Different strains of Toxoplasma gondii induce different cytokine responses in CBA/Ca mice

To investigate the role that cytokines may have in the development of toxoplasmic encephalitis (TE), the levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12 ]p40[), IL-10, IL-6, IL-4, and IL-2 in serum were examined in CBA/Ca mice infected with a type II strain (ME49 or FORT) of Toxoplasma gondii. These strains caused severe (ME49) or mild (FORT) TE in CBA/Ca mice. From weeks 1 to 8 of infection, the levels of IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha in serum were significantly higher in the ME49-infected mice than in the FORT-infected mice, suggesting a role for these cytokines in the severity of TE in CBA/Ca mice. Since the ME49 and FORT strains are of the same type, our results suggest a role for the parasite in the development of severe TE through the increased production of proinflammatory cytokines and indicate that not all type II strains cause TE.     

Early neutrophil but not eosinophil or platelet recruitment to the lungs of allergic horses following antigen exposure

Previous studies have shown that bronchoalveolar lavage fluid from horses with allergic respiratory disease and showing clinical symptoms contains increased numbers of neutrophils. In some cases, the eosinophil count is also increased. In this study the time course of changes in lung function and the accumulation of radiolabelled leucocytes and platelets in the lungs of allergic and normal horses has been examined during a 7 hr allergen exposure. Antigen challenge had no effect on pleural pressure or the distribution of radiolabelled neutrophils, eosinophils or platelets in normal horses. In contrast, in 6/8 allergic horses, there was an increase in pleural pressure and neutrophil accumulation in the lungs, both of which were evident after 4-5 hr. However, during the 7 hr challenge period radiolabelled eosinophils were detected in the lungs of only 1/6 horses exhibiting an increase in pleural pressure and in 1/7 horses that failed to show a change in airway function despite a clinical history of allergic respiratory disease. Antigen challenge did not alter the distribution of radiolabelled platelets in the five allergic horses tested. These results demonstrate that increased pleural pressure is not accompanied by eosinophil or platelet accumulation in the lungs of horses with allergic respiratory disease following exposure to antigen. However, changes in airway function can be associated with neutrophil accumulation but can also take place in the absence of this cell recruitment. This raises the possibility that the presence of neutrophils in the lung is not a prerequisite for changes in lung function.     

Effect of hyperlipidemia on thymocyte sensitivity to apoptosis in CBA and C57Bl/6 mice

Effects of experimental hyperlipidemia on apoptosis and proliferation of thymocytes in response to mitogens were studied in CBA and C57Bl/6 mice. The concentrations of cholesterol in the serum and thymocyte membranes increased in both mouse strains. Spontaneous and dexamethasone-induced apoptosisin vitro and the proliferative response to phytohemagglutinin and concanavalin A were enhanced in thymocytes from C57Bl/6 mice and suppressed in cells from CBA mice. These data suggest opposite reactions of thymocyte to increased serum cholesterol concentration in these two strains, associated with stimulation and suppression of cell activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 200–202, August, 2000     

Effect of 2-mercaptoethanol on some metabolic indices of ageing of CBA/Ca inbred mice

It has been shown in several studies that 2-mercaptoethanol (2-ME) improves the life span and aspects of life performance of laboratory animals. To obtain further details on the beneficial effects of 2-ME, a long-term study has been performed on male CBA/Ca inbred mice treated with this antioxidant. Four month-old mice were each given 4 micrograms of 2-ME in physiological saline via intraperitoneal (i.p.) injection 3 x per week. Measurements were made of the following: cold tolerance (heat performance), apparent total body protein turnover (T1/2), changes in the major lipid and fatty acid compositions of the liver, superoxide dismutase activity and formation of malondialdehyde and observations on a range of pathological changes. It was found that the basal rectal temperatures of the treated mice were higher and in the oldest group, heat performance capacity was better than those of the controls. After about 1 year of age the apparent biological half-life time of total body protein (T1/2) was observed to be shorter in the treated mice. Significant increases were observed to occur in the proportions of the polyunsaturated fatty acids in the lipids of the liver in the mice injected with 2-ME. Although no differences were observed in the superoxide dismutase activities, malondialdehyde concentrations in the livers of the experimental mice were significantly increased. Autopsy data showed that Dunn-sarcomas associated with amyloidoses occurred more frequently in the untreated mice.     

