠肝组织HBcAg表达较低。结论我们成功建立了CXCR3基因敲除小鼠慢性HBV复制模型,可用于进一步研究CXCR3基因及其配体与HBV感染的关系。     

Interstrain differences in susceptibility to non-alcoholic steatohepatitis

Background and Aim: The pathophysiological mechanisms leading to the development of non‐alcoholic steatohepatitis (NASH) remain unclear. There are differences in the susceptibility to NASH between the different species and sexes. The investigation of the precise mechanism of interstrain differences may provide new means by which the pathophysiological mechanisms of NASH may be understood. Methods: C57BL/6N and C3H/HeN mice were administered a methionine‐ and choline‐deficient (MCD) diet to establish a dietary model of NASH. Results: An elevation of the serum alanine aminotransferase and increased infiltration of inflammatory cells were predominant in C57BL/6N mice at 8 weeks. The increase in the steatosis and lipid contents in the liver was greater in C57BL/6N mice than in C3H/HeN mice. The indices of lipid peroxidation demonstrated by F2‐isoprostanes or 8‐hydroxy‐2′‐deoxyguanosine also increased in the livers of C57BL/6N mice. Furthermore, Sirius red staining revealed an increase in the degree of fibrosis in C57BL/6N mice given the MCD diet. As a result, the C57BL/6N strain had a higher susceptibility to NASH than the C3H/HeN mice. The carnitine palmitoyltransferase 1A (in β‐oxidation) mRNA and mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (in ketogenesis) mRNA were downregulated in the C57BL/6N mice in comparison with C3H/HeN mice. There were no differences in the expression of microsomal triglyceride transfer protein or sterol regulatory element binding protein 1 between the C57BL/6N and C3H/HeN mice. Conclusion: There were interstrain differences in susceptibility to NASH observed in a rodent dietary model. Further evaluations of the precise molecular mechanism of this interstrain difference may provide some indications of the pathophysiological mechanisms of NASH in humans.     

Organ-specific and systemic autoimmune diseases originate from defects in hematopoietic stem cells.

Transplantation of bone marrow cells from nonobese diabetic (NOD) mice, a model for type 1 diabetes mellitus, to C3H/HeN mice, which express I-E alpha molecules and have aspartic acid at residue 57 of the I-A beta chain, induced insulitis followed by overt diabetes in the recipient C3H/HeN mice more than 40 weeks after bone marrow transplantation. When cyclosporin A, which perturbs T-cell functions, was injected intraperitoneally into [NOD----C3H/HeN] chimeric mice daily for 1 month, the chimeric mice developed insulitis and overt diabetes within 20 weeks following bone marrow transplantation. Transplantation of bone marrow cells from (NZW x BXSB)F1 mice, which develop lupus nephritis, myocardial infarction, and idiopathic thrombocytopenic purpura, into C3H/HeN or C57BL/6J mice induced in the recipient strains both lupus nephritis and idiopathic thrombocytopenic purpura more than 3 months after transplantation. Transplantation of a stem-cell-enriched population from (NZW x BXSB)F1 mice into normal mice also induced autoimmune disease in the recipients. These results indicate that both systemic autoimmune disease and organ-specific autoimmune disease originate from defects that reside within the stem cells; the thymus and environmental factors such as sex hormones appear to act only as accelerating factors.     

Differential susceptibility of inbred mouse strains to Helicobacter pylori infection

