Rapidly fatal leishmaniasis in resistant C57BL/6 mice lacking TNF

The resolution of infections with the protozoan parasite Leishmania major in mice requires a Th1 response that is closely associated with the expression of IL-12, IFN-gamma, and inducible NO synthase. Previous Ab neutralization studies or the use of mice deficient for both TNF receptors suggested that TNF plays only a limited role in the control of parasite replication in vivo. In this study we demonstrate that resistant C57BL/6 (B6.WT) mice locally infected with L. major rapidly succumb to progressive visceral leishmaniasis after deletion of the TNF gene by homologous recombination. A reduction of the parasite inoculum to 3000 promastigotes did not prevent the fatal outcome of the disease. An influence of the altered morphology of secondary lymphoid organs in C57BL/6-TNF(-/-) (B6.TNF(-/-)) mice on the course of disease could be excluded by the generation of reciprocal bone marrow chimeras. Although infected B6.TNF(-/-) mice mounted an L. major-specific IFN-gamma response and expressed IL-12, the onset of the immune reaction was delayed. After in vitro stimulation, B6.TNF(-/-) inflammatory macrophages released 10-fold less NO in response to IFN-gamma than B6.WT cells. However, in the presence of a costimulus, e.g., L. major infection or LPS, the production of NO by B6.WT and B6.TNF(-/-) macrophages was comparable. In vivo, inducible NO synthase protein was readily detectable in skin lesions and draining lymph nodes of B6.TNF(-/-) mice, but its expression was more disperse and less focal in the absence of TNF. These are the first data to demonstrate that TNF is essential for the in vivo control of L. major.     

Restraint increases prolactin and REM sleep in C57BL/6J mice but not in BALB/cJ mice

Sleep is generally considered to be a recovery from prior wakefulness. The architecture of sleep not only depends on the duration of wakefulness but also on its quality in terms of specific experiences. In the present experiment, we studied the effects of restraint stress on sleep architecture and sleep electroencephalography (EEG) in different strains of mice (C57BL/6J and BALB/cJ). One objective was to determine if the rapid eye movement (REM) sleep-promoting effects of restraint stress previously reported for rats would also occur in mice. In addition, we examined whether the effects of restraint stress on sleep are different from effects of social defeat stress, which was found to have a non-REM (NREM) sleep-promoting effect. We further measured corticosterone and prolactin levels as possible mediators of restraint stress-induced changes in sleep. Adult male C57BL/6J and BALB/cJ mice were subjected to 1 h of restraint stress in the middle of the light phase. To control for possible effects of sleep loss per se, the animals were also kept awake for 1 h by gentle handling. Restraint stress resulted in a mild increase in NREM sleep compared with baseline, but, overall, this effect was not significantly different from sleep deprivation by gentle handling. In contrast, restraint stress caused a significant increase in REM sleep compared with handling in the C57BL/6J mice but not in BALB/cJ mice. Corticosterone levels were significantly and similarly elevated after restraint in both strains, but prolactin was increased only in the C57BL/6J mice. In conclusion, this study shows that the restraint stress-induced increase in REM sleep in mice is strongly strain dependent. The concomitant increases in prolactin and REM sleep in the C57BL/6J mice, but not in BALB/cJ mice, suggest prolactin may be involved in the mechanism underlying restraint stress-induced REM sleep. Furthermore, this study confirms that different stressors differentially affect NREM and REM sleep. Whereas restraint stress promotes REM sleep in C57BL/6J mice, we previously found that in the same strain, social defeat stress promotes NREM sleep. As such, studying the consequences of specific stressful stimuli may be an important tool to unravel both the mechanism and function of different sleep stages.     

