In vitro sensitization to transplantation antigens. VI. Inhibition of sensitization

We have previously reported that C57BL/6 lymph node cells cultured with C3H/He macrophage monolayers are subsequently able to transfer specific allograft immunity to recipient C57 mice. The present communication is an investigation of the requirement for the functional integrity of cells to mediate allosensitization in vitro and to transfer allograft immunity. Our results indicate that C3H macrophage monolayers subjected to X-irradiation, actinomycin D, or antimacrophage serum pretreatment can no longer sensitize C57 lymph node cells in vitro; supernatants of C3H macrophage cultures do not substitute for monolayered cells and cannot sensitize C57 lymph node cells. The present data also indicate that the integrity of the lymph node cells is required after sensitization in vitro: X-irradiated or sonicated allosensitized lymph node cells do not enable recipient mice to accelerate C3H allograft rejection. The results of this report, therefore, suggest that intact, functionally normal cells are required to sensitize, and to transfer allosensitization.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

Studies on the induction of tolerance to alloantigens. II. The generation of serum factor(s) able to transfer alloantigen-specific tolerance for delayed-type hypersensitivity by portal venous inoculation with allogeneic cells

The administration of C3H/He spleen cells into allogeneic BALB/c mice via portal venous (p.v.) route resulted in C3H/He alloantigen-specific tolerance for delayed-type hypersensitivity (DTH) responses. When serum from these tolerant BALB/c mice were transferred into naive syngeneic BALB/c mice, the recipient mice lost the capability of generating DTH responses as induced by s.c. immunization with C3H/He cells. Tolerance was transferred only by serum from BALB/c mice inoculated p.v. with C3H/He cells, but not by serum from C3H/He mice inoculated p.v. with C3H/He cells, or BALB/c mice inoculated i.v. with C3H/He cells. This tolerogenic activity in serum from p.v. inoculated BALB/c mice was C3H/He alloantigen specific, because the transfer of the serum did not interfere with the development of anti-C57BL/6 DTH responses in recipient BALB/c mice. Such a serum factor(s) was inducible as early as 1 wk after the inoculation of C3H/He cells into BALB/c mice and not associated with anti-C3H/He alloantibody activity. Moreover, anti-C3H/He or C57BL/6-specific tolerogenic factor(s) prepared in the respective BALB/c or C3H/He mice was successfully transferred into totally allogeneic recipient mice, indicating no requirement of H-2, as well as non-H-2 restriction for the function of serum tolerogenic factor(s). Thus this study demonstrates that p.v. inoculation of allogeneic cells generates serum factor(s) able to transfer in H-2 and non-H-2-unrestricted manners the in vivo tolerance of the alloreactivity specific for alloantigens used for p.v. inoculation.     

Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius

Dobson C., Brindley P. J. and Sitepu P. 1982. Influence of serum donor and recipient mouse genotype on the passive transfer of protective immunity with serum against Nematospiroides dubius. International Journal for Parasitology12: 567–572. Different strains of serum donor mice showed variations in innate immunity to primary infections with Nematospiroides dubius. Different levels of anti-N, dubius antibodies were detected in sera from these mouse strains; there was no correlation between antibody titre and numbers of worms recovered. Serum from donor wild and six laboratory strains of mice protected female Quackenbush (Q) recipients against N. dubius infections; donor mouse strain influenced the degree of protection conferred and donor serum antibody titre related to the degree of stunting of worm growth in recipient mice. Five laboratory strains of mice developed different levels of protective immunity following multiple experimental infections with N. dubius. Antibody titres in these mice were strongly correlated with the percentage protection observed after 1–4 infections: Q and CBA mice produced more anti-N. dubius antibody and were better protected than DBA/2, BALB/c and C3H mice. However BALB/c, C3H and CBA mice attained similar anti-N. dubius antibody titres after a single infection with N. dubius but serum from BALB/c gave better protection when transferred to female Q recipients than that from the other two strains. This suggested qualitative differences in the protective antibodies in sera between mouse strains. Five mouse strains were passively immunized with a uniform dose of serum from female Q donors: DBA/2 female recipients showed the least, BALB/c and C3H females were intermediate, and Q and CBA female mice attained the greatest level of passive protection against N. dubius. A close positive correlation existed between the degree of actively acquired and the level of passively acquired protection between the five strains of mice.     

