Kinetics of antibody response in BALB/c and C57BL/6 mice bitten by Phlebotomus papatasi

Background

Phlebotomine sand flies are blood-sucking insects transmitting Leishmania parasites. In bitten hosts, sand fly saliva elicits specific immune response and the humoral immunity was shown to reflect the intensity of sand fly exposure. Thus, anti-saliva antibodies were suggested as the potential risk marker of Leishmania transmission. In this study, we examined the long-term kinetics and persistence of anti-Phlebotomus papatasi saliva antibody response in BALB/c and C57BL/6 mice. We also tested the reactivity of mice sera with P. papatasi salivary antigens and with the recombinant proteins.

Methodology/Principal Findings

Sera of BALB/c and C57BL/6 mice experimentally bitten by Phlebotomus papatasi were tested by ELISA for the presence of anti-saliva IgE, IgG and its subclasses. We detected a significant increase of specific IgG and IgG1 in both mice strains and IgG2b in BALB/c mice that positively correlated with the number of blood-fed P. papatasi females. Using western blot and mass spectrometry we identified the major P. papatasi antigens as Yellow-related proteins, D7-related proteins, antigen 5-related proteins and SP-15-like proteins. We therefore tested the reactivity of mice sera with four P. papatasi recombinant proteins coding for most of these potential antigens (PpSP44, PpSP42, PpSP30, and PpSP28). Each mouse serum reacted with at least one of the recombinant protein tested, although none of the recombinant proteins were recognized by all sera.

Conclusions

Our data confirmed the concept of using anti-sand fly saliva antibodies as a marker of sand fly exposure in Phlebotomus papatasi–mice model. As screening of specific antibodies is limited by the availability of salivary gland homogenate, utilization of recombinant proteins in such studies would be beneficial. Our present work demonstrates the feasibility of this implementation. A combination of recombinant salivary proteins is recommended for evaluation of intensity of sand fly exposure in endemic areas and for estimation of risk of Leishmania transmission.     

Forestomach ulcers in Crj:B6C3 (C57BL/6NCrj x C3H/HeNCrj) F1 mice

An unusually high incidence of forestomach ulcers was observed in a mouse strain that is used frequently for long-term toxicology studies. Examination of 98 untreated male and 98 untreated female B6C3F1 hybrid mice, the majority of which were between 105 and 113 weeks of age, revealed forestomach ulcers in 52% of the males and 54% of the females. Glandular stomach ulcers were uncommon, being found in only four female mice. The incidence of the ulcers increased with age. The etiology of the lesion is unknown.     

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.     

C57BL/6 and BALB/c bronchoalveolar macrophages respond differently to exercise.

Macrophages from prototypical Th1 strains (e.g., C57BL/6) and Th2 strains (e.g., BALB/c) are classified as M-1 and M-2 phenotypes. We investigated the different phagocytic responses between M-1 and M-2 bronchoalveolar macrophages (BAMs) under resting and two various exercise conditions. At rest, M-1 BAMs showed higher phagocytic capacity of unopsonized particles, higher expression of MARCO (macrophage receptor with collagenous structure), and higher generation of NO than M-2 BAMs. Severe exercise, but not moderate exercise, significantly enhanced both phagocytosis of unopsonized particles and expression of MARCO in M-2 BAMs. In contrast, M-1 BAMs were unaffected by either exercise protocol. The phagocytosis of unopsonized particles was largely mediated by MARCO, especially in M-1 BAMs. Secreted products from cultured M-2 BAMs isolated after severe exercise, but not those from M-1 BAMs, enhanced BAM phagocytosis. The cultured M-1 BAMs secreted phagocytosis inhibitors, and this effect could be blocked by NO antagonists. Moreover, the extent of phagocytosis suppression induced by M-1 BAM-secreted products correlated with their production of nitrite/nitrate. Exogenous NO donors as well as NO derivatives, nitrite and nitrate, suppressed the BAM phagocytosis. We propose that while the severe exercise-enhanced phagocytosis in M-2 BAMs was largely mediated by MARCO up-regulation and secretion of stimulators, the lack of exercise effect in M-1 BAMs could be partially due to the constitutive secretion of NO-related suppressors. In conclusion, genetically different mice use different strategies in regulating BAM activity under resting conditions and in response to various exercise paradigms.     