In vitro investigation of host resistance to Toxoplasma gondii infection in microglia of BALB/c and CBA/Ca mice

Toxoplasmic encephalitis (TE) is a life-threatening disease of immunocompromised individuals and has increased in prevalence as a consequence of AIDS. TE has been modeled in inbred mice, with CBA/Ca mice being susceptible and BALB/c mice resistant to the development of TE. To better understand the innate mechanisms in the brain that play a role in resistance to TE, nitric oxide (NO)-dependent and NO-independent mechanisms were examined in microglia from BALB/c and CBA/Ca mice and correlated with the ability of these cells to inhibit Toxoplasma gondii replication. These parameters were measured 48 h after stimulation with lipopolysaccharide (LPS) gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), or combinations of these inducers in T. gondii-infected microglia isolated from newborn mice. CBA/Ca microglia consistently produced less NO than did BALB/c microglia after stimulation with LPS or with IFN-gamma plus TNF-alpha, and they inhibited T. gondii replication significantly less than did BALB/c microglia. Cells of both strains treated with IFN-gamma alone significantly inhibited uracil incorporation by T. gondii, and N(G)-monomethyl-L-arginine (NMMA) treatment did not reverse this effect. In cells treated with IFN-gamma in combination with other inducers, NMMA treatment resulted in only partial recovery of T. gondii replication. This IFN-gamma-dependent inhibition of replication was not due to generation of reactive oxygen species or to increased tryptophan degradation. These data suggest that NO production and an IFN-gamma-dependent mechanism contribute to the inhibition of T. gondii replication after in vitro stimulation with IFN-gamma plus TNF-alpha or with LPS. Differences in NO production but not in IFN-gamma-dependent inhibition of T. gondii replication were observed between CBA/Ca and BALB/c microglia.     

Renal tubulointerstitial lesions in CBA/J mice

Renal tubulointerstitial lesions (RTL) were observed with high frequency in CBA/J mice more than 2 months old. RTL were characterized by interstitial infiltrates of lymphocytes and macrophages in the corticomedullary zone. Multinucleated structures sometimes resembling giant cells were present, and there was destruction of tubules and tubular basement membranes in areas of infiltration. Glomeruli appeared normal. RTL were first seen in 12 to 22 CBA/J mice 2.5 to 3 months old. By the age of 7 to 9 months, 35 to 45 mice were affected, and all 24 mice 12 months old or older had RTL. CBA/J mice had these unique renal lesions whether they were purchased and examined immediately, were obtained as weanlings andreared in our quarters or those of another institution, or were fourth generation descendants of purchased breeders. The propensity to develop RTL has been present in this strain for at least 2 years. RTL were not observed in C57BL/6J mice housed for 14 months with affected animals or in a survey of CBA/HUmc, C57BL/6J, A/J, BALB/cJ, or C3H/HeJ mice. Immunofluorescent examination of CBA/J kidneys appears to rule out antitubular basement membrane autoantibodies or immune complexes in the pathogenesis of RTL.     

Effect of hyperlipidemia on thymocyte sensitivity to apoptosis in CBA and C57B1/6 mice

Effects of experimental hyperlipidemia on apoptosis and proliferation of thymocytes in response to mitogens were studied in CBA and C57B1/6 mice. The concentrations of cholesterol in the serum and thymocyte membranes increased in both mouse strains. Spontaneous and dexamethasone-induced apoptosisin vitro and the proliferative response to phytohemag-glutinin and concanavalin A were enhanced in thymocytes from C57B1/6 mice and suppressed in cells from CBA mice. These data suggest opposite reactions of thymocyte to increased serum cholesterol concentration in these two strains, associated with stimulation and suppression of cell activity. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 130, No. 8, pp. 200–202, August, 2000     

Ectopic lung transplantation induces the accumulation of eosinophil progenitors in the recipients'' lungs through an allergen- and interleukin-5-dependent mechanism