Leishsmania (Leishmania) amazonensis infection: Muscular involvement in BALB/c and C3H.HeN mice

Recent studies have provided some insights into Leishsmania (Leishmania) amazonensis muscular infection in dogs, although, muscular disease due to leishmaniasis has been poorly documented. The aim of our study was to evaluate involvement of Leishmania in muscular infection of two distinct mouse strains (BALB/c and C3H.He), with different genetic backgrounds. BALB/c mice, susceptible to Leishmania infection, showed, at the beginning of infection, a great number of infected macrophages among muscle fibers; however, in C3H.He resistant mice, muscle fibers were less damaged than in BALB/c mice, but some parasitized macrophages could be seen among them. A follow up of the infection showed an intense inflammatory infiltrate mainly composed of infected macrophages in BALB/c muscles and the presence of amastigotes within muscle fibers; while C3H.He mice exhibited a moderate inflammatory infiltrate among skeletal muscle fibers and an absence of amastigotes. Total destruction of muscles was observed in BALB/c mice in the late phase of infection (day 90) while C3H.He mice showed a process of muscle repair. We concluded that: (1) the muscles of BALB/c mice were more affected by leishmaniasis than those of C3/H.He mice; (2) Leishmania amastigotes are capable of infecting muscular fibers, as observed in BALB/c mice; (3) as inflammatory infiltrate is less intense in C3H.He mice these animals are capable of restoring muscular fibers.     

Trypanosoma cruzi: Correlation of resistance and susceptibility in infected inbred mice with the in vivo primary antibody response to sheep red blood cells

Nonspecific immune responses during the course of murine Trypanosoma cruzi infection were examined in mouse strains genetically resistant or susceptible to the Brazil strain of T. cruzi. Spleen cells from infected susceptible (C3H) or resistant [C57 B1/10 and FI (C3H × C57)]mice at various points during the course of infection exhibited a reduced response to concanavalin A and lipopolysaccharide in vitro. Since this reduced response occurred in both susceptible and resistant mice, it was not predictive of resistance or susceptibility in vivo. We next examined the kinetics of in vivo primary antibody response to sheep red blood cells (SRBC) in infected C3H and C57 mice. C3H mice exhibited inhibition of the direct plaque-forming cell assay (d-PFC) which persisted until death. In contrast C57 mice exhibited no inhibition of the response at Day 5 and subsequently a markedly augmented response was observed. Other strains of mice were similarly investigated: all the susceptible mice examined (A/J, BALB/c) showed inhibition or depression of the primary antibody response and resistant mice [B10Br, C57B1/10, SJL, F1 (C3H × C57)]demonstrated either no inhibition or considerable augmentation of this response. CS7 mice resistant to the Brazil strain were susceptible to the Tulahuén strain. The mice in this latter group exhibited a markedly significant inhibition of the in vivo primary antibody response to SRBC. Culture forms of the Brazil strain protected C3H mice from a virulent challenge. This immunization resulted in a markedly augmented antibody response. The data reported herein are consistent with the notion that inhibition of the primary antibody response to SRBC correlates with susceptibility whereas no inhibition or, indeed, augmentation of the response correlates with natural as well as acquired resistance.     

Defect in macrophage effector function confers Salmonella typhimurium susceptibility on C3H/HeJ mice

The defective allele of the endotoxin response locus (Lps^d) renders mice (e.g., C3H/HeJ strain) both endotoxin hyporesponsive and susceptible to Salmonella typhimurium. In this study, the mechanism of Lps^d-regulated susceptibility to murine typhoid was examined. C3H/ HeJ mice became significantly more resistant to S. typhimurium by reconstitution with bone marrow from syngeneic C3H/HeN mice (Lpsⁿ, salmonella resistant). Thus, the Lps^d resistance defect appeared to reside in a radiosensitive bone marrow-derived cell(s). At least one of the abnormal cell types appeared to be a macrophage because C3H/HeJ mice preinfected with Mycobacterium bovis (BCG) were, in contrast to controls, able to restrict early salmonella replication in their spleens and displayed a signficant increase in mean time to death. In contrast, no deficiency in uptake of salmonellae by C3H/HeJ macrophages was observed. These results indicate that the early deaths of C3H/HeJ mice following S. typhimurium challenge reflect a failure of their macrophages to limit the growth of these gram-negative bacteria.     