Performance of BALB/c and C57BL/6 mice under an incremental repeated acquisition of behavioral chains procedure

BALB/c (n = 8) and C57BL/6 (n = 11) male mice were trained under an incremental repeated acquisition (IRA) procedure using two distinct training procedures: forward and backward chaining. A new metric for assessing progress on the IRA procedure, progress quotient (PQ), quantified progress as the product of chain length and number of reinforcers earned during a session divided by the total number of reinforcers earned. BALB/c mice progressed further, had higher overall responding, earned more reinforcers, and acquired the response sequences faster than the C57BL/6 mice on both training procedures. There were only minimal effects of training procedure for either strain. The strain differences found between BALB/c and C57BL/6 mice confirm the importance of genetic background to behavior. C57BL/6 mice may be deficient in learning as compared with BALB/c mice but other contributing factors probably include overall responding, motivation, and more rapid satiation or habituation to sucrose reinforcement by the C57BL/6 mice. PQ is a sensitive and valid measure of progress for use in studies of mastery-based incremental repeated acquisition and BALB/c mice perform this challenging learning task better than do C57BL/6 mice.     

In vitro studies of the natural humoral antitumor reactivity of BALB/c and C57BL/6J mice

Summary Antitumor antibodies were produced in vitro by spleen cells harvested from 12- to 18-month-old C57BL/6J and BALB/c mice with a naturally occurring complement-dependent serum cytotoxicity for tumor cells. The antibodies were of the IgM class and had a complement-dependent cytotoxic reactivity on EL4 cells, which was not absorbed by normal thymus cells. No natural antibodies were produced by untreated spleen cells from 1- or 3-month-old mice of the same two strains, which had no natural serum reactivity. However, after a treatment with an anti-Thy-1 serum and complement before culturing, spleen cells from 3-month-old mice, but not 1-month-old mice, produced the natural antitumor cytotoxic antibodies in vitro. When antibody-producing spleen cells from 12-month-old mice were cultured in vitro together with spleen cells from the unreactive 3-month-old syngeneic mice, inhibition of the antibody production occurred. This inhibiting capacity increased between 1 and 6 months of age and was abrogated by a treatment with an anti-Thy-1 serum and complement or with an anti-Ly-2 serum, whereas the passage of the inhibiting spleen cells on an anti-IgG column did not modify the inhibiting capacity. The in vitro data confirm previous in vivo findings on the nature, specificity and systems of regulation of the natural antitumor cytotoxic antibodies.     

Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice.

There has been significant utilization of the technique described by Hamelmann et al. (Am J Respir Crit Care Med 156: 766-775, 1997) in which a parameter, enhanced pause (Penh), related to airways responsiveness is noninvasively measured by unrestrained plethysmography (UP). Investigating this technique, we sought to answer these questions: 1). How do changes in Penh compare with changes in traditional plethysmographic and lung mechanical parameters? 2). How do UP parameters perform in two different mouse strains? Awake immunized and control BALB/c (n = 16) and C57BL/6 (n = 14) mice were placed in the UP chamber and exposed to doses of aerosolized methacholine while the following parameters were measured at each concentration: inspiratory time (Ti), expiratory time (Te), total time (Ttot), Ti/Ttot, peak inspiratory pressure, peak expiratory pressure, Pause, Penh, tidal volume (Vt), Vt/Ti, Vt/Te, and Vt/Ttot. The next day, lung resistance (Rl) and compliance (Cl) were invasively measured in the same animals. For the BALB/c, the parameters with the highest magnitude of correlation coefficient vs. Rl are (in order) 1). Cl, 2). Pause and Penh, 3). parameters of breathing frequency (Te, Ttot, Ti), and 4). parameters related to Vt (inspiratory pressure, expiratory pressure). Flow parameters (Vt/Ttot, Vt/Te, Vt/Ti) and duty cycle parameters (Ti/Ttot) had insignificant correlations. This ordering is significantly different in C57BL/6 mice, in which the parameters with the largest correlations are 1). Cl, 2). parameters of breathing frequency, and 3). flow parameters. Pause, Penh, Vt, and duty cycle parameters had insignificant correlations. These data show that Penh is problematic in the sense that it is strain specific; it behaves very differently in BALB/c and C57BL/6 mice. We suggest that UP parameters largely originate as part of reflex control of breathing processes, rather than in the lung mechanics and conclude that it is inappropriate to use UP parameters in general, and Penh specifically, as substitute variables for invasive mechanical indexes such as Rl.     

Comparison of BALB/c and B6-albino mouse strain blastocysts as hosts for the injection of C57BL6/N-derived C2 embryonic stem cells

Embryonic stem (ES) cells from a C57BL/6N (B6N) background injected into B6(Cg)-Tyrc-2J/J (B6-albino) recipient blastocysts are commonly used for generating genetically modified mouse models. To understand the influence of the recipient blastocyst strain on germline transmission, BALB/cAnNTac and B6-albino germline transmission rates were compared using the C57BL6/N-derived C2 ES cell line. A total of 92 ES cell clones from 27 constructs were injected. We compared blastocyst yield, birth rate, chimera formation rate, and high-percentage (>50 %) male chimera formation rate. For germline transmission, we analyzed 24 clones from 19 constructs, which generated high-percentage male chimeras from both donor strains. B6-albino hosts resulted in higher mean blastocyst yields per donor than did BALB/c ones (3.6 vs. 2.5). However, BALB/c hosts resulted in a higher birth rate than B6-albino ones (36 vs. 27 %), a higher chimera formation rate (50 vs. 42 %), a higher high-percentage male chimera rate (10 vs. 8 %), and a higher germline transmission rate (65 vs. 49 %), respectively. Our data suggest that BALB/c is a suitable blastocyst host strain for C2 ES cells and has an advantage over the B6-albino strain for receiving the injection of C2 ES cells.     