BACKGROUND: Host factors play an important part in the pathogenesis of Helicobacter pylori-associated disease. The aim of this study was to screen various inbred strains of mice for genetic differences in susceptibility to H. pylori infection. METHODS: Mice of strains BALB/cJ, C.B-17-Prkdc(scid), C3H/HeJ, C3H/HeN, C57BL/6J, C57BL/6J-I110(tm/Cgn), DBA/2J, and FVB/N were inoculated intragastrically with H. pylori SS1. At 1, 4 and 6 months after inoculation, mice were necropsied, and bacterial cultures and histologic studies of the stomachs were performed. RESULTS: Significant differences in the level of colonization by H. pylori were observed among inbred strains at each time of infection. These differences were most distinct at 4 months after inoculation with highest levels in strains C3H/HeJ and C3H/HeN and lowest in strains FVB/N, C57BL/6J and C57BL/6J-I110(tm/Cgn). Infected mice revealed a mild increase in inflammatory cells compared with controls at 1 and 4 months, but not at 6 months after inoculation. The host strain effect on gastric disease was fairly mild, with two exceptions. Firstly, infected I110(tm/Cgn) mice developed a severe, hyperplastic gastritis, indicating that interleukin-10 is an important regulator of the inflammatory response to H. pylori. Secondly, infected C3H/HeN mice had a propensity to develop lymphoid aggregates in the gastric mucosa. CONCLUSIONS: The strain differences described here will be useful for the design of genetic mapping studies in mice to elucidate the genes controlling gastric infection by H. pylori. Our results further show that genetically altered mice are a valuable tool for identifying candidate genes possibly contributing to susceptibility to H. pylori infection.     

Rickettsial infection in murine models activates an early anti-rickettsial effect mediated by NK cells and associated with production of gamma interferon

Natural killer (NK) cell activity was significantly increased on days 2-6 of infection in the Rickettsia conorii-infected C3H/HeN mice and on day 2 in the Rickettsia typhi-infected C57BL/6 mice. Depletion of NK cell activity utilizing anti-NK1.1 monoclonal antibody enhanced the susceptibility of normally resistant C57BL/6 mice to infection with R. typhi, and depletion of NK cell activity with antibody to asialo GM1 enhanced the susceptibility of C3H/HeN mice to infection with R. conorii. Serum gamma interferon was increased in R. conorii-infected C3H/HeN mice compared with NK cell-depleted, infected mice during the early course of infection. Additionally, the NK cell activating cytokine IL-12 was elevated in the sera of infected mice during the time period representing enhanced NK cell activity compared with uninfected mice. Thus, it appears that NK cells contribute to the early anti-rickettsial immune response, likely via a mechanism involving gamma interferon.     

水动力注射法构建乙型肝炎病毒感染小鼠模型

目的建立尽量接近人类自然条件下发生的急性病毒性肝炎感染动物模型,研究其发病机制和治疗方法。方法选择中国地区高流行的人HBV C基因型具有复制能力的1.2倍HBV基因组为材料,构建可高效表达HBV基因组的重组腺病毒相关病毒载体pZAC2.1-HBV。利用尾静脉水动力注射法将pZAC2.1-HBV注入小鼠。结果瞬间转染人肝癌细胞系HepG2细胞后,在细胞上清和裂解液中检测到HBsAg。ELISA方法检测小鼠血清中HBsAg和HBeAg的水平可持续表达14天,随后抗-HBs、抗-HBe出现。同时在14天之内可检测到血清中HBV-DNA特异片段。结论采用水动力注射法成功建立HBV感染急性小鼠模型。     

Disaccharidase activity in the small intestine of susceptible and resistant mice after primary and challenge infections with Giardia muris.

The activities of four disaccharidases were examined in resistant (C57Bl/6) and susceptible (C3H/HeN) mice during the primary infection with Giardia muris and after challenge with either trophozoite extract or cysts. Significant decreases in lactase, sucrase, trehalase, and maltase activities in C57Bl/6 mice and lactase and sucrase activities in C3H/HeN mice in the anterior 25% of the small intestine were observed on day 10 after infection. The activities of maltase, sucrase, trehalase, and lactase in the jejunum of C3H/HeN mice were significantly reduced after challenge with trophozoite extract, when compared with the uninfected or infected, but not challenged animals. Decreases in enzyme activities of C3H/HeN mice were evident as early as 12 hours after challenge with the extract. The resistant C57Bl/6 mice showed little change in disaccharidase activity after challenge with trophozoite extract. On the other hand, challenge with cysts resulted in a few decreases in disaccharidase activities in both strains of mice: C57Bl/6 mice showed decreases in the duodenum, while disaccharidases of C3H/HeN mice had lower activity more posteriorly. Thus, challenge with parasite antigen results in a more severe disaccharidase deficiency in susceptible hosts when compared with resistant ones.     