Lipopolysaccharide-induced suppression of antibody-directed cell-mediated cytotoxicity to chicken red blood cells

Prior treatment with commercially prepared and acetone-extracted lipopolysaccharide (LPS) was found to suppress the expression of antibody-directed, cell-mediated cytotoxicity (ADCC) to chicken red blood cells (CRBC) by spleen cells from C57/BL10, C3H, and BALB/c mice. The in vitro incubation with commercial LPS suppressed ADCC-CRBC activity of spleen cells from both C3H/HeJ and C3H/HeN mice. Only the C3H/HeN strain was suppressed when treated with purified LPS. ADCC-CRBC activity of neonatal spleen cells could be suppressed after a 3-hr in vivo incubation with LPS while adult spleen cells required a minimum of 15 hr preincubation.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.     

The pattern of methacholine responsiveness in mice is dependent on antigen challenge dose

Background

The relative contributions of the cytotoxic phenotype of P. aeruginosa expressing type III secretory toxins and an immunocompromised condition lacking normal Toll-like receptor 4 (TLR4) signaling in the pathogenesis of acute lung injury and sepsis were evaluated in a mouse model for Pseudomonas aeruginosa pneumonia. By using lipopolysaccharide-resistant C3H/HeJ mice missing normal TLR4 signaling due to a mutation on the tlr4 gene, we evaluated how TLR4 signaling modulates the pneumonia caused by cytotoxic P. aeruginosa expressing type III secretory toxins.

Methods

We infected C3H/HeJ or C3H/FeJ mice with three different doses of either a cytotoxic P. aeruginosa strain (wild type PA103) or its non-cytotoxic isogenic mutant missing the type III secretory toxins (PA103ΔUT). Survival of the infected mice was evaluated, and the severity of acute lung injury quantified by measuring alveolar epithelial permeability as an index of acute epithelial injury and the water to dry weight ratios of lung homogenates as an index of lung edema. Bacteriological analysis and cytokine assays were performed in the infected mice.

Results

Development of acute lung injury and sepsis was observed in all mouse strains when the cytotoxic P. aeruginosa strain but not the non-cytotoxic strain was instilled in the airspaces of the mice. Only C3H/HeJ mice had severe bacteremia and high mortality when a low dose of the cytotoxic P. aeruginosa strain was instilled in their lungs.

Conclusion

The cytotoxic phenotype of P. aeruginosa is the critical factor causing acute lung injury and sepsis in infected hosts. When the P. aeruginosa is a cytotoxic strain, the TLR4 signaling system is essential to clear the batcteria to prevent lethal lung injury and bacteremia.     

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

Abstract We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21–28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

Congenic Strains Confirm the Pleiotropic Effect of Chromosome 4 QTL on Mouse Femoral Geometry and Biomechanical Performance

A pleiotropic quantitative trait locus (QTL) for bone geometry and mechanical performance in mice was mapped to distal chromosome 4 via an intercross of recombinant congenic mice HcB-8 and HcB-23. To study the QTL in isolation, we have generated C3H.B10-(rs6355453-rs13478087) (C.B.4.3) and C3H.B10-(rs6369860-D4Mit170) (C.B.4.2) congenic strains that harbor ~20 Mb and ~3 Mb, respectively, of chromosome 4 overlapping segments from C57BL/10ScSnA (B10) within the locus on a C3H/DiSnA (C3H) background. Using 3-point bend testing and standard beam equations, we phenotyped these mice for femoral mid-diaphyseal geometry and biomechanical performance. We analyzed the results via 2-way ANOVA, using sex and genotype as factors. In the C.B.4.3 strain, we found that homozygous B10/B10 male mice had smaller cross sectional area (CSA) and reduced total displacement than homozygous C3H/C3H mice. Sex by genotype interaction was also observed for maximum load and stiffness for C3H/C3H and B10/B10 mice, respectively. In C.B.4.2 strain, we found that homozygous B10/B10 mice had lower total displacement, post-yield displacement (PYD), stiffness, yield load and maximum load than mice harboring C3H allele. Sex by genotype interaction was observed in B10/B10 mice for perimeter, outer minor axis (OMA) and CSA. There were no significant differences in tissue level mechanical performance, which suggest that the QTL acts primarily on circumferential bone size. These data confirm the prior QTL mapping data and support other work demonstrating the importance of chromosome 4 QTL on bone modeling and bone responses to mechanical loading.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21-28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