IL-10 gene knockout attenuates allergen-induced airway hyperresponsiveness in C57BL/6 mice

Intratracheal administration of interleukin-10 (IL-10) has been reported to inhibit allergic inflammation but augment airway hyperresponsiveness (AHR). In the present study, airway and smooth muscle responsiveness to methacholine (MCh) were compared in wild-type (WT) and IL-10-deficient (IL-10-KO) mice to investigate the role of endogenous IL-10 in AHR development. Naive WT and IL-10-KO mice exhibited similar dose-dependent increases in airway resistance (Raw) to intravenous MCh. Sensitization and challenge with ragweed (RW) induced a twofold increase in responsiveness to intravenous MCh in WT mice, but hyperresponsiveness was not observed in similarly treated IL-10-KO mice. Likewise, tracheal rings from RW-sensitized and -challenged WT mice exhibited a fourfold greater responsiveness to MCh than IL-10-KO tracheal preparations. Measurements of airway constriction by whole body plethysmography further supported the Raw and tracheal ring data (i.e., AHR was not observed in the absence of IL-10). Interestingly, factors previously implicated in the development of AHR, including IL-4, IL-5, IL-13, IgA, IgG1, IgE, eosinophilia, and lymphocyte recruitment to the airways, were upregulated in the IL-10-KO mice. Treatment with recombinant murine IL-10 at the time of allergen challenge reduced the magnitude of inflammation but reinstated AHR development in IL-10-KO mice. Adoptive transfer of mononuclear splenocytes to IL-10-sufficient severe combined immunodeficient mice indicated that lymphocytes were an important source of the IL-10 impacting AHR development. These results provide evidence that IL-10 expression promotes the development of allergen-induced smooth muscle hyperresponsiveness.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

Strain-specific effects of riboflavin supplementation on zymosan-induced peritonitis in C57BL/6J, BALB/c and CBA mice

AimsWe investigated the effects of riboflavin (vitamin B2) on the kinetics of zymosan-induced peritonitis in three strains of mice.Main methodsPeritonitis was induced in males of C57BL/6J, BALB/c and CBA mice by intraperitoneal injection of zymosan (40 mg/kg) or zymosan supplemented with riboflavin (50 mg/kg). During the first 45 min of inflammation the pain symptoms were scored. At the selected time points (4, 6, 8, 10, 24, and 30 h) the mice were sacrificed and peritoneal exudates were retrieved. Leukocytes, among them polymorphonuclear cells (PMNs) and macrophages (Mac3⁺ cells) were counted. Levels of inducible nitric oxide synthase (iNOS) were measured in cell pellets while supernatants were used for measurements of nitric oxide, cytokine/chemokines (IL-6, IL-10, MCP-1, IFNγ, TNF-α, and IL-12p70), and matrix metalloproteinase-9 (MMP-9).Key findingA riboflavin ip injection induced pain symptoms itself, but reduced zymosan-induced pain in C57BL/6J and CBA strains of mice when coinjected with zymosan. In comparison with the mice injected with zymosan only, riboflavin coinjection prolonged inflammation in C57BL/6J mice due to prolonged macrophage accumulation; inhibited peritoneal leukocytes (PTL) accumulation in BALB/c due to inhibited influx of macrophages and PMNs; and inhibited PTL accumulation in CBA mice due to delayed PMN influx. These effects corresponded with the delayed (C57BL/6J) or inhibited (BALB/c and CBA) expression of iNOS in PTL lysates, and with the prolonged (C57BL/6) or inhibited (BALB/c) intraperitoneal accumulation of MMP-9. Moreover, cytokine accumulation was affected in a strain-specific way.SignificanceRiboflavin is antinociceptive during yeast-induced peritonitis, but its anti-inflammatory effects are strain-specific.     

Differential effects of age on circulating and splenic leukocyte populations in C57BL/6 and BALB/c male mice

Background  

Despite several reports on age-related phenotypic changes of the immune system's cells, studies that use a multipoint age comparison between the specific and innate immune cell populations of prototypical Th1- and Th2-type polarized mouse strains are still lacking.     

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.     

