To drive or be driven: the path of a mouse model of recurrent pregnancy loss.

This review is an example of the use of an animal model to try to understand the immune biology of pregnancy. A well-known model of recurrent spontaneous pregnancy loss is put in clinical, historical, and theoretical context, with emphasis on T cell biology.

Impact of mouse pregnancy on thymic T lymphocyte subsets.

It has been reported that fetal lymphoid progenitor cells are acquired during gestation and are able to develop in the maternal mouse thymus into functional T cells. Moreover, previous pregnancies increase the number of fetal cells in the mother. In the present study, we investigated whether mouse pregnancy induces changes in T lymphocyte subsets in the maternal thymus. We determined the T lymphocyte subsets in two allogeneic cross-breedings, namely CBA/J×BALB/c (normal) and CBA/J×DBA/2 (abortion prone), and investigated the effects of the age and parity of the female, as well as pregnancy outcome, on thymocyte populations. In addition, hormonal effects were evaluated in a syngeneic combination (CBA/J×CBA/J). We found that during pregnancy both hormonal and allogeneic stimuli induced a reduction in the CD4(+)CD8(+) subset with an increase in the CD4(+)CD8(-) population. Only young females of the normal combination exhibited an increase in the CD4(-)CD8(+) population. All young mice showed an increase in CD4(+)CD25(+)FoxP3(+) T cells. Interestingly, the γδT thymus pool was increased in all females of the normal allogeneic pregnancy only, suggesting the participation of this pool in the observed beneficial effect of multiparity in this cross-breeding. Our results demonstrate that allogeneic pregnancies induce important variations in maternal thymocyte subpopulations depending on the age of the female and the male component of the cross-breeding.

Morphofunctional characteristics of the immune system in CBA and C57Bl/6g mice.

We studied peculiarities of morphofunctional organization of the immune system in C57Bl/6g and CBA mice differing by their susceptibility to various types of infectious agents. The revealed differences in the structure of lymphoid organs, T lymphocyte subpopulation ratio and their differentiation into Th1/Th2 cells after mitogen stimulation drove us to a conclusion on genetically determined regularities in the development of the immune response in these animal strains.

The persistence of paternal antigens in the maternal body is involved in regulatory T-cell expansion and fetal-maternal tolerance in murine pregnancy.

PROBLEM: Mammalian pregnancy is a state of immunological tolerance and CD4(+) CD25(+) regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen-specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens. METHOD OF STUDY: We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy. RESULTS: Presence of paternal and maternal MHC class II(+) cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3(+) cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal-maternal interface. CONCLUSION: Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi-allogeneic fetus until the time point of birth.

Differential rates of apoptosis and recruitment limit eosinophil accumulation in the lungs of asthma-resistant CBA/Ca mice.

The accumulation of eosinophils is a common feature of allergic airway inflammation and correlates with disease severity. In an ovalbumin (OVA)-induced murine model of allergic lung disease, CBA/Ca mice develop much lower levels of lung eosinophilia, lung oedema, mucus hypersecretion and airways obstruction than BALB/c and C57BL/6 strains. In this study these strains have been examined to identify mechanisms that control the recruitment and survival of eosinophils in the allergic lung. Following immunization with OVA, CBA/Ca mice developed a robust systemic allergic response, with high levels of total and OVA-specific IgE and increases in peripheral blood eosinophils. Lung eotaxin-1 levels and expression of CD18 on eosinophils recovered by bronchoalveolar lavage (BAL) were least pronounced in CBA/Ca mice, whereas mRNA for L-selectin was highest in eosinophils from C57BL/6 mice. Apoptosis of BAL eosinophils ex vivo was most pronounced in the CBA/Ca strain. BALB/c mice expressed the highest levels of the eosinophil growth and survival factor interleukin (IL)-5 in the lungs and BAL eosinophils from these animals expressed more of the anti-apoptotic proteins Bcl-xL and Bcl-2 than cells from the other strains. A combination of lower levels of recruitment and rapid apoptosis may therefore limit the accumulation of eosinophils and pathology in the lungs of CBA/Ca mice. In addition, although the level of pathology that developed in C57BL/6 and BALB/c mice was similar, some of the underlying mechanisms are likely to differ.

Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma.

Acoustic injury results in destruction of hair cells and numerous nonsensory cells of the cochlea. How these injured structures undergo repair is not well understood. This study was designed to examine the cochlea for the presence of mononuclear phagocytes after tissue injury caused by noise damage. We used octave band noise (8--16 kHz) at three levels (106, 112, and 120 dB) for 2 hours and studied the mice at 1, 3, 7, and 14 days after noise exposure to determine how noise affected hearing thresholds, hair cell number, and tissue injury in the cochlea. Furthermore, we assessed the cochlea for presence of inflammation by performing immunohistochemistry for CD45, common leukocyte antigen. We counted the number of CD45(+) cells that were present in the cochlea at the above-mentioned time points after noise. CD45 is present on all bone marrow-derived white blood cells and is not otherwise expressed in the inner ear. We found that, after noise exposure, there is a large increase in CD45(+) cells. These marrow-derived cells are concentrated in the spiral ligament and spiral limbus, areas that are known to be susceptible to acoustic injury. It is possible that this inflammatory response plays a role in propagating cellular damage in these areas. Immunohistochemistry demonstrates that these cochlear cells are derived from the monocyte/macrophage lineage and serve a phagocytic function in the inner ear.

Immune reactions in different mouse strains.

The responses of immunocompetent cells to thymus-dependent antigen differ in mice of different strains. Immunization stimulated phagocytic activity of peritoneal macrophages in CBA/CaLac, DBA/2, and BALB/c mice and suppressed it in CC57W mice. By the formation of antibody-producing cells in the spleen in response to thymus-dependent antigen DBA/2 and CBA/CaLac mice can be classified as high responders, BALB/c mice as medium-responders, and C57Bl/6 and CC57W mice as low responders.

Skin allograft maintenance in a new synchimeric model system of tolerance.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Estudo comparativo das infecções por Leishmania major e Leishmania amazonensis em camundongos isogênicos CBA / Comparative study of infections with L. major or L. amazonensis CBA mice

A maioria dos trabalhos sobre o modelo murino na leishmaniose tegumentar utiliza diferentes linhagens de camundongos que são resistentes ou susceptíveis a uma determinada espécie de leishmânia ou trata de manipulações da resposta imune tornando camundongos susceptíveis, resistentes a determinada leishmânia, ou tornando resistentes, susceptíveis. No presente estudo avalia-se comparativamente, resistência e susceptibilidade à infecção por leishmânia, utilizando o modelo de infecção de camundongos isogênicos CBA infectados com L. amazonensis (L.a.), para as quais são susceptíveis, e infectados com L. major (L.m.), para as quais são resistentes. Nós comparamos a resposta imune-inflamatória nesses dois grupos através da avaliação do curso da infecção pelo monitoramento do tamanho das lesões e avaliação da quantidade de parasitos em cortes histológicos através de imunohistoquímica. A resposta tissular foi estudada em cortes histológicos das patas e dos linfonodos de drenagem no intervalo de três a 70 dias após a infecção. A produção de IFN-y, IL-4 e IL-10 foi avaliada pelo método ELlSA e a produção de NO pelo método de Griess, em sobrenadantes de culturas de células do linfonodo de drenagem. Os camundongos CBA infectados por L.m. controlam a infecção e curam, enquanto os infectados por L.a. exarcebam a infecção e morrem. Os padrões de resposta tissular no local da infecção e no linfonodo de drenagem são distintos. Nos animais resistentes ocorre inflamação mista com formação de granuloma e fibrose, enquanto nos susceptíveis ocorre reação macrofágica monomórfica, sem granulomas e fibrose. O IFN-y foi predominante produzido pelas células do linfonodo dos animais infectados por L.m., enquanto que os níveis de IL-4 foram mantidos mais alto no grupo de animais infectados por L.a., após o 7º dia de infecção. Os perfis distintos de resposta correspondem a padrões distintos de resposta tissular e estão relacionados...

