Altered splenic T cell function of BALB/cByJ mice infected with mouse hepatitis virus or Sendai virus

Mouse hepatitis virus and Sendai virus are among the most common viruses naturally infecting laboratory mice. Concanavalin A-stimulated in vitro proliferative responses of splenocytes were examined after infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus (MHV-JHM) or Sendai virus. Mice were exposed to these viruses by presumed natural routes (per os or intranasally). Immunodepression was marked but transient among BALB/cByJ mice exposed to MHV-JHM. Among mice exposed to Sendai virus and examined over a 21-day period, spleen cells from only one mouse, sacrificed 10 days postinoculation, exhibited a severely impaired ability to respond to concanavalin A. Lymphokine production by spleen cells from control and infected mice was then assessed. IL 2 was either absent or present at very low levels in culture supernates of concanavalin A-unresponsive spleen cells from MHV-JHM-infected mice. Spleen cells from the single Sendai virus-infected mouse also produced very low levels of IL 2. In contrast, IL 1 was detected in supernatants of all spleen cell cultures derived from control, MHV-JHM-infected, or Sendai virus-infected mice. There was not a clear correlation between concanavalin A responsiveness and the ability of spleen cells to produce interferon-gamma. These results stress the importance of using laboratory mice of known microbiological status for immunologic experiments.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Infection of BALB/cByJ mice with the JHM strain of mouse hepatitis virus alters in vitro splenic T cell proliferation and cytokine production.

Earlier studies from this laboratory showed that infection of BALB/cByJ mice by a natural route with mouse hepatitis virus, strain JHM (MHV-JHM), results in functional splenic T cell suppression in vitro. This was evidenced by reduced concanavalin A-driven spleen cell proliferation and interleukin (IL)2 production measured after conventional intervals of cell culture (72 and 24 h, respectively). The purpose of the present work was to determine whether MHV-induced T cell dysfunction is kinetic or absolute and whether production of other T-cell derived cytokines is defective. Bioassays revealed that production of IL2, gamma interferon, and IL4 by spleen cells from acutely infected mice is suppressed and that some of the defects are kinetic as well as absolute. Proliferative responses of both CD4+ and CD8+ T cells were depressed, but neither cell type contained infectious virus. Cells that proliferated poorly in response to concanavalin A were fully capable of responding to specific virus stimulation. These results further emphasize the potential complications that MHV infection may pose to immunologic research using mice.     

Genetic determinants of resistance to ectromelia (mousepox) virus-induced mortality.

Resistance to ectromelia (mousepox) virus-induced mortality was examined in crosses between susceptible DBA/2J, A/J, and BALB/cByJ mice and resistant C57BL/6J and AKR/J mice. Depending on the cross, resistance to mousepox virus was shown to be determined by one or more independently assorting autosomal loci with dominant alleles for resistance in AKR/J and C57BL/6J mice and recessive alleles in A/J, BALB/cByJ, and DBA/2J mice. A sexual dimorphism in resistance to disease was also observed.     

Differential susceptibility to actively induced experimental allergic encephalomyelitis and experimental allergic orchitis among BALB/c substrains

Experimental allergic orchitis (EAO) and experimental allergic encephalomyelitis (EAE) are animal models of organ-specific autoimmune disease. In this study, BALB/cByJ and BALB/cAnNCr mice were susceptible to both autoimmune diseases whereas BALB/cJ subline mice were resistant. Disease resistance in BALB/cJ mice did not appear to be a reflection of either (i) a nonspecific generalized impairment of cellular immunity or (ii) an alteration in the phenotypic expression of Bordetella pertussis-induced histamine sensitization, a phenotype which has been shown to be associated with susceptibility to both diseases. Susceptibility to both EAE and EAO was inherited as a dominant trait in F1 hybrid animals. Segregation analysis in a (BALB/cByJ X BALB/cJ) X BALB/cJ backcross population suggested that disease resistance may be associated with a single genotypic difference in a common regulatory gene affecting susceptibility to both diseases. Linkage analysis of the backcross population failed to demonstrate an association of disease resistance with the mutant raf-1b allele carried by BALB/cJ mice. The results of these studies support previous observations that multiple genotypic differences may in fact exist in mice of the BALB/cJ subline and that such differences play a significant role in the genetic control of susceptibility to EAE and EAO.     

