Reciprocal influence of the graft vs. host reaction and pregnancy]

The graft versus host reaction (GVHR) was induced in mouse females-hybrids F1 (CBA X C57BL/6) by intravenous injection of suspension of the lymphoid cells of the spleen and of lymphoid nodes from C57BL/6 mouse females. Pregnancy resulted from interbreeding of the test females with syngenic males 1--5 days before, and 1--10, 10--20, 30--40 and more than 40 days after the moment of the lymphoid cells injection, aggravated the GVHR induced transplantation disease. At the same time the GVHR under these conditions decreased the percentage of pregnant animals and brought to child-bearing disfunction of the test animals (stillbirth, death of pregnant females, miscarriage). In some of the test mice aggravation of the GVHR was observed after delivery. Survival of the progeny decreased.     

Alloreactive lymphocytes from T cell receptor (beta-chain) transgenic mice do not mediate a graft-versus-host reaction.

The injection of mature T cells into a tolerant or immunocompromised allogeneic host animal produces a graft versus host response (GVHR) that can result in splenomegaly, immunosuppression and death of the host animal. We demonstrate here that lymphocytes from T cell receptor beta-chain (TCR-beta) transgenic mice, in which the expression of the transgene inhibits endogenous beta- and gamma-gene rearrangements and thus causes abnormal T cell development, are unable to mediate a GVHR. The GVHR was measured after the injection of lymphocytes from transgenic mice into normal F1 mice and also after transplantation of bone marrow and lymphocytes from transgenic mice into lethally irradiated F1 recipients. In both systems, cells from transgenic mice failed to produce a significant GVHR. Cells from the transgenic mice were able to recognize the foreign histocompatibility Ag of the host in vitro and in vivo although the transgenic mice rejected skin grafts more slowly than controls. Thus, lymphocytes from transgenic mice were unable to produce a GVHR despite the presence of alloreactive T cells. These results suggest that lymphocytes from TCR-beta transgenic mice fail to mediate a GVHR either because lymphocytes with a single transgenic TCR-beta chain have a limited ability to recognize allogeneic cells in vivo or because the transgenic mice lack lymphocyte subsets that are important for the mediation of a GVHR.     

Status of T- and B-lymphocytes in long living CBA--F1 (CBA X C57BL/6) chimeras]

Semiallogeneic chimeras were produced by injecting 3 X 10(7) spleen cells of mice CBA (H--2k, Mlsd) to lethally irradiated mice (CBA X C57BL/6)F1. Two days later recipients were given cyclophosphamide (CP), 2 mg per mouse, to prevent death of graft versus host reaction (GVHR). For 1.5--2 months after the creation of chimerism in 23 of 26 mice under study all cells producing antibodies to SRBC were represented by donor cells of H-2 phenotype; 3 mice were partial chimeras. Spontaneous blast transformation in the cultures of chimera spleen did not exceed the control level, and in the mixed lymphocyte culture chimera cells failed to proliferate on addition of irradiated lymphocytes (CBA X C57BL/6) F1. At the same time chimera gave intensive blast transformation to the irradiated lymphocytes of the third line of mice DBA/2 (H--2d, Mlsa). Among the chimera spleen cells no killers capable of destroying target cells of donor or recipient origin were revealed. Similar results were obtained in vivo: chimera cells gave no positive local GVHR after administration to mice (CBA X C57BL/6) F1. Prolonged chimerism was accompanied by a reactivity of donor T-lymphocytes to the recipient transplantation antigens. A blocking factor was revealed in the blood serum of chimeras. The substitution of donor lymphocytes for the recipient cells begins after 3 to 5 months. At the same period donor T-cell population reconstitutes partially the responsiveness to the recipient antigens and the blocking factor disappears from chimeras blood.     