Autoimmunity to heat shock protein 60 and antigen-specific production of interleukin-10.

The immunopathologic sequelae of chlamydial infection are correlated with immune responses to the Chlamydia trachomatis heat shock protein 60 (hsp60). One pathogenic mechanism that may explain this association is the induction of autoimmune responses to self hsp60, since these two proteins share a high degree of amino acid sequence identity. To investigate the conditions under which autoimmune responses can be generated against self hsp60, groups of CBA mice were immunized with recombinant mouse hsp60, recombinant chlamydial hsp60, or both proteins. The data show that autoimmune responses characterized by strong T-cell proliferation and high titers of antibody to self hsp60 are induced only by concurrent immunization with mouse and chlamydial hsp60. Immunization with mouse hsp60 alone induced lymphocytes that secreted high levels of interleukin-10 (IL-10) but did not proliferate in response to in vitro stimulation with mouse hsp60; coimmunization with mouse and chlamydial hsp60s induced lymphocytes that proliferated strongly in response to mouse hsp60, secreted 6-fold less IL-10, and exhibited a 12-fold increase in the ratio of gamma interferon/IL-10 production. Switches in cytokine production patterns may mediate the pathogenesis of hsp60-associated diseases such as C. trachomatis immunopathology.

Differences in immunological response to a T. gondii protein (SAG1) derived peptide between two strains of mice: effect on protection in T. gondii infection.

This study presents the analysis of the immunogenicity, antigenicity and protective effects of a peptide derived from the major surface antigen of Toxoplasma gondii, SAG1. This synthetic peptide carrying three predicted H-2k restricted T cell epitopes was used to immunize mice. The protective effect of the peptide was evaluated in CBA/J and C57BL/6 mice using the decrease in brain cyst load as evidence of protection. Immunization of C57BL/6 mice yielded high antibody titres but had no protective effect after oral challenge. Immunized CBA/J, mice which responded with a lower titre, showed a 35% reduction in cyst burden after oral challenge. Both strains yielded antibodies which recognized the cognate SAG1 protein on immunoblot assay. Using the BIAcore, system, it was shown that at lower titres the CBA/J mouse sera recognized the native SAG1 protein more effectively than the C57BL/6 mouse sera, yielding much higher anti-peptide titres. Lymphoproliferation assays using the peptide experimentally confirmed the predicted T-cell epitopes and showed that they were also recognized by cells of T. gondii infected mice. The anti-peptide subclass analysis suggested a Th1 orientation in CBA/J mice, whereas a Th2 orientation was observed in C57BL/6 mice. Finally, fine analysis of sequences recognized under MHC class I indicated the existence of a T-cell epitope in the H-2k haplotype (CBA/J mice) but not in the H-2b haplotype (C57BL/6 mice). This study provides a structural basis to the understanding of the vaccination response to one of the T. gondii antigens in different strains of mice.

V beta 8.2 transgene expression interferes with development of experimental autoimmune thyroiditis in CBA k/q but not k/k mice.

The thyroiditogenic T cell receptor (TCR) repertoire is not yet well defined in murine experimental autoimmune thyroiditis (EAT). Our recent work has shown that, while V beta 8+ T cells have no major role in EAT induction with mouse thyroglobulin (MTg), V beta 13 may be involved. To examine the effect of skewing the TCR repertoire on EAT development, CBA (H2k) mice were mated with B10.Q mice harboring an ovalbumin-specific V beta 8.2 TCR transgene (trg), and the trg+ mice were backcrossed to CBA. FACS analysis showed that peripheral blood T cells from trg+ mice had about 76 and 90% V beta 8.2+ cells in the CD4+ and CD8+ subsets, respectively, compared with about 15 and 11% in trg- sibs. The transgenic CBA k/k and k/q mice were immunized with MTg and sacrificed 28 days later. In all trg+ mice, anti-MTg titers and T cell proliferative responses to MTg were significantly lowered. However, thyroid infiltration was distinctly different in the two strains of transgenic mice; a significant decrease was seen primarily in k/q, but not k/k, trg+ mice. Thus, skewing the TCR repertoire to overexpress an irrelevant TCR revealed the possession of a less flexible thyroiditogenic TCR repertoire in k/q, but not k/k, mice.