Insights into the Mechanisms of Protective Immunity against Cryptococcus neoformans Infection Using a Mouse Model of Pulmonary Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4⁺ T cells, CD11c⁺ cells, and Gr-1⁺ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.     

Sex and Genetic Factors Determine Osteoblastic Differentiation Potential of Murine Bone Marrow Stromal Cells

Sex and genetic factors determine skeletal mass, and we tested whether bone histomorphometric parameters were sexually dimorphic in femurs from 1 to 6 month old C57BL/6 mice. Trabecular bone volume declined more rapidly in female mice than in male littermates because of enhanced bone resorption. Although bone formation was not different between sexes, female mice exhibited a higher number of osteoblasts than male littermates, suggesting that osteoblasts from female mice may have a reduced ability to form bone. To determine the impact of sex on osteoblastogenesis, we investigated the potential for osteoblastic differentiation of bone marrow stromal cells from C57BL/6, Friend leukemia virus-B (FVB), C3H/HeJ and BALB/c mice of both sexes. Bone marrow stromal cells from female FVB, C57BL/6 and C3H/HeJ mice exhibited lower Alpl and Osteocalcin expression and alkaline phosphatase activity, and formed fewer mineralized nodules than cells from male littermates. Proliferative capacity was greater in cells from male than female C57BL/6, but not FVB, mice. Sorting of bone marrow stromal cells from mice expressing an α-Smooth muscle actin-green fluorescent protein transgene, revealed a higher yield of mesenchymal stem cells in cultures from male mice than in those from female littermates. Sex had a modest impact on osteoblastic differentiation of mesenchymal stem cells. To determine the influence of sex and genetic factors on osteoblast function, calvarial osteoblasts were harvested from C57BL/6, FVB, C3H/HeJ and BALB/c mice. Alpl expression and activity were lower in osteoblasts from C57BL/6 and C3H/HeJ, but not FVB or BALB/c, female mice than in cells from littermates. Sex had no effect on osteoclastogenesis of bone marrow cultures of C57BL/6 mice, but osteoblasts from female mice exhibited higher Rankl and lower Opg expression than cells from male littermates. In conclusion, osteoblastogenesis is sexually dimorphic and influenced by genetic factors.     

Pregnancy rates, prenatal and postnatal survival of offspring, and litter sizes after reciprocal embryo transfer in DBA/2JHd, C3H/HeNCrl and NMRI mice

Success of embryo transfer is often a limiting factor in transgenic procedures and rederivation efforts, and depends on the genetic background of the donor and recipient strains used. Here we show that embryo transfer to DBA/2J females is possible, and present data on pre- and postnatal success rates after reciprocal embryo transfer using the inbred DBA/2J and C3H/HeN, and outbred NMRI strains. The highest embryo yield was achieved in outbred NMRI females, but embryo yields were similar in DBA/2J and C3H/HeN mice following superovulation despite poor estrus cycle synchronization in DBA/2J females. In-strain transfer of DBA/2J blastocysts (transfer of embryos to recipients from the same strain) resulted in pregnancy rates (57.1%) similar to those obtained following in-strain transfer of C3H/HeN (60.0%) and NMRI mice (83.3%), although the prenatal survival rate of blastocysts was low. Moreover, from the pups born only half survived the postnatal period after transfer of DBA/2J and C3H/HeN blastocysts to DBA/2J recipients. These problems were not observed when transferring NMRI-blastocysts to C3H/HeN and DBA/2J mothers. The number of blastocysts transferred also had a positive effect on the success of embryo transfer. In conclusion, C3H/HeN and DBA/2J females can be used as recipients for embryo transfer procedures for certain donor strains like NMRI, as one major determinant seems to be the genetic background of the embryos transferred. We also recommend to increase the number of DBA/2J blastocysts transferred, and to foster the DBA/2J pups to other DBA/2J mothers postnatally for in-strain transfer of DBA/2J mice.     