Background Airway challenge of ovalbumin‐sensitized mice induces intrapulmonary accumulation of eosinophil progenitors. Objective To evaluate whether allergen‐challenged lungs release factors promoting intrapulmonary accumulation of haemopoietic cells, and define the role of allergic lung injury, we developed a transplantation model. Methods Lung tissue from allergen‐challenged, sensitized donors was ectopically grafted in syngeneic recipients, and haemopoietic progenitors inside the lungs of the recipients were quantified. Results In BALB/c mice, accumulation of progenitors occurred only when: (a) donors were sensitized and airway challenged with homologous allergen; (b) and recipients were sensitized. Grafts from the appropriate donors released biologically active IL‐5, which was effective in sensitized recipients. The effect of the appropriate donor–recipient combination was prevented by neutralizing anti‐IL‐5 antibody. Grafts from unchallenged, sensitized donors synergized with recombinant IL‐5 in sensitized recipients. Unlike BALB/c, grafts from naïve IL‐5 transgenic CBA/Ca mice (whose lungs contained a large number of progenitors, independently of sensitization and challenge) were effective in non‐transgenic, ovalbumin‐sensitized recipients. Conclusion This shows that: (a) intrapulmonary accumulation of progenitors is independent of immunological injury; (b) grafts systemically release IL‐5, which is required for progenitor accumulation in the recipients' lungs; (c) and sensitization is required for full responsiveness to IL‐5 and for generation of lung‐derived signals that synergize with IL‐5.     

嗜酸性粒细胞募集在支气管哮喘发病中的作用

支气管哮喘是一种气道慢性炎症性疾病,伴有嗜酸性粒细胞增多、杯状细胞肥大、黏液分泌增多、可逆性的气道阻塞及对吸入变应原和非特异性刺激的高反应性等特点,其中嗜酸性粒细胞募集和随后的激活在支气管哮喘发病中起着十分重要的作用。本文就嗜酸性粒细胞募集在支气管哮喘发病机制中的研究进展作一综述,为支气管哮喘的治疗提供一线新的曙光。     

Cortisone-induced immunotolerance to nematode infection in CBA/Ca mice. I. Investigation of the defect in the protective response.

The parasitic nematode Trichuris muris is expelled from the large intestine of CBA/Ca mice after approximately 20 days of infection. Short term treatment with cortisone acetate prevented expulsion and allowed the establishment and survival of an adult worm population for at least 70 days. This tolerance to the primary infection persisted after removal of the adult worms by chemotherapy and allowed the full development of a secondary infection. High levels of blast activity were seen in the mesenteric lymph node on days 30 and 40 of a primary infection in tolerant mice. Serum taken from tolerant mice passively conferred immunity on naive mice, but cells transferred from such donors were ineffective. Cells from immune mice were not capable of eliciting expulsion of an established primary infection in tolerant mice but did cause a significant reduction in worm numbers when given on the same day as a secondary infection. Cells from naive mice had no effect upon infections in tolerant mice. It is suggested that, in this system, corticosteroid treatment deletes an accessory cell population causing tolerization of immunocompetent T cells.     

Individual and typological behavioral characteristics of CBA/CaLac mice

Individual characteristics of higher nervous activity were studied in CBA/CaLac mice. The animals were divided into groups by the parameters of drinking response conditioning in a complex spatial environment. As distinct from bad learners, good learners exhibited higher orientation and exploratory activity in the open field, rapid adaptation, and low ability to retain the responses. Changes in conditioned reflex activity during neuroses were more pronounced in good learners. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 124–127, August, 2007     

Monocyte recruitment into the lungs in pneumococcal pneumonia

The recruitment of monocytes into the alveolar spaces is crucial for clearing infections and resolving the inflammatory response. We have previously reported the effect of acute pneumonia on monocyte transport through the bone marrow, and the present study concerns their clearance from the blood and migration into the lung airspaces. Dividing monocytes were labeled with the thymidine analog, 5'-bromo-2'-deoxyuridine (BrdU). Whole blood containing the labeled monocytes (MO(BrdU)) was transfused from either donor rabbits with pneumonia or from uninfected controls into recipients with pneumonia, where they were detected in blood and tissues using a double immunostaining method. The results show that MO(BrdU) from infected animals rapidly disappeared from the circulation (P < 0.05), preferentially sequestered in the infected lung tissue within 1 h (22.0 +/- 3.3% versus 6.0 +/- 0.4%, pneumonic region versus peripheral blood, P < 0.05), and accumulated to a greater degree in pneumonic airspaces than control monocytes 48 h after transfusion (3.9 +/- 0.5% versus 1.1 +/- 0.1%, P < 0.05). We conclude that immature monocytes released from the marrow by pneumonia sequester rapidly in lung microvessels but their migration in pneumonic airspaces is delayed.     

Chemokine production and leukocyte recruitment to the lungs of Paracoccidioides brasiliensis-infected mice is modulated by interferon-gamma

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.     