Influence of the Ity/Lsh/Bcg gene on the development of suppressor cell precursors in the early phase of the infection of mice with Mycobacterium lepraemurium.

In vitro inducible suppressor cell precursors were detected in the spleen of BALB/c but not in DBA/2 mice infected intraperitoneally with 10(8) Mycobacterium lepraemurium bacilli, thus suggesting that their development is genetically controlled. Two pairs of mouse strains congenic at the Ity/Lsh/Bcg locus (BALB/c-C.D2 and B10.A-B10.A.Bcgr) were used to investigate whether this phenomenon is influenced by this gene known to control the relative susceptibility of mice to Myco. lepraemurium infection. This seems likely, as the detection of culture-induced suppressor activity was delayed for 5-6 weeks in C.D2 and B10.A.Bcgr mice infected intravenously with 10(4) Myco. lepraemurium bacilli. However, despite the retardation in the detection of suppressor cell precursors, the level of in vitro induced suppressor activity at onset in spleen cell suspensions of mice carrying the resistant allele was higher than in cell cultures derived from susceptible mice. As the resistant allele has a different effect when found on BALB/c or DBA/2 background, other genetic factors are apparently involved in the development of suppressor cell precursors. We finally observed that, in spleen cell cultures from intravenously infected Ity/Lsh/Bcg congenic mice on the BALB/c background, adherent and non-adherent cells were required in the inductive phase of suppressor cell development, whereas in vitro induced suppressor activity was found exclusively in the adherent cell fraction. Given these properties, we thus conclude that suppressor cell precursors detected in the spleen of these intravenously infected mice are similar to those previously observed in C3H mice infected intraperitoneally with a thousand times more bacilli.

[The effect of serotonin antibodies on the development of the alcohol abstinence syndrome in CBA mice]. / Vliianie antitel k serotoninu na razvitie alkogol'nogo abstinentnogo sindroma u myshei linii CBA.

Experiments on CBA mice with alcohol withdrawal syndrome showed a reduction of abstinence after injection of serotonin antibodies in dose 5 and 10 mg intraperitoneally. One more result was a decrease of horizontal locomotory activity after injection of 5 mg of serotonin antibodies to mice with 24 hour withdrawal syndrome.

Occurrence of a soluble nonspecific suppressor factor in the serum early after birth.

A nonspecific suppressor factor has been identified in serum of newborn rats and calves. This factor, designated SUF-s, was shown to interfere--across species barriers--with lymphocyte responses in vitro and in vivo. SUF-s interferes in vitro with T- and B-cell proliferation induced by different mitogens and IL-2. Our findings indicate that the activity of SUF-s in vitro, which is of a reversible nature, is directed at an early event in the cascade of T-cell activation. SUF-s does not affect intrinsically regulated proliferation, such as that of tumor cells or established cell lines. In vivo, SUF-s prevents graft-vs-host disease induced by transplantation of allogeneic bone marrow cells in lethally irradiated mice. Using of affinity chromatography, hydrophobic interaction chromatography, and gel filtration, a 15,000-fold purification of the suppressive factor was attained. The moiety engaged in suppression was identified as a 20- to 40-kDa protein. The biological activity is destroyed at temperatures above 70 degrees C, by proteolytic enzyme digestion and under alkaline conditions but was resistant to acidic and reducing conditions. Judged by its biological activity and some of its physical properties, SUF-s is most likely distinct from other described suppressor factors or known cytokines with suppressor activity, such as IL-4, IL-10, interferon-gamma, transforming growth factor-beta or alpha-fetoprotein.

Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge.