The granulopoietic effect of human urinary colony stimulating factor on normal and cyclophosphamide treated mice

A practically endotoxin-free colony stimulating factor from human urine (CSFHU) was prepared and its granulopoietic effect on normal and cyclophosphamide treated mice was examined. When normal C57BL/6N mice were injected intraperitoneally with 2.5 X 10(6) units/kg of the CSFHU daily for a 5-day period, the numbers of progenitor cells (CFUC) in the femur and spleen were significantly increased. The CFUC in the femur and spleen reached a maximum at day 3 (270%) and day 5 (250%) after the initial injection, respectively. The increase in number of CFUC in the femur exhibited a dose-dependency with respect to the CSFHU and a significant increase was observed even at 4 X 10(5) U/kg (P less than 0.05). However, neither granulocytosis nor monocytosis occurred in normal C57BL/6N mice injected with the CSFHU. In cyclophosphamide induced leukopenic C3H/HeN mice, daily injections of the CSFHU at 2.5 X 10(6) U/kg for 5 days stimulated the restorative granulocyte production (P less than 0.05) as well as the CFUC recovery in both the femur and spleen. These findings suggested that the CSFHU might be involved in granulocyte production in vivo.     

Aged mice exhibit greater mortality concomitant to increased brain and plasma TNF-alpha levels following intracerebroventricular injection of lipopolysaccharide

BACKGROUND: Age-related defects in the development of peripheral inflammatory responses have been observed in rodents and humans. OBJECTIVE: We examined the effects of age on a centrally injected endotoxin-induced cytokine production and cellular activation in mice. METHODS: Male C57BL/6J (B6) mice, C3H/HeN mice, and C3H/HeJ mice received an intracerebroventricular injection of lipopolysaccharide (LPS) and were sacrificed at various times (2, 4, 8 h) thereafter. ELISA for IL-1beta, IL-6, IL-12, and TNF-alpha were conducted on forebrain tissue homogenates as well as plasma samples, and lectin staining to detect activated microglia was prepared for selected brain slices. RESULTS: Intracerebroventricular injection of LPS in B6 mice produced an age-associated increase in mortality which was paralleled with a significant increase in brain and plasma levels of TNF-alpha. AntiTNF-alpha- and IL-6-immunoreactive cells possessed macrophagelike morphologies and were observed along the LPS injection tract and scattered throughout the hilus of the dorsal hippocampus and cerebral cortices. This LPS-mediated response was found to be specific in that the LPS-hyporesponsive mouse strain (C3H/HeJ) failed to demonstrate significant brain or plasma levels of TNF-alpha after LPS administration compared to C3H/HeN mice. CONCLUSION: These results suggest that the age-related increases in TNF-alpha production and mortality following the intracerebroventricular administration of LPS may be due to an increased endotoxin hypersensitivity of brain microglia/macrophages within aged animals.     

Murine model of Kawasaki disease induced by mannoprotein-beta-glucan complex, CAWS, obtained from Candida albicans

Intraperitoneal administration of CAWS (water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans) to mice induces coronary arteritis similar to Kawasaki disease. We analyzed differences in the production of cytokines involved in the occurrence of coronary arteritis among mouse strains, C3H/HeN, C57BL/6, DBA/2 and CBA/J. The incidence of arteritis was 100% in C57BL/6, C3H/HeN and DBA/2 mice, but only 10% in CBA/J mice. The coronary arteritis observed in DBA/2 mice was the most serious, with several mice expiring during the observation period. The CAWS-sensitive strains revealed increased levels of IL-6 and IFN-gamma during the course of a specific response to CAWS by spleen cells. In contrast, IL-10 levels were observed to increase markedly in CAWS-resistant CBA/J mice, but not the CAWS-sensitive strains. However, TNF-alpha levels were more elevated only in DBA/2 mice. The difference in disease development and cytokine production strongly suggests that the genetic background of the immune response to CAWS contributes to the occurrence of coronary arteritis.     