Allogeneic pregnancy in B-lymphocyte deprived CBA/Ca mice — Effects on maternal lymphoid organs and fetal survival

A number of female CBA/Ca (H2-k) mice were injected with rabbit-anti mouse IgM every 2 days throughout their life cycle in order to prevent their development of immunocompetent B-lymphocytes. The efficiency of the anti IgM-treatment was good in the group of mice used, and all experimental mice almost completely lacked 1) surface Ig-staining cells (immunoperoxidase ABC method), 2) serum IgG (ELISA technique), and 3) Ig-secreting cells (protein A plaque assay). All experimental mice contained a functioning T-cell pool, as recorded $\text{in vitro}$ by conA-stimulation and one-way MLC. At an age of 40–60 days these mice were allowed to mate allogeneically with C57/B1 (H-2b) males. No harmful effects of the anti-IgM treatment on the first pregnancy cycle were found, and B-cell-deprived mice delivered as many healthy litters as did the controls. It was also observed that the enlargement of the spleen and uterus-draining nodes was of the same magnitude in both experimental and control mice at the end of the first pregnancy.     

Elemental signals regulating eosinophil accumulation in the lung

Summary: In this review we identify the elemental signals that regulate eosinophil accumulation in the allergic lung. We show that there are two interwoven mechanisms for the accumulation of eosinophils in pulmonary tissues and that these mechanisms are linked to the development of airways hyperreactivity (AHR). Interleukin-(IL)-5 plays a critical role in the expansion of eosinophil pools in both the bone marrow and blood in response to allergen provocation of the airways. Secondly, IL-4 and IL-13 operate within the allergic lung to control the transmigration of eosinophils across the vascular bed into pulmonary tissues. This process exclusively promotes tissue accumulation of eosinophils. IL-13 and IL-4 probably act by activating eosinophil-specific adhesion pathways and by regulating the production of IL-5 and eotaxin in the lung compartment. IL-5 and eotaxin co-operate locally in pulmonary tissues to selectively and synergistically promote eosinophilia. Thus, IL-5 acts systemically to induce eosinophilia and within tissues to promote local chemotactic signals. Regulation of IL-5 and eotaxin levels within the lung by IL-4 and IL-13 allows Th2 cells to elegantly co-ordinate tissue and peripheral eosinophilia. Whilst the inhibition of either the IL-4/IL-13 or IL-5/ eotaxin pathways resulted in the abolition of tissue eosinophils and AHR, only depletion of IL-5 and eotaxin concurrently results in marked attenuation of pulmonary inflammation. These data highlight the importance of targeting both IL-5 and CCR3 signalling systems for the resolution of inflammation and AHR associated with asthma.
S.M. is a Postdoctoral Fellow funded by a grant from the Human Frontiers Foundation to P.S.F. and M.E.R. J.M. is supported by the German Research Association (grant MA 2241/1-1) and S.P.H by a NH&MRC CJ Martin Postdoctoral Fellowship.     

Immunological memory in CBA and CBA/N mice after vaccination with Mycobacterium bovis (BCG)

In inbred CBA and CBA/N mice immunological memory was induced by subcutaneous injection of Mycobacterium bovis (BCG). Experiments with adoptive transfer of spleen T cells and ionomycin-resistant T cells (memory cells) between CBA and CBA/N mice in various combinations showed that immunological memory was not formed in CBA/N mice, but can be induced by adoptive transfer of cells from CBA mice.     

Germline mutation rates and the long-term phenotypic effects of mutation accumulation in wild-type laboratory mice and mutator mice