Fetal death persists through recurrent pregnancies in mice following Ljungan virus infection

OBJECTIVES: Laboratory mice infected with Ljungan virus (LV) early in pregnancy suffer from perinatal death. Here we investigate the persistence of that effect through the outcome of consecutive pregnancies in LV‐infected mice. STUDY DESIGN: CD‐1 mice were infected while pregnant and their adult female offspring were followed in parallel with uninfected control mice during repeated pregnancies. Three mating attempts resulted in two or three pregnancies per dam. The outcome of the last pregnancy was carefully monitored. RESULTS: Both the dams infected as adults and their adult female offspring suffered perinatal deaths during the last pregnancy which occurred approximately 6 months after the original LV exposure and acute infection. The non‐infected control animals experienced no perinatal death. CONCLUSIONS: Perinatal death persists across recurrent pregnancies in this mouse model of LV infection, both in animals infected as adults and in females exposed to the virus in utero. This implies that LV persists in mice long after intial infection, and is maintained in a quiescent state but can remain pathogenic in later pregnancies. Birth Defects Res (Part B) 83:507‐510, 2008. © 2008 Wiley‐Liss, Inc.     

Differences in thymus dependency among the alloreactive T-cell subpopulations in their development

Effects of thymectomy at various times after birth (Tx-1, Tx-3, Tx-7, Tx-14) on mixed lymphocyte reaction (MLR), cell-mediated cytotoxicity (CMC), and graft-versus-host reaction (GVHR) were examined by experiments with spleen cells obtained at 8 weeks of age. All of MLR, CMC, and GVHR were detected in spleen cells of Tx-14 mice. However, the ability of spleen cells to induce GVHR was abolished by thymectomy at 7 days after birth. On the other hand, MLR and CMC were not affected in such mice. In Tx-1 or Tx-3 mice, only MLR was detected in spleen cells. These results suggest that thymus dependency of T cells responsible for MLR are lower in their maturation than those for CMC and that thymus dependency of T cells responsible for GVHR are the highest among these T-cell subpopulations.     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Heparin exerts anti-apoptotic effects on uterine explants by targeting the endocannabinoid system

Miscarriage caused by Gram-negative bacteria infecting the female genital tract is one of the most common complications of human pregnancy. Intraperitoneal administration of LPS to 7-days pregnant mice induces embryo resorption after 24 h. Here, we show that LPS induced apoptosis on uterine explants from 7-days pregnant mice and that CB1 receptor was involved in this effect. On the other hand, heparin has been widely used for the prevention of pregnancy loss in women with frequent miscarriage with or without thrombophilia. Besides its anticoagulant properties, heparin exerts anti-inflammatory, immunomodulatory and anti-apoptotic effects. Here, we sought to investigate whether the administration of heparin prevented LPS-induced apoptosis in uterine explants from 7-days pregnant mice. We found that heparin enhanced cell survival in LPS-treated uterine explants and that this effect was mediated by increasing uterine FAAH activity. Taken together, our results point towards a novel mechanism involved in the protective effects of heparin.     

Suppression of interferon synthesis during the "graft-versus-host" reaction in F1(CBAXC57BL/6) mice]

The development of the graft-versus-host reaction (GVHR) in the F1(1CBA X C57BL/6 hybrid mice after the transplantation of spleen cells from the C57BL/6 parent donor resulted in a strong inhibition of the serum interferon production induced by the intraperitoneal injection of the Newcastle disease virus. In vitro with the mouse bone marrow cells during the development of the GVHR the interferon response was first reduced and then disappeared completely. The described phenomenon could therefore serve as an index of the development of the GVHR.     

Effects of the chromatographic fractions of the pig placental trophoblast on graft-versus-host reaction