Control of hemopoiesis in mice by sensitized L3T4+ Lyt2-lymphocytes during infection with bacillus Calmette-Guérin.

When injected intravenously with bacillus Calmette-Guérin (BCG; 10(7) viable units), C57BL/6 mice rapidly develop a transient anemia associated with an increased number of granulocytes and monocytes, whereas C3H/He mice do not. Because these two features are lacking in C57BL/6 nude mice we postulated that T lymphocytes can regulate hemopoiesis during infection. To assess further the role in hemopoiesis of T lymphocytes present in bone marrow of C57BL/6 and C3H/He mice, the frequency of BCG-specific T lymphocytes and their surface marker phenotype were determined by limiting dilution analysis and use of monoclonal antibodies. The number of BCG-specific T lymphocytes was estimated to be 50- to 100-fold higher in bone marrow of C57BL/6 than in that of C3H/He mice. Although L3T4+ Lyt2-and L3T4- Lyt2+ BCG-specific T lymphocytes were generated in mice of both strains, in C57BL/6 mice L3T4+ cells were induced preferentially from day 1 through day 5 after infection in correlation with hemopoietic changes. The relation between T-cell immune response and hemopoietic changes was substantiated by results obtained after in vivo treatment with monoclonal antibodies. Selective depletion of L3T4+ T cells by in vivo injection of anti-L3T4 monoclonal antibodies (GK 1-5) inhibited the development of the anemia and the related increased production of phagocytes in C57BL/6 mice receiving BCG.     

Successful liver allografts in mice by combination with allogeneic bone marrow transplantation.

Successful liver allografts were established by combination with allogeneic bone marrow transplantation. When liver tissue of BALB/c (H-2d) or C57BL/6J (H-2b) mice was minced and grafted under the kidney capsules of C3H/HeN (H-2k) mice, it was rejected. However, when C3H/HeN mice were irradiated and reconstituted with T-cell-depleted BALB/c or BALB/c nu/nu bone marrow cells, or with fetal liver cells of BALB/c mice, they accepted both donor (stem-cell)-type (BALB/c) and host (thymus)-type (C3H/HeN) liver tissue. Assays for both mixed-lymphocyte reaction and induction of cytotoxic T lymphocytes revealed that the newly developed T cells were tolerant of both donor (stem-cell)-type and host (thymus)-type major histocompatibility complex determinants. We propose that liver allografts combined with bone marrow transplantation should be considered as a viable therapy for patients with liver disease such as liver cirrhosis and hepatoma.     

RNA干扰在动物体内抗乙型肝炎病毒的效果

目的以乙型肝炎病毒(HBV)C区为靶位,动物实验体内观察RNA干扰抗HBV的效果。方法以流体动力学法建立HBV感染的动物模型,将pcDNA 3．1-HBV和体外细胞实验证明有效的小干扰 RNA(siRNA)尾静脉共注射BALB／c小鼠;用时间分辨免疫荧光分析法检测小鼠血清中乙型肝炎表面抗原(HBsAg)水平,用荧光定量聚合酶链反应法检测血清HBV DNA水平,用逆转录聚合酶链反应法检测 HBV C-mRNA,用免疫组织化学法检测肝组织HBsAg和乙型肝炎核心抗原。结果在小鼠体内,siRNA 能有效抑制HBV的复制和表达,干扰效果至少持续3 d。结论靶向HBV C区的siRNA在动物体内能有效抗HBV。     