Cisplatin-induced apoptotic cell death in mouse hybrid neurons is blocked by antioxidants through suppression of cisplatin-mediated accumulation of p53 but not of Fas/Fas ligand

Peripheral neuropathy following cisplatin treatment is a major limiting factor in cisplatin chemotherapy of cancer patients. We investigated the pathomechanism underlying cisplatin neuropathy using a mouse dorsal root ganglion neuron-neuroblastoma hybrid cell line (N18D3) developed in our laboratory. DNA fragmentation, a characteristic feature of apoptosis, was induced in hybrid neurons following treatment with cisplatin. Accumulation of p53, Fas, and Fas ligand (Fas-L) was also demonstrated in these neurons. Preincubation with N-acetylcysteine (NAC), a precursor of glutathione, blocked cisplatin-induced apoptosis completely, whereas Trolox, a vitamin E analogue, blocked it partially. Cisplatin-induced p53 accumulation was suppressed by NAC treatment, whereas p53 accumulation was retarded by Trolox treatment. In contrast, neither NAC nor Trolox showed any inhibitory effect on cisplatin-induced Fas/Fas-L accumulation. These results suggest that the neuroprotective effects of antioxidants against cisplatin-induced neurotoxicity in hybrid neurons are mediated mainly through the inhibition of p53 accumulation but not of Fas/Fas-L accumulation by these antioxidants.     

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Background Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial. Methods We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG₁ peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Results Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Conclusions Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.     

T cell epitope-based peptide-DNA dual vaccine induces protective immunity against Schistosoma japonicum infection in C57BL/6J mice

Schistosomiasis is a major public health problem that primarily affects developing countries. Although schistosomicidal drugs exist, the development of an efficacious vaccine would potentially be the most powerful means of controlling this disease. Previous studies have shown that vaccination with selected protective epitopes successfully induced partial protection and/or reduced female fecundity in animal models. Thus, we investigated whether the T cell epitope P5 from the host-interactive tegument of Schistosoma japonicum 22.6 (S. japonicum) could act as a protective epitope. The protective potential of P5 in a vaccine against S. japonicum was determined by using a T cell epitope based peptide-DNA dual vaccine (PDDV). In our experiments, the vaccine construct (P5-18K-PDDV) contains the peptide of the T cell epitope (P5) and plasmid DNA, encoding P5 and adjuvant GM-CSF. We show that P5-18K-PDDV induced both cell-mediated and humoral immune responses in vivo and achieved partial protection against S. japonicum infection in C57BL/6J mice. Histopathological studies reveal that P5-18K-PDDV immunized mice had substantially reduced liver pathology compared to the control groups. Together, these results suggest that P5 could be used as a vaccine immunogen for both worm killing and disease prevention against S. japonicum.     

The superior olivary complex in C57BL/6 mice.

The cellular and cytoarchitectural features of the lateral superior olive, the medial superior olive, the superior paraolivary nucleus and the medial, lateral and ventral nuclei of the trapezoid body are described in C57BL/6 mice using Nissl, Bodian and Golgi techniques. Principal, spindle and marginal cells are present in a well-defined lateral superior olive. The dendrites of these cells run primarily within rostrocaudal sheets as in the cat. The principal cells of the medial nucleus of the trapezoid body are similar to the principal cells in the cat. Large multipolar cells characterize the lateral nucleus of the trapezoid body and bipolar cells with a medial-lateral orientation are found in the medial superior olive. The largest neurons are found in the superior paraolivary nucleus and the lateral superior olive, and the medial and ventral nuclei of the trapezoid body. While brain weight and neuronal packing density change with development, the characteristic location of cell groups and the shape and Nissl-staining pattern of neurons in the youngest brains examined were essentially unchanged in the adult mice, although dendritic maturation had occurred. The homologies of the C57BL/6 superior olivary complex nuclei with the same areas described in other mouse strains, rat and cat are discussed. This study expands our understanding of the organization of the superior olivary complex in an inbred strain of Mus musculus and relates it to other species. The data about changes occurring during postnatal maturation may aid in the interpretation of behavioral and physiological studies of neonatal plasticity of the auditory system.     

Mouse adenovirus type 1 causes a fatal hemorrhagic encephalomyelitis in adult C57BL/6 but not BALB/c mice.