Interferon and Host Resistance to Rauscher Virus-induced Leukemia

A random bred strain of mice (CD-1) was shown to develop resistance to Rauscher leukemia virus (RLV) as the animals matured. Resistant adult mice developed relatively high-serum levels of interferon (150 to 2,000 units per ml) in contrast to susceptible 21-day-old animals in which interferon levels were undetectable or low (less than 20 to 200 units per ml). A similar correlation between resistance and interferon levels was observed in comparisons between resistant CD-1 and susceptible BALB/c mice. The F(1) hybrids of CD-1 x BALB/c and BALB/c x CD-1 matings manifested an intermediate degree of susceptibility and interferon production. The difference in interferon production by CD-1 and BALB/c mice was specific for the RLV-host interaction, since both strains produced equal serum levels of interferon in response to Sindbis and Newcastle disease viruses. The mortality of CD-1 suckling mice infected with Rauscher leukemia virus was decreased by treatment with interferon. These data demonstrate an association between interferon production by the host and the observed relative resistance of the CD-1 strain of adult mice to the subsequent malignant transformation. This virus-host relationship provides an excellent model for further study of factors affecting the development of virus-induced leukemia.     

Plasmodium chabaudi chabaudi: B-1 cell expansion correlates with semiresistance in BALB/cJ mice

The largest obstacle impeding the development of an effective malaria vaccine is the incomplete understanding of how the immune response is regulated during infection. B-1a cells, a poorly understood subcategory of B lymphocytes, produce nonpathologic autoantibodies of low affinity which have been shown to have distinct immunoregulatory capabilities. What the exact activity of B-1a cells are during the course of malaria has yet to be determined. By use of flow cytometry, it was observed that B-1a cells significantly expand by day 3 postinfection in the spleen and peritoneum of Plasmodium chabaudi chabaudi semiresistant BALB/cJ mice, but not until day 8 postinfection in the spleen of P. chabaudi chabaudi fully susceptible BALB/cByJ mice. The activation of B-1a cells was also demonstrated by the measurement of natural autoantibody IgM production from the serum and cultured peritoneal B-1a cells. Infected semiresistant BALB/cJ mice generated higher levels of anti-ssDNA IgM antibodies than infected fully susceptible BALB/cByJ mice. The preliminary data presented here suggest a possible roll of B-1 cells in contributing to the successful survival of murine malarial infection.     

Kinetics of ectromelia virus (mousepox) transmission and clinical response in C57BL/6j, BALB/cByj and AKR/J inbred mice

Research was undertaken to answer basic questions on susceptibility, clinical response and transmission of ectromelia virus in selected strains of inbred mice. C57BL/6J and AKR/J were found to be markedly more resistant to a virulent strain of ectromelia virus (isolated during the 1979-80 outbreak at the National Institutes of Health) than C57LJ, BALB/cByJ, DBA/2J, A.By/SNJ and C3H/HeJ when infected by footpad inoculation. In C57BL/6J and AKR/J the LD50 was about 7 logs higher than the ID50. With one exception, C57LJ, the LD50 and ID50 titers in the other strains were about equal. In C57LJ the LD50 titer was intermediate. Following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and up to 29 days from BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days respectively after infection. Virus was isolated from the spleen in 2 of 5 BALB/cByJ mice and 1 of 7 C57BL/6J mice tested 95 days after gastric inoculation. Following footpad inoculation, BALB/cByJ mice consistently transmitted virus to cage mates before death at 10-12 days. C57BL/6J mice transmitted between days 8 and 17, but not beyond. Virus was maintained in C57BL/6J mice by exposure to infected cage mates for seven passages, which was the most attempted. Clinical signs in infected C57BL/6J mice were usually subtle or inapparent.     

Experimental allergic orchitis in mice

Inbred strains of mice were studied for their susceptibility to the induction of experimental allergic orchitis after sensitization with mouse testicular homogenate in complete Freund's adjuvant accompanied by injections of extract from Bordetella pertussis. Susceptibility to autoimmune orchitis was found to be linked to the major histocompatibility complex in BALB/c and C57BL/10 mice and mapped to genes encoded within the H-2D ^dregion. In five of six groups of bidirectional (susceptible × resistant) F₁ hybrids, H-2D ^d-linked susceptibility was inherited as a dominant autosomal trait. However, in (BALB/cByJ × DBA/2J)F₁ and (DBA/2J × BALB/cByJ)F₁ hybrids, dominant autosomal resistance to the induction of autoimmune orchitis was observed. Backcross analysis between the resistant F₁ hybrid and the susceptible BALB/cByJ parent suggests that a single independently segregating DBA/2J locus is capable of negating H-2D ^d-linked susceptibility, and controls resistance to the induction of autoimmune orchitis.Abbreviations used in this paper BP extract Bordetella pertussis extract - CFA complete Freund's adjuvant - EAO experimental allergic orchitis - Ir immune response - MHC major histocompatibility complex - MLH mouse liver homogenate - MTH mouse testis homogenate - PI pathology index     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Estrous cycle effects on behavior of C57BL/6J and BALB/cByJ female mice: implications for phenotyping strategies