Helicobacter pylori infection in wild-type and cytokine-deficient C57BL/6 and BALB/c mouse mutants

Helicobacter pylori causes gastroduodenal ulcer disease in humans. T lymphocytes and their cytokines are thought to play a substantial role in the control of H. pylori infection. To determine the importance of T helper (Th) cytokines and background genes we investigated the natural course of H. pylori infection in BALB/c and C57BL/6 wild-type or mutant mice deficient for either interleukin (IL)-4 or interferon (IFN)-gamma. H. pylori SPM 326 persisted for at least six months in C57BL/6 but was cleared by BALB/c wild-type mice nine weeks postinfection. H. pylori was recovered more frequently from IFN-gamma(-/-) BALB/c and IFN-gamma( -/-) C57BL/6 mice than from the respective wild-type animals. In contrast, IL-4 deficiency had no detectable effect on H. pylori recovery rates from either strain of mice. Our data suggest a protective role of IFN-gamma by mediating inflammation in murine H. pylori infection. In addition, our data emphasize that background genes which differ between BALB/c and C57BL/6 mice regulate the clearance of H. pylori.     

Macrophage cholesterol efflux to free apoprotein A-I in C3H and C57BL/6 mice

Cholesterol efflux from peritoneal macrophages of mice C57BL/6 susceptible and C3H resistant to atherosclerosis was compared, using apoprotein A-I as acceptor. The elicited macrophages were labeled with 3H-cholesterol and cholesterol enriched by incubation for 24 h with acetylated LDL. After incubation for 6 or 24 h, 3H-cholesterol efflux to free apoA-I (10 microg/ml) was significantly higher with macrophages derived from C3H mice compared to C57BL/6 mice. The cells were also pretreated with 0.3-0.45 mM cyclic AMP, 10 microM 9-cis-retinoic acid or 10 microM 22(R)-hydroxycholesterol, RXR and LXR ligands. Treatment with cyclic AMP, RXR, or LXR ligands, resulted in enhancement of 3H-cholesterol efflux in both strains. Under all conditions, 3H-cholesterol efflux was significantly higher in C3H compared to C57BL/6 macrophages. In conclusion, the higher cholesterol efflux from C3H macrophages could contribute toward the resistance of this strain to diet-induced atherosclerosis despite hypercholesterolemia.     

Enhanced glucose tolerance in pancreatic-derived factor (PANDER) knockout C57BL/6 mice

Pancreatic-derived factor (PANDER; also known as FAM3B) is a uniquely structured protein strongly expressed within and secreted from the endocrine pancreas. PANDER has been hypothesized to regulate fasting and fed glucose homeostasis, hepatic lipogenesis and insulin signaling, and to serve a potential role in the onset or progression of type 2 diabetes (T2D). Despite having potentially pivotal pleiotropic roles in glycemic regulation and T2D, there has been limited generation of stable animal models for the investigation of PANDER function, and there are no models on well-established genetic murine backgrounds for T2D. Our aim was to generate an enhanced murine model to further elucidate the biological function of PANDER. Therefore, a pure-bred PANDER knockout C57BL/6 (PANKO-C57) model was created and phenotypically characterized with respect to glycemic regulation and hepatic insulin signaling. The PANKO-C57 model exhibited an enhanced metabolic phenotype, particularly with regard to enhanced glucose tolerance. Male PANKO-C57 mice displayed decreased fasting plasma insulin and C-peptide levels, whereas leptin levels were increased as compared with matched C57BL/6J wild-type mice. Despite similar peripheral insulin sensitivity between both groups, hepatic insulin signaling was significantly increased during fasting conditions, as demonstrated by increased phosphorylation of hepatic PKB/Akt and AMPK, along with mature SREBP-1 expression. Insulin stimulation of PANKO-C57 mice resulted in increased hepatic triglyceride and glycogen content as compared with wild-type C57BL/6 mice. In summary, the PANKO-C57 mouse represents a suitable model for the investigation of PANDER in multiple metabolic states and provides an additional tool to elucidate the biological function and potential role in T2D.KEY WORDS: Pancreatic-derived factor, FAM3B, Knockout model, Glycemic regulation, Hepatic insulin signaling, Glucose tolerance     

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.     

Differences in anxiety-related behavior between apolipoprotein E-deficient C57BL/6 and wild type C57BL/6 mice

The influence of ApoE gene deletion on the anxiety state has not been previously investigated. The elevated plus maze was used in this study to determine differences in anxiety-related behavior between apoE-deficient and wild type C57BL/6 mice. The apoE-deficient mice demonstrated less anxiety on the elevated plus maze by spending more time in the open arms of the elevated plus maze compared to wild type mice (p<0.001). Additionally, female apoE-deficient mice visited the open arm of the maze more often than their apoE-deficient male counterpart (p<0.05). The anxiety state and/or sex are possible variables to be considered when designing physiological and/or behavioral studies involving mice that are apoE-deficient.