Mouse adenovirus type 1 (MAV-1) produces a lethal disease in newborn or suckling mice characterized by infectious virus and viral lesions in multiple organs. Previous reports of MAV-1 infection of adult mice generally described serologic evidence of infection without morbidity or mortality. However, our current results demonstrate that MAV-1 causes a fatal illness in adult C57BL/6(B6) mice (50% lethal dose, [LD50], 10(3.0) PFU) but not in adult BALB/c mice at all of the doses tested (LD50, > or = 10(5.0) PFU). Adult (BALB/c x B6)F1 mice were intermediately susceptible (LD50, 10(4.5) PFU). Clinically, the sensitive B6 mice showed symptoms of acute central nervous system (CNS) disease, including tremors, seizures, ataxia, and paralysis. Light microscopic examination of CNS tissue from the B6 animals revealed petechial hemorrhages, edema, neovascularization, and mild inflammation in the brain and spinal cord. Analysis by electron microscopy showed evidence of inflammation, such as activated microglia, as well as swollen astrocytic endfeet and perivascular lipid deposition indicative of blood-brain barrier dysfunction. Outside of the CNS, the only significant pathological findings were foci of cytolysis in the splenic white pulp. Assessment of viral replication from multiple tissues was performed by using RNase protection assays with an antisense MAV-1 early region 1a probe. The greatest amounts of viral mRNA in MAV-1-infected B6 animals were located in the brain and spinal cord. Less viral message was detected in the spleen, lungs, and heart. No viral mRNA was detected in BALB/c mouse tissue, with the exception of low levels in the heart. Viral titers of organ tissues were also determined and were concordant with RNase protection findings on the brain and spinal cord but failed to demonstrate significant infectious virus in additional organs. Our experiments demonstrate that MAV-1 has a striking tropism for the CNS that is strain dependent, and this provides an informative in vivo model for the study of adenoviral pathogenesis.     

High-efficiency CAG-FLPe deleter mice in C57BL/6J background.

The increasing popularity of conditional knockout (KO) technology has resulted in the demand for efficient FLP deleter mice. In addition, FLP deleters are needed in genetic backgrounds that are suited to behavioral studies. We generated CAG-FLPe transgenic (Tg) mice with the C57BL/6J genetic background, which is one of the most commonly-used strains in behavioral studies. We assessed the recombination efficiency of the CAG-FLPe-Tg lines by crossing them with a mouse line carrying a FRT-PGK-neo-FRT cassette. Four of five independent CAG-FLPe lines induced recombination in most (91%-100%) of their progenies, although a small fraction (0%-30%, depending on the line) showed mosaic recombination patterns. These animals are highly potent as deleters of FRT cassettes and are useful for behavioral studies involving conditional KO mice.     

Delayed matching-to-position performance in C57BL/6N mice

Delayed matching-to-sample is one of the most frequently employed behavioral tasks for assessing spatial working memory in animals. Although the advantages of the task have been widely acknowledged and it is used in the study of a variety of species, its application to mice has been rare. In the present study, we reported the efficacy of a delayed matching-to-position task in C57BL mice lever-pressing in an operant-conditioning chamber. Each trial started with the press of a back lever, followed by the presentation of either a left or right front lever. When the ratio requirement for presses to the front lever (sample) was met, a delay interval started. Delay interval continued until the mice made the first response after the elapse of the programmed delay interval. This was followed by the presentation of a choice of left or right front levers. The choice of the same front lever as the sample was reinforced, whereas the other was not. The proportion of correct choices showed a delay-dependent decrement. A higher ratio of response requirement to the sample resulted in increased accuracy, but the duration of the intertrial interval had no effect. Preceding trials also influenced response accuracy, indicating proactive interference. Overall, the results replicated the effects of parametric manipulations reported in other species, and thus, our findings validate the efficacy of the task for assessing spatial working memory in laboratory mice.     

Substrains matter in phenotyping of C57BL/6 mice

The inbred mouse strain C57BL/6 has been widely used as a background strain for spontaneous and induced mutations. Developed in the 1930s, the C57BL/6 strain diverged into two major groups in the 1950s, namely, C57BL/6J and C57BL/6N, and more than 20 substrains have been established from them worldwide. We previously reported genetic differences among C57BL/6 substrains in 2009 and 2015. Since then, dozens of reports have been published on phenotypic differences in behavioral, neurological, cardiovascular, and metabolic traits. Substrains need to be chosen according to the purpose of the study because phenotypic differences might affect the experimental results. In this paper, we review recent reports of phenotypic and genetic differences among C57BL/6 substrains, focus our attention on the proper use of C57BL/6 and other inbred strains in the era of genome editing, and provide the life science research community wider knowledge about this subject.     