The trophoblast has a significant role in regulation of immune reactions at the materno-fetal interface by producing biologically active substances. In our previous studies five fractions with immunomodulatory activities were isolated by gel chromatography from trophoblast of pig placentas. To confirm the immunomodulatory effect of these trophoblast fractions on allogeneic in vivo systems and to obtain more evidence for the relevance of their activity on the maternofetal interface, their effect was studied on graft-versus-host reaction (GVHR). To assess the GVHR, the primary and secondary popliteal lymph nodes assay was used in mice. In the primary GVHR, 100 microg protein of Fraction 2-5, mixed with 5 x 10(6) allogeneic spleen cells (C57BL/6), were injected into one of the foot pads of recipient (BALB/c) mice. The secondary GVHR was induced in F1 (BALB/c x C57BL/6) mice by injection of spleen cells of BALB/c mice intraperitoneally preimmunized with allogeneic cells. The GVHR was measured by the weight of lymph nodes and by the lymphocyte proliferation. Flow cytometric analyses of the cells in the nodes with GVHR and under the influence of Fraction 4 or 5 were performed using monoclonal antibodies. In the primary GVHR, Fraction 4 or 5, injected simultaneously with allogeneic spleen cells, significantly suppressed the lymph nodes reactivity. Fractions 4 and 5 inhibited the ability of the spleen cells of mice intraperitoneally preimmunized with allogeneic cells to induce secondary GVHR in F1 mice. The Fraction 2 and 3 had no effect on GVHR. The results revealed that a group of proteins with Mr 37-7 kDa, isolated from trophoblast of pig placenta, strongly suppressed popliteal lymph node reactivity in the primary and secondary GVHR. The data provide convincing evidence for these fractions in vivo activity, for their effect across the species barrier and suggest the relevance of the same reactions on the materno-fetal interface.     

Characteristics of early autoimmune reaction in vaccinated and revaccinated animals

Experiments were conducted on albino mongrel female mice weighing 18 to 22 g. Development of early autoimmune reaction in response to the primary administration of microbial antigens was revealed; its intensity and duration depended on the dose of the antigen. Injection of physiological saline of the same or greater volume led only to the short-lived increase in the count of the autoantibody synthesizing cells in the spleen tissue. In repeated contact with the given antigen, there was noted a marked intensification of the reaction to homologous antigen, and a depression--to heterologous one in 7 to 14 days. Reaction to the administration of physiological saline alone failed to alter.     

Sequential changes of thymocyte surface antigens with presence or absence of graft-vs-host reaction following allogeneic bone marrow transplantation

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2^b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.     

Hyaluronic capsule as one of the factors of hemolytic streptococcal pathogenicity]

Experiments were conducted on albino mice. It was shown that treatment of streptococci with testicular hyaluronidase or a single administration of the enzyme to infected animals failed to inhibit the infectious process. Injection of hyaluronidase solution to every 3 or 4 hours depressed the development of the process and increased the percentage of survived animals during its first stages. A marked intensification of the hyaluronidase action inhibiting the infectious process was observed under conditions of a moderately active or passive immunity and also in the case of preliminary treatment of streptococci with homologous immune serum. The data obtained permit to regard the hyaluronic capsule in the hemolytic streptococci as one of the pathogenicity factors of this microbial species providing survival of the causative agent after its entrance into the macroorganism.     

Mls-1a-induced peripheral tolerance to host minor histocompatibility antigens in radiation bone marrow chimeras. Modification of T cell repertoire associated with active suppression and permanent presentation of host antigens.

Minor histocompatibility Ag (mHAg) can be responsible for the development of graft vs host reaction (GVHR) after bone marrow transplantation. In a mouse model, B10.D2 donor immunization against Mls-1a prevents lethal GVHR developed by CD4+ T cells against DBA mHAg in irradiated (DBA/2 x B10.D2)F1 hosts. Such F1 hosts become 100% chimeric and show long term survival (LS mice). The cellular mechanisms underlying the tolerance in LS mice was investigated. It was found that a state of tolerance can be induced in thymectomized F1 hosts. Although spleen cells from LS mice are able to initiate lethal GVHR in third-party H-2k-incompatible hosts, no GVHR is observed in secondary hosts incompatible for specific DBA/2 mHAg. Mixed lymphocyte experiments in vitro confirm that T cells from LS mice are unresponsive toward specific DBA/2 mHAg, although they are able to proliferate in response to H-2 or Mls-1a Ag. The responsiveness to Mls-1a correlates with the presence of V beta 6+ cells in LS mice, probably derived from mature T cells present in the donor inoculum. The tolerance in LS mice is not due to the lack of DBA/2 mHAg presentation; instead, permanent presentation of Ag (Ag I and Ag II) previously described as being responsible for lethal GVHR is consistently observed. A significant protection against GVHR is obtained by transferring normal B10.D2 cells together with spleen cells from LS mice, clearly indicating the contribution of active suppression in the state of tolerance; this is further confirmed by in vitro results obtained in limiting dilution assays. It is concluded that tolerance in chimeric LS mice 1) is due to a peripheral (thymus-independent) mechanism; 2) is specific for mHAg; 3) correlates with unresponsiveness of the repertoire to host mHAg, without alteration of the repertoire for H-2 and Mls-1a Ag; and 4) is associated with an active suppression and with a permanent presentation of at least two mHAg responsible for GVHR mortality.     