Systematic behavioral phenotyping of genetically modified mice is a powerful method with which to identify the molecular factors implicated in control of animal behavior, with potential relevance for research into neuropsychiatric disorders. A number of such disorders display sex differences, yet the use of female mice in phenotyping strategies has been a rare practice because of the potential variability related to the estrous cycle. We have now investigated the behavioral effects of the estrous cycle in a battery of behavioral tests in C57BL/6J and BALB/cByJ inbred strains of mice. Whereas the performance of BALB/cByJ female mice varied significantly depending on the phase of the estrous cycle in the open field, tail flick and tail suspension tests, the behavior of C57BL/6J females, with the exception of the tail suspension performance, remained stable across all four phases of the estrous cycle in all of the tests including open field, rotarod, startle reflex and pre-pulse inhibition, tail flick and hot plate. We also found that irrespective of the estrous cycle, the behavior of C57BL/6J females was different from that of BALB/cByJ groups in all of the behavioral paradigms. Such strain differences were previously reported in male comparisons, suggesting that the same inter-group differences can be revealed by studying female or male mice. In addition, strain differences were evident even for behaviors that were susceptible to estrous cycle modulations, although their detection might necessitate the constitution of large experimental groups.     

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Markers for heightened monitoring, imminent death, and euthanasia in aged inbred mice

The goal of this study was to identify objective criteria that would reliably predict spontaneous death in aged inbred mice. We evaluated male and female AKR/J mice, which die at a relatively young age due to the development of lymphoma, as well as male C57BL/6J and BALB/cByJ mice. Mice were implanted subcutaneously with an identification chip that also allowed remote measurement of body temperature. Temperatures and body weights were measured weekly until spontaneous death occurred or until euthanasia was performed for humane reasons. In AKR/J mice, hypothermia and weight loss began about 4 wk prior to death and increased gradually during that antemortem interval. In C57BL/6J and BALB/cByJ mice, these declines began earlier and were more prolonged prior to death. However, C57BL/6J and BALB/cByJ mice developed a relatively precipitous hypothermia during the 2 wk prior to death. For all 3 strains, the derived composite score of temperature × weight, expressed as a percentage of stable values for each mouse, was similarly informative. These changes in individual and composite measures can signal the need for closer observation or euthanasia of individual mice. Validated markers of clinical decline or imminent death can allow the use of endpoints that reduce terminal distress, do not significantly affect longevity or survival data, and permit timely collection of biologic samples.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Inactivation of Cryptosporidium parvum Oocysts in Fresh Apple Cider by UV Irradiation

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm². Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.     

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.     

Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation

The kinetics of IL-1 (alpha and beta) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1alpha and -1beta (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i. ). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1alpha and -1beta (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1beta was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1beta polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Spink2 Modulates Apoptotic Susceptibility and Is a Candidate Gene in the Rgcs1 QTL That Affects Retinal Ganglion Cell Death after Optic Nerve Damage

The Rgcs1 quantitative trait locus, on mouse chromosome 5, influences susceptibility of retinal ganglion cells to acute damage of the optic nerve. Normally resistant mice (DBA/2J) congenic for the susceptible allele from BALB/cByJ mice exhibit susceptibility to ganglion cells, not only in acute optic nerve crush, but also to chronic inherited glaucoma that is characteristic of the DBA/2J strain as they age. SNP mapping of this QTL has narrowed the region of interest to 1 Mb. In this region, a single gene (Spink2) is the most likely candidate for this effect. Spink2 is expressed in retinal ganglion cells and is increased after optic nerve damage. This gene is also polymorphic between resistant and susceptible strains, containing a single conserved amino acid change (threonine to serine) and a 220 bp deletion in intron 1 that may quantitatively alter endogenous expression levels between strains. Overexpression of the different variants of Spink2 in D407 tissue culture cells also increases their susceptibility to the apoptosis-inducing agent staurosporine in a manner consistent with the differential susceptibility between the DBA/2J and BALB/cByJ strains.