Leishmania mexicana and Leishmania tropica: Cross immunity in C57BL/6 mice

Leishmania mexicana and Leishmania tropica infection were comparatively studied in C57BL/6 mice. Infection with 10⁴ amastigotes of L. mexicana was followed by the appearance of a single lesion which ulcerated in 8 weeks and healed in 24 weeks. Mice infected with 10⁴ amastigotes of L. tropica developed less severe lesions which healed in 18 weeks. In both cases healing was accompanied by a delayed hypersensitivity response and an in vitro lymphocyte reactivity to leishmanial antigens. Mice recovered from a primary infection with L. mexicana or L. tropica were resistant to both homologous and heterologous challenge. In vitro and in vivo immunological tests indicated that L. mexicana and L. tropica share antigenic determinants which are involved in cell-mediated immune responses to these parasites.     

Age-related fibrillar deposits in brains of C57BL/6 mice

The present article reviews findings regarding the age-related occurrence of clusters of unusual granules in the brains of C57BL/6 (B6) mice and discusses the potential relevance of this phenomenon as a model of specific aspects of brain aging in humans. The granules occur predominatly in the hippocampus of B6 mice and represent aggregations of fibrillar material that are mostly associated with astrocytes. The deposits become evident at about 4 to 6 mo of age, and increase markedly in both number and size thereafter. Similar structures have been observed in adult senescence accelerated mice (SAM) and have been noted, although very rarely, in older mice from other strains. The deposits appear to manifest dominant genetic heritability. Heparan sulfate proteoglycan and laminin or related molecules have been identified as components of the granular material. Although the deposits do not represent senile plaques with β-amyloid deposition, they might mimic the deposition of extracellular matrix molecules that is thought to be an early event in amyloidogenesis in the aged brain and in Alzheimer's disease.     

Aging and chronic estradiol exposure impair estradiol-induced cornification but not proliferation of vaginal epithelium in C57BL/6J mice

Long-term exposure of adult female rodents to estrogen has many deleterious effects on reproductive neuro-endocrine structure and function, but its effects on peripheral target tissues are not well known. This study was designed to determine whether chronic exposure of young mice to estradiol (E2) alters the response of the vagina to E2, and if so, whether aging potentiates this alteration. Eight-week-old mice were ovariectomized (ovx) and given subcutaneous Silastic or polyethylene (PE) implants containing E2. Silastic implants produced supra-physiologic E2 levels, while E2 levels in PE-implanted mice were within the physiologic range. Initially all E2-exposed mice showed vaginal cornification (CORN). However, CORN soon began to decline and was virtually absent 3-5 mo after implantation, despite evidence of continued, albeit reduced, release of E2 from the implants. Mice were reimplanted with new E2 implants to determine whether the loss of CORN resulted from an altered response to E2 or from a decreased release of E2 from the implants. Vaginas of mice previously exposed to either Silastic (high E2) or PE (low E2) implants failed to cornify in response to new E2 implants, whereas vaginas of mice that had been initially exposed to implants without E2 cornified in response to identical E2 implants. When old (23 mo) acutely ovx mice were given E2-containing Silastic implants, the peak level and duration of CORN were only one-third and one-fifth, respectively, of that seen in young mice. Non-cornifying epithelia from both young and old chronically E2-exposed mice were as hyperplastic and active mitotically as cornifying epithelia, indicating that the loss of CORN was not a result of decreased epithelial proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol-induced adult anovulatory syndrome in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments

Long-term effects of elevated plasma estradiol (E2) on ovarian and neuroendocrine functions were examined in 4-month-old cycling female C57BL/6J mice injected s.c. with 0.2 or 0.05 mg estradiol valerate (EV), or oil. Within 7 days, EV-injected mice became permanently acyclic, exhibiting the persistent vaginal cornification (PVC) characteristic of reproductive senescence in rodents. Four months after injection, ovaries from EV-injected mice exhibited no corpora lutea, but ovulated in response to an injection of human chorionic gonadotropin (hCG) (as do older, spontaneously PVC mice). When grafted into young mice, ovaries from EV-injected mice supported as many estrous cycles as ovaries from oil-injected controls. EV did not alter the suppression of luteinizing hormone (LH) by E2, LH response to injected LH releasing hormone (LHRH), or plasma prolactin (Prl). However, EV-injected mice exhibited impairments in LH regulation similar to those seen in old, acyclic mice. Plasma LH 30 days after ovariectomy was 40% lower, and E2-induced LH surges were 60% lower, in EV-injected mice versus controls. Furthermore, EV-injected mice were unable to support estrous cycles given young ovarian grafts, in contrast to controls. Effects of sustained but physiological levels (15-20 pg/ml) of plasma E2, were examined in intact cycling mice given sham or E2 implants. Six weeks after implantation, the implants were removed; only 50% of the E2-implanted mice subsequently exhibited estrous cycles, compared with 100% of sham-implanted controls. Furthermore, those E2-implanted mice which did cycle had fewer cycles than controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione deficient C57BL/6J mice are not sensitized to ozone-induced lung injury

In this study we examined the role of the antioxidant glutathione (GSH) in pulmonary susceptibility to ozone toxicity, utilizing GSH deficient C57BL/6J mice that lack the expression of glutamate-cysteine ligase modifier subunit (GCLM). Gclm(−/−) knockout mice had 70% GSH depletion in the lung. Gclm(+/+) wild-type and Gclm(−/−) mice were exposed to either 0.3 ppm ozone or filtered air for 48 h. Ozone-induced lung hyperpermeability, as measured by total protein concentration in bronchoalveolar lavage fluid, was surprisingly lower in Gclm(−/−) mice than in wild-type mice. Lung hyperpermeability did not correlate with the degree of neutrophilia or with inflammatory gene expression. Pulmonary antioxidant response to ozone, assessed by increased mRNA levels of metallothionein 1 and 2, α-tocopherol transporter protein, and solute carrier family 23 member 2 (sodium-dependent vitamin C transporter) was greater in Gclm(−/−) mice than in Gclm(+/+) mice. These results suggest that compensatory augmentation of antioxidant defenses in Gclm(−/−) mice may confer increased resistance to ozone-induced lung injury.     

P-selectin glycoprotein ligand 1 is not required for the development of experimental autoimmune encephalomyelitis in SJL and C57BL/6 mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.     

Epstein-Barr virus nuclear antigen 1 does not cause lymphoma in C57BL/6J mice

To test whether transgenic Epstein-Barr virus nuclear antigen 1 (EBNA1) expression in C57BL/6 mouse lymphocytes causes lymphoma, EBNA1 expressed in three FVB lineages at two or three times the level of latent infection was crossed up to six successive times into C57BL/6J mice. After five or six crosses, 14/36, (38%) EBNA1 transgenic mice, 11/31 (36%) littermate EBNA1-negative controls, and 9/25 (36%) inbred C57BL/6J mice housed in the same facility had lymphoma. These data indicate that EBNA1 does not significantly increase lymphoma prevalence in C57BL/6J mice.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.     

Sex-specific strategies in spatial orientation in C57BL/6J mice

Sex differences in spatial learning are found in many species of mammals and even in invertebrates. Results from laboratory mouse studies, however, have been inconsistent in comparison to studies of humans, laboratory rats and wild rodent species. Here we re-examined this question in C57BL/6J mice that were exposed to enriched environments using two tasks, an object recognition task and a place learning task where mice were motivated by exploratory drive, not aversive conditioning or food restriction. Using these methods, we found a female advantage for object recognition, similar to the female advantage found in humans and laboratory rats. In the place learning task, male performance was unimpaired by intra-maze cue deletion but impaired by extra-maze cue masking. Female mice, in contrast, were able to navigate accurately under both cue conditions. In summary, by utilizing testing and housing methods that were more species appropriate, we found sex-specific patterns of cue encoding and place learning in better accordance with prior results from other mammalian species. The implication of these results is that the C57BL/6J mouse is an appropriate model for the study of cognitive sex differences in mammals.