To determine if the food-grade bacterium Lactococcus lactis holds promise as a vaccine antigen delivery vector we have investigated whether this bacterium can be made to produce high levels of a heterologous protein antigen. A regulated expression system has been developed which may be generally suitable for the expression of foreign antigens (and other proteins) in L. lactis. The system utilizes the fast-acting T7 RNA polymerase to transcribe target genes, and provides the first example of the successful use of this polymerase in a Gram-positive bacterium. When the performance of the expression system was characterized using tetanus toxin fragment C (TTFC) up to 22% of soluble cell protein was routinely obtained as TTFC. Mice immunized subcutaneously with L. lactis expressing TTFC were protected from lethal challenge with tetanus toxin. These results show for the first time that L. lactis is able to express substantial quantities of a heterologous protein antigen and that this organism can present this antigen to the immune system in an immunogenic form.

The expression of CD45RB on antigen-responsive CD4+ lymphocytes: mouse strain polymorphism and different responses to distinct antigens.

The levels of CD45RB expression by HGG-specific CD4+ cells residing in the Ag-draining lymph nodes of HGG-primed CBA/CaJ mice were analyzed. When sorted populations of CD4+, CD45RBhi, and CD4+, CD45RBlo cells were cultured with HGG and Ag-presenting cells, the majority of the proliferative response was found in the CD45RBlo fraction early after in vivo priming (Day 6), and this pattern remained stable through 12 days postpriming. To determine whether this segregation of responsiveness was consistent in other mouse strains, HGG-primed C57BL/6J mice were similarly analyzed. In contrast to findings with the CBA/CaJ strain, the CD4+, CD45RBhi cell fraction obtained from C57BL/6J mice was the predominant responding population early after in vivo priming (Day 6); however, there was a parallel increase in responsiveness of CD4+, CD45RBhi, and CD4+, CD45RBlo cells by Day 12. Thus, there was not a decrease in CD45RBhi expression with a concommitant increase in CD45RBlo expression in CD4+ cells proliferating to HGG. Despite the heterogeneity in CD45RB expression by the primed CD4+ cells of the two strains, the entire proliferative response to HGG early after priming resided in the fraction bearing high levels of membrane CD44, thus arguing for the existence of CD45RBhi, CD44hi and CD45RBlo, CD44hi cells during the early phase of the response. In both mouse strains the CD4+, CD45RBhi subset of primed lymph node cells produced significant levels of IL-2 in response to HGG and APC, whereas no significant IL-2 or IL-4 production was detectable in HGG-stimulated CD45RBlo cells of either strain. The CD4+, CD45RBhi subset also proliferated more vigorously in response to polyclonal activation than the CD4+ CD45RBlo fraction. To examine whether the patterns of CD45RB expression on HGG-primed cells from C57BL/6J mice were common to other antigens, the response profiles were examined after in vivo priming with a second antigen, KLH. In contrast to studies with HGG as the Ag, the proliferative response to KLH in C57BL/6J mice was evenly divided among the CD45RBhi and CD45RBlo fractions on Day 8 after priming, but shifted markedly to the CD45RBlo fraction by Day 12 after priming. Taken together, these data show that the patterns of CD45RB expression on primed populations of CD4+ cells can exhibit mouse strain polymorphism and can differ depending on the choice of antigen for immunization.

Anti-Mlsa-like effect of CBA/J anti DBA2 antibody in in vitro MLRs between Mls incompatible combinations.

Antisera from CBA/J (H-2k,Mlsa) mice hyperimmunised with DBA2 (H-2d,Mlsa) and/or AKR (H-2k,Mlsa) splenocytes, can block in vitro MLRs against Mlsa determinants. These two antisera are furthermore specifically cytotoxic against targets expressing H-2 components but not against Mlsa-bearing targets. In these experimental models the anti-Mlsa-like effects of CBA/J anti-DBA2 and CBA/J anti AKR antibodies seem to be associated with an autoreactive immune response in CBA/J mice elicited by DBA2 and AKR splenocytes, respectively. In this report we propose that anti-Mlsa antibodies can also be generated in a situation in which Mlsa is presented as 'altered self' in conjunction with allo-determinants.