The germline mutation rate is an important parameter that affects the amount of genetic variation and the rate of evolution. However, neither the rate of germline mutations in laboratory mice nor the biological significance of the mutation rate in mammalian populations is clear. Here we studied genome-wide mutation rates and the long-term effects of mutation accumulation on phenotype in more than 20 generations of wild-type C57BL/6 mice and mutator mice, which have high DNA replication error rates. We estimated the base-substitution mutation rate to be 5.4 × 10⁻⁹ (95% confidence interval = 4.6 × 10⁻⁹–6.5 × 10⁻⁹) per nucleotide per generation in C57BL/6 laboratory mice, about half the rate reported in humans. The mutation rate in mutator mice was 17 times that in wild-type mice. Abnormal phenotypes were 4.1-fold more frequent in the mutator lines than in the wild-type lines. After several generations, the mutator mice reproduced at substantially lower rates than the controls, exhibiting low pregnancy rates, lower survival rates, and smaller litter sizes, and many of the breeding lines died out. These results provide fundamental information about mouse genetics and reveal the impact of germline mutation rates on phenotypes in a mammalian population.Germline mutations are the ultimate source of congenital diseases, individual phenotypic variations, and evolutionary phenotypic changes. The per generation de novo mutation rate affects genetic variability and the speed of evolution (Kimura 1983; Drake et al. 1998). Advancements in high-throughput sequencing have made it possible to determine the mutation rates in various organisms. For example, the mean germline base-substitution mutation rate is calculated at 1.2 × 10⁻⁸ per nucleotide per generation for humans (Conrad et al. 2011; Kong et al. 2012; Campbell and Eichler 2013), 1.2 × 10⁻⁸ for chimpanzees (Venn et al. 2014), 2.8 × 10⁻⁹ for Drosophila melanogaster (Keightley et al. 2014), and 2.7 × 10⁻⁹ for Caenorhabditis elegans (Denver et al. 2009). The per generation mutation rate varies between species (Lynch 2010a) and within them; for example, the rate in humans varies several-fold between individuals, and is influenced by age, sex, and other genetic or environmental factors (Conrad et al. 2011; Kong et al. 2012; Campbell and Eichler 2013).More recently, the importance of understanding the risks and effects of germline mutagens, such as environmental chemicals and radiation, on the future health of animal populations, including humans, has become clear (Yauk et al. 2015). However, only limited information about the long-term effects of mutation-rate differences is currently available. For example, we know that an increased mutation rate will increase the frequency of congenital disease. However, the overall phenotypic effects of accumulated mutations on future populations living under higher mutation-rate conditions are largely unknown. Furthermore, the range of germline mutation rates that will permit the long-term survival of mammalian populations is unclear. Thus, here we established a new experimental model for assessing germline mutation rates and their phenotypic effects on future populations living under higher mutation-rate conditions.For this model, we raised two lines of mice, wild-type C57BL/6 mice (control mice) and homozygous Pold1^exo/exo mice (mutator mice), for more than 20 generations with phenotypic inspection, and established a set of mutation accumulation (MA) lines. Pold1^exo/exo mice lack the 3′-5′ exonuclease activity of DNA polymerase delta (of which a catalytic subunit is encoded by the Pold1 gene) on a C57BL/6 background (Uchimura et al. 2009) and have a high rate of DNA replication errors. DNA polymerase delta, a major enzyme in DNA replication and genome maintenance, contributes to faithful DNA synthesis, mainly lagging-strand synthesis, through its intrinsic 3′-5′ exonuclease proofreading activity (Burgers 2009; Prindle and Loeb 2012). Disrupting its exonuclease activity increases the spontaneous mutation (mainly base-substitution) rates in yeast (Simon et al. 1991) and mouse somatic cells (Albertson et al. 2009) and increases tumor susceptibility in mice (Goldsby et al. 2002) and humans (Palles et al. 2013). In the current study, ∼30% of the mutator mice died from thymic lymphoma at 3–8 mo of age, but the tumor rate did not change over the generations studied. The mice were bred by one-to-one natural mating between siblings, without artificial selection. At the time of this writing, we had studied up to 24 generations of these breeding lines (Fig. 1).Open in a separate windowFigure 1.Pedigrees of control and mutator mice in a long-term breeding study. All breeding lines that were separated by five or more generations from the original line are shown: blue, wild-type; black, surviving mutator lines; and red, extinct mutator lines. Green arrows indicate whole-genome sequencing was performed on mice from the indicated generations. The first appearance of a conspicuous phenotypic anomaly is shown for each breeding line. Gray shading indicates body weight was analyzed for the generations indicated (see Fig. 3, legend).For the current study, we performed whole-genome sequencing of the mouse MA lines and estimated the per generation mutation rate in the control and mutator mice. This is the first application of high-throughput sequencing to determine the spontaneous germline mutation rate of wild-type mice and provides the first direct estimate of a genome-wide germline mutation rate in laboratory mice. We also recorded detailed phenotypic data through several generations and found that the different germline mutation rates between the mutator and control mice had a significant impact on the health of their descendants. Our findings indicate that the combination of genome sequencing and MA analyses in mice is a promising tool for understanding the biological significance of mutation rate and de novo mutations in a population at the whole-genome level.