Effect of atherogenic diet on reverse cholesterol transport in vivo in atherosclerosis susceptible (C57BL/6) and resistant (C3H) mice

Mice susceptible (C57BL/6) or resistant (C3H) to atherosclerosis induced by a high cholesterol-cholate containing diet (A-diet) were used to study reverse cholesterol transport (RCT) in vivo as measured by loss of cholesterol from a depot created by injection of cationized LDL into the rectus femoris muscle. Plasma total and HDL-cholesterol (HDL-C), total and HDL phospholipid (HDL-PL) levels in chow fed C3H male and female mice were higher than in C57BL/6 mice. After one month on A-diet, plasma cholesterol more than doubled in both strains and genders. The decrease in HDL-C and HDL-PL was twice as great in C57BL/6 as in C3H female mice, while in male C3H mice there was no decrease. The loss of exogenous cholesterol mass (ECM) after injection of cationized LDL was more rapid in C3H than in C57BL/6 mice. In chow fed mice, ECM retained in muscle on day 12 was 37% in C57BL/6 and 20% in C3H females; in males it was 39% and 18% in C57BL/6 and C3H, respectively. On A-diet, 76% were retained in C57BL/6 and 28% in C3H females; these values were 59% and 28% in C57BL/6 and C3H males. Thus, the slow clearance of ECM (which represents RCT) in C57BL/6 mice on A-diet, that could be related to a marked decrease of HDL-PL, might contribute towards their susceptibility to atherosclerosis.     

Differences in hepatic fibrosis in ICR, C3H, and C57BL/6 mice infected with Schistosoma mansoni

The collagen content of the liver, measured as hepatic hydroxyproline, was examined for a period of up to 52 weeks following Schistosoma mansoni infection. Hepatic fibrosis was much more marked in S. mansoni-infected mice of an outbred ICR strain than in C57BL/6J mice, while C3H/HeN mice occupied an intermediate position. The marked difference in hepatic fibrosis in ICR and C57BL/6J mice correlated with more rapid in vitro synthesis of collagen by the livers of infected ICR mice. Strains of mice exhibiting high and low levels of fibrosis provide an excellent tool for examining mechanisms of murine schistosomal hepatic fibrosis and its genetic regulation.     

Separation of hematopoietic stem cells into two populations and their characterization.

Two populations of hematopoietic stem cells (HSC) in mouse bone marrow (BM) are defined on the basis of the presence or absence of interleukin-3 (IL-3) receptor-associated antigen (IL-3RAA). HSC were purified by depletion of mature lymphoid-lineage cells followed by collection of the low-density fraction and sorting of wheat germ agglutinin-binding (WGA+) cells using a fluorescein-activated cell sorter. WGA+ cells were further separated into two populations (IL-3RAA+/WGA+ and IL-3RAA-/WGA+) by a monoclonal antibody (MoAb) against IL-3RAA. IL-3RAA+/WGA+ cells formed CFU-S on day 8; this population consisted mainly of cells in the cycling phase. IL-3RAA-/WGA+ cells form CFU-S on day 12; this population consisted mainly of dormant cells (cells in the G0 phase). When two populations obtained from C3H/HeN mice were injected into lethally irradiated (C57BL/6 x C3H/HeN)F1 mice, donor-derived cells in the peripheral blood (PB) appeared significantly earlier in mice injected with IL-3RAA+/WGA+ cells than in those injected with IL-3RAA-/WGA+ cells, whereas the reconstruction efficiency of IL-3RAA-/WGA+ cells had overtaken that of IL-3RAA+/WGA+ cells 6 weeks after injection. Long-term observation showed no significant difference between these two populations, however. The radioprotective ability (RPA) (30-day survival) of these two populations was therefore compared. The RPA of IL-3RAA-/WGA+ cells was significantly higher than that of IL-3RAA+/WGA+ cells. These findings therefore suggest that the former population is more primitive.     