Severity of chronic experimental Chagas' heart disease parallels tumour necrosis factor and nitric oxide levels in the serum: models of mild and severe disease

Heart tissue inflammation, progressive fibrosis and electrocardiographic alterations occur in approximately 30% of patients infected by Trypanosoma cruzi, 10-30 years after infection. Further, plasma levels of tumour necrosis factor (TNF) and nitric oxide (NO) are associated with the degree of heart dysfunction in chronic chagasic cardiomyopathy (CCC). Thus, our aim was to establish experimental models that mimic a range of parasitological, pathological and cardiac alterations described in patients with chronic Chagas’ heart disease and evaluate whether heart disease severity was associated with increased TNF and NO levels in the serum. Our results show that C3H/He mice chronically infected with the Colombian T. cruzi strain have more severe cardiac parasitism and inflammation than C57BL/6 mice. In addition, connexin 43 disorganisation and fibronectin deposition in the heart tissue, increased levels of creatine kinase cardiac MB isoenzyme activity in the serum and more severe electrical abnormalities were observed in T. cruzi-infected C3H/He mice compared to C57BL/6 mice. Therefore, T. cruzi-infected C3H/He and C57BL/6 mice represent severe and mild models of CCC, respectively. Moreover, the CCC severity paralleled the TNF and NO levels in the serum. Therefore, these models are appropriate for studying the pathophysiology and biomarkers of CCC progression, as well as for testing therapeutic agents for patients with Chagas’ heart disease.     

Leptin Receptor (Lepr) Is a Negative Modulator of Bone Mechanosensitivity and Genetic Variations in Lepr May Contribute to the Differential Osteogenic Response to Mechanical Stimulation in the C57BL/6J and C3H/HeJ Pair of Mouse Strains

This study investigated the role of leptin receptor (Lepr) signaling in determining the bone mechanosensitivity and also evaluated whether differences in the Lepr signaling may contribute to the differential osteogenic response of the C57BL/6J (B6) and C3H/HeJ (C3H) pair of mouse strains to mechanical stimuli. This study shows that a loading strain of ∼2,500 μϵ, which was insufficient to produce a bone formation response in B6 mice, significantly increased bone formation parameters in leptin-deficient ob⁻/ob⁻ mice and that a loading strain of ∼3,000 μϵ also yielded greater osteogenic responses in Lepr-deficient db⁻/db⁻ mice than in wild-type littermates. In vitro, a 30-min steady shear stress increased [³H]thymidine incorporation and Erk1/2 phosphorylation in ob⁻/ob⁻ osteoblasts and db⁻/db⁻ osteoblasts much greater than those in corresponding wild-type osteoblasts. The siRNA-mediated suppression of Lepr expression in B6 osteoblasts enhanced (but in osteoblasts of C3H (the mouse strain with poor bone mechanosensitivity) restored) their anabolic responses to shear stress. The Lepr signaling (leptin-induced Jak2/Stat3 phosphorylation) in C3H osteoblasts was higher than that in B6 osteoblasts. One of the three single nucleotide polymorphisms in the C3H Lepr coding region yielded an I359V substitution near the leptin binding region, suggesting that genetic variation of Lepr may contribute to a dysfunctional Lepr signaling in C3H osteoblasts. In conclusion, Lepr signaling is a negative modulator of bone mechanosensitivity. Genetic variations in Lepr, which result in a dysfunctional Lepr signaling in C3H mice, may contribute to the poor osteogenic response to loading in C3H mice.