Study of the immunotropic activity of aminoglycoside antibiotics

The action of some aminoglycoside antibiotics on the immune system was studied on both intact mice and the animals with immune deficiency caused by administration of cyclophosphamide. The following tests were used: local hemolysis (the Herne test), lymphocyte transformation (LT), delayed hypersensitivity to sheep red blood cells and the local graft versus host reaction (GVHR). Amikacin was shown to have no significant action on the activity of lymphocytes in the intact mice and stimulated both cellular (LT and GVHR) and humoral (the Herne test) immunity in the animals with lowered immunological reactivity. Sisomicin had no significant action on the immune system of the animals. Gentamicin suppressed the immune response only in the intact mice. Kanamycin and streptomycin induced inhibition of humoral and cellular immunity in both the intact mice and animals with immune deficiency. On the basis of the results it was concluded that gentamicin, amikacin and sisomicin may be used in the treatment of diseases developing in the presence of immune deficiency whereas streptomycin and kanamycin should be recommended when inhibition of the immunity is needed.     

Appearance of a syndrome similar to graft versus host reaction in C57 B1/6 mice bearing skin allogenic graft.

A syndrome similar to GVHR is described in mice of C57B1/6 strain during the WHT/Ht skin allografts rejection period. In all the cases the described thymus involution was associated with the hypertrophy of lymph nodes. Their volume increase is due to the high number of blastic pyroninophilic and plasma cells concomitant with small lymphocytes depletion in the cortical area, and with a very pronounced hypertrophy of medullary cords by presence of a high number of plasmocytes and blastic cells. These changes have been noticed only in some animals sacrified during the first days after grafting and never later one. In agreement with the scarce data of the literature, we think that the immunocompetent passenger lymphocyte comprised in the skin grafts constitute an immunologic organ able to induce a GVHR at the beginning of the period of graft survival on the host. This GVHR, generally mild and without clinical and microscopic signs, becomes obvious only in animals which, for unknown reasons, present a low immunologic defense capacity. In these animals the described process seems to be reversible.     

Changes in lymphocyte and macrophage subsets due to morphine and ethanol treatment during a retrovirus infection causing murine AIDS

Infection by LP-BM5 murine leukemia virus (MuLV) suppressed significantly the percentage of peripheral blood cells showing surface markers for macrophages, lymphocytes and activated lymphoid cells. Chronic administration of a 7% (36% calories) ethanol diet or injection of 1.9 mg/mouse/day of morphine for a 7 day period were followed by 3 week periods of abstinence and then 1 week periods of consumption of 5% ethanol diets or morphine injection to female C57BL/6 mice resulted in changes in the numbers of macrophages and lymphocyte subsets. The number of lymphocytes of various subsets were not significantly changed by the ethanol exposure except those showing activation markers which were reduced. The percentage of peripheral blood cells showing markers for macrophage functions and their activation were significantly reduced after "binge" use of ethanol. Ethanol retarded suppression of cells by retroviral infection. However by 25 weeks of infection there was a 8.6% survival in the ethanol fed mice infected with retrovirus which was much less than virally infected controls (45.0%). Morphine treatment also increased the percentage of cells with markers for macrophages and activated macrophages in virally infected mice, while suppressing them in uninfected mice. The second and third morphine injection series suppressed lymphocyte T-helper and T-suppressor cells, but not total T cells. However, suppression by morphine was significantly less during retroviral disease than suppression caused by the virus only. At 25 weeks of infection 44.8% of morphine treated, infected mice survived. Morphine treatment also caused deaths such that the survival in morphine treated, retrovirally infected was higher than would have been expected if the death rate in virally infected, and morphine injected animals were combined during combined treatment. Thus these drugs of abuse can modulate peripheral blood lymphoid subsets, suppression caused by retroviral infection, and survival.     