Short-term exposure to nitrogen dioxide enhances susceptibility to murine respiratory mycoplasmosis and decreases intrapulmonary killing of Mycoplasma pulmonis

In C57BL/6N and C3H/HeN mice known to be free of all murine pathogens and matched for age, sex, microbiologic, and environmental factors, exposure to NO2 for 4 h prior to exposure to infectious aerosols of Mycoplasma pulmonis resulted in potentiation of murine respiratory mycoplasmosis (MRM). In the C57BL/6N mice, NO2 increased the incidence of death, incidence of gross lung lesions, and incidence of microscopic lung lesions, but did not increase the incidence of infection in the lungs. Nitrogen dioxide affected the C3H/HeN mice (a strain known to be more susceptible than the C57BL/6N strain to MRM) similarly, with the exception that the incidence of death and microscopic lesions were not affected in this strain at the concentrations of M. pulmonis used. Exposure to the oxidant also increased the severity of microscopic lesions and the numbers of Mycoplasma organisms in the lungs of both mouse strains. Thus, NO2 appeared to affect host lung defense mechanisms responsible for limiting the extent of infection. The NO2 exposure level required to produce potentiation varied with the genetic background of the host, the number of Mycoplasma organisms administered, and the end point measured. In further experiments in C57BL/6N mice, exposure to 5 or 10 ppm of NO2 for 4 h prior to infection with aerosolized, radiolabeled M. pulmonis reduced clearance of these organisms from the lungs over a 72-h time period. Nitrogen dioxide exposure did not change the rate of physical removal of Mycoplasma organisms from the lung. Reduced clearance was due to impaired intrapulmonary killing of Mycoplasma organisms in NO2-exposed mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bipedal ambulation induces experimental scoliosis in C57BL/6J mice with reduced plasma and pineal melatonin levels

In addition to the induction of scoliosis in chickens by pinealectomy (PINX), we previously demonstrated that removal of the pineal gland also produces scoliosis in bipedal rats, which can be inhibited by melatonin treatment. Using C57BL/6J mice with genetically low circulating melatonin levels, the main objective of the present study was to investigate whether bipedal ambulation in C57BL/6J mice has the same effects on spinal deformity as those seen in pinealectomized bipedal rats. The present study consisted of two phases. The aim of the first experiment was to determine whether the C57BL/6J mouse strain actually exhibits depressed plasma concentrations and/or pineal contents of melatonin during both the light and the dark phase of the light:dark cycle. The aims of the second experiment were to evaluate; (i) whether bipedal ambulation alone in the C57BL/6J mouse induces scoliosis, and (ii) whether PINX with bipedal ambulation in another mouse strain, i.e. C3H/HeJ, which normally exhibits diurnal fluctuations in melatonin synthesis and secretion, has effects similar to those of bipedal ambulation alone in C57BL/6J mice. C3H/HeJ mice, serving as controls, showed significant increases in both plasma concentrations and pineal contents of melatonin during the dark phase when compared with the light phase. In contrast, there were no differences in either circulating levels or pineal contents of melatonin between the light and dark phases in C57BL/6J mice. Moreover, plasma melatonin levels were below the detection limit of the assay in both phases and pineal melatonin was < 10% of that in C3H/HeJ mice. Bipedal ambulation for 40 wk in C57BL/6J mice induced scoliosis at a rate of 64.3%, and two of nine scoliotic mice showed two sites of spinal deformity. This type of ambulation in C3H/HeJ mice resulted in scoliosis at a lower rate (25%), and affected animals had only a single scoliotic site. However, PINX combined with bipedal ambulation in C3H/HeJ mice produced scoliosis at a rate (70%) similar to that seen in C57BL/6J mice, and some double deformations were induced. These results confirm our previous observations in rats, and also support our hypothesis that melatonin as well as the bipedal ambulation appear to play a critical pathogenic role in scoliosis in experimental mammals.