Effect of prodigiosin and its combination with immunodepressants on the graft versus host reaction in mice

The local (lymph node) graft-versus-host reaction (GVHR) in F1 (CBA X C57BL/6) mice and the lethal GVHR in C57BL/6 mice were induced by transfer of lymph node cells of CBA mice with skin allotransplants from C57BL/6 mice. Prednisolone in combination with asathioprin (imuran) administered to CBA mice inhibited the GVHR. Prodigiosan used alone was not active, while in combination with immunodepressants it increased their inhibitory effect. Adhesive cells with a suppressive activity were detected in the spleen of mice treated with prodigiosan. Such cells were capable of suppressing the capacity of syngeneic lymphocytes for inducing the GVHR.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Selected contribution: Effects of gender on reduced-size liver ischemia and reperfusion injury.

Hepatic resection with concomitant periods of ischemia and reperfusion (I/R) is a common occurrence in resectional surgery as well as reduced-size liver transplantation (e.g., split liver or living donor transplantation). However, the I/R induced by these types of surgical manipulations may impair liver regeneration, ultimately leading to liver failure. The objectives of the study were to develop a murine model of reduced-size liver I/R and assess the role of gender in this model of hepatocellular injury. We found that 100% of female mice survived the surgery indefinitely, whereas all male mice had greater initial liver injury and died within 5 days after surgery. The protective effect observed in females appeared to be due to ovarian 17beta-estradiol, as ovariectomy of females or administration of a selective estrogen antagonist to female mice resulted in enhanced liver injury and greater mortality following reduced-size liver I/R. Conversely, 17beta-estradiol-treated male mice exhibited less hepatocellular damage and survived indefinitely. Taken together, these data demonstrate an estrogen-mediated protective pathway(s) that limits or attenuates hepatocellular injury induced by reduced-size liver I/R.     

Depletion of CD8+ cells exacerbates organ-specific autoimmune diseases induced by CD4+ T cells in semiallogeneic hosts with MHC class II disparity

Autoimmune diseases are known to be induced in some donor-recipient combinations of mice undergoing the graft-vs-host reaction (GVHR). In this paper, we report on the development of primary biliary cirrhosis (PBC)-like hepatic lesions and also on pancreatic insulitis in (B6 x bm12)F1 mice injected with B6 CD4+ T cells. At the sites of these lesions, cellular infiltration around ductal structure was observed. Immunohistochemical studies revealed that both CD4+ and CD8+ T cells were present in the lesions of the liver and pancreas. To clarify the role of the CD8+ T cells, which were probably of host origin, we used a mAb against the Lyt-2 molecule. Both the PBC-like hepatic lesions and pancreatic insulitis were exacerbated by eliminating CD8+ T cells from mice with MHC class II GVHR. Also, autoantibodies against the pyruvate dehydrogenase-E2 component, which has been recently found to contain an immunodominant site (autoepitope) for B cell reactivity in patients with PBC, were detected in the sera of these mice by ELISA and their presence was confirmed by immunoblotting procedures. Our findings suggest that similar mechanisms as in GVHR caused by MHC class II disparity are active in the development of PBC. It should also be noted that, in addition to the hepatic lesions, insulitis closely resembling that seen in the nonobese diabetic mouse was induced in our experimental system. The results suggest that our model provides a unique opportunity to study organ-specific autoimmune diseases. Because the effector in our experimental system was defined to be CD4+ T cells responding to Iabm12 Ag, our findings support the hypothesis that an excessive immune response directed against Ia Ag can produce autoimmune disease.