Induction of heterotopic and orthotopic cartilage and bone formation in mice

Heterotopic cartilage, bone and bone-marrow formation was achieved in mice by transplantation of a variety of xenogeneic established cell lines, by the transitional epithelium or by implants of demineralized bone matrix. The pattern and the sequence of events were always the same, regardless of the inducer used; viz., hyaline cartilage appeared 6-7 days after implantation, and endochondral bone formation followed. However, in cases of allogeneic implants of transitional epithelium into species other than the mouse, an intramembranous osteogenesis was the main mode of bone formation. When the yield of induced bone was high enough, a true myelopoiesis developed after three weeks. Heterotopically-induced bones had a relatively short life-span. Periosteal membranes of bones at the sites of sarcomes induced by M-MSV responded with rapid and extensive proliferation, with subsequent bone and, sometimes, hyaline cartilage deposition. This phenomenon was observed in long and cranial bones. However, bone induced heterotopically by demineralized bone matrix did not respond in such a way to the presence of M-MSV-induced sarcoma, suggesting that the connective tissue-encapsulated heterotopic bone was not a functioning periosteum. M-MSV-induced sarcoma also stimulates proliferation of elastic cartilage.     

Validation of Bone Conversion in Osteoconductive and Osteoinductive Bone Substitutes

Bone reconstruction can be performed with an autogeneic graft from various donor regions. Osteoconductive and osteoinductive bone substitutes originate from substances of diverse chemical and morphological types and can have a synthetic or a biological derivation. Alongside autogeneic bone transplants and allogenic and xenogeneic bone implants, alloplastic bone replacements of synthetic or semi-synthetic origin are being used for defect reconstruction. In an animal model in rabbits five bone substitutes and one autogeneic graft were surgically incorporated into identical bone defects (10times 10 mm in size) in six anatomically defined regions of the skull. With scintigraphic and histological methods, the metabolic dynamics of the bone is examined as it reacts to the transplantation of autogeneic bone or to implanted bone replacement material. The different autogeneic, xenogeneic and alloplastic bone replacement materials can be differentiated according to the functional quality of the new tissue and the dynamics of the bone conversion thus induced. In the comparison of mineralized, osteoconductive bone subsitutes (TCP, HA, calcium carbonate ceramics) with demineralized, osteoinductive implants (DBM new, DBM old) and autogeneic bone grafts, the bone inducing matrices show the largest quantity of new bone formation, making possible a volume-constant reconstruction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mixed xenogeneic chimerism induces donor-specific humoral and cellular immune tolerance for cardiac xenografts

Xenotransplantation has been suggested as a potential solution to the critical shortage of donor organs. However, success has been limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. Mixed xenogeneic bone marrow chimeras resulting from the transplantation of a mixture of host and donor marrow (B10 mouse + F344 rat --> B10 mouse) results in donor-specific cross-species transplantation tolerance for subsequent nonvascularized skin and islet grafts. Furthermore, compared with fully xenogeneic chimeras (rat --> mouse), mixed xenogeneic chimeras exhibit superior immunocompetence for infectious agents in vivo and in vitro, suggesting that the immune system is intact. The ability to establish long-term humoral and cellular tolerance for primarily vascularized xenografts in vivo, in the setting of both recipient and donor Ig and effector cell production, has not previously been characterized. Mixed xenogeneic chimeras exhibit donor-specific humoral tolerance as evident by the absence of anti-donor Ab and Ab-dependent donor-specific cytotoxicity in vitro and intravascular IgM deposition within donor-strain (F344) cardiac xenografts in vivo. F344 cardiac xenografts are accepted (median > or =180 days) without clinical or histologic evidence of rejection, suggesting cellular tolerance. In contrast, MHC-disparate third-party mouse (B10.BR) and rat (ACI or WF) grafts are rejected (median of 23 and 41 days, respectively) in association with extensive mononuclear cell infiltration and vascular deposits of mouse IgM. These results demonstrate that mixed xenogeneic chimerism establishes donor-specific humoral and cellular tolerance and permits the successful transplantation of even primarily vascularized xenografts in the setting of intact Ab production.     

Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning

Hematopoietic stem cells have been successfully employed for tolerance induction in a variety of rodent and large animal studies. However, clinical transplantation of fully allogeneic bone marrow or blood-borne stem cells is still associated with major obstacles, such as graft-versus-host disease or cytoreductive conditioning-related toxicity. Here we show that when rat embryonic stem cell-like cells of WKY origin are injected intraportally into fully MHC-mismatched DA rats, they engraft permanently (>150 days) without supplementary host conditioning. This deviation of a potentially alloreactive immune response sets the basis for long-term graft acceptance of second-set transplanted WKY cardiac allografts. Graft survival was strictly correlated with a state of mixed chimerism, which required functional thymic host competence. Our results provide a rationale for using preimplantation-stage stem cells as vehicles in gene therapy and for the induction of long-term graft acceptance.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Blockade of CD28-B7, but not CD40-CD154, prevents costimulation of allogeneic porcine and xenogeneic human anti-porcine T cell responses

Despite increasing use of swine in transplantation research, the ability to block costimulation of allogeneic T cell responses has not been demonstrated in swine, and the effects of costimulatory blockade on xenogeneic human anti-porcine T cell responses are also not clear. We have compared the in vitro effects of anti-human CD154 mAb and human CTLA4IgG4 on allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses. Both anti-CD154 mAb and CTLA4IgG4 cross-reacted on pig cells. While anti-CD154 mAb and CTLA4IgG4 both inhibited the primary allogeneic pig MLRs, CTLA4IgG4 (7.88 microg/ml) was considerably more inhibitory than anti-CD154 mAb (100 microg/ml) at optimal doses. Anti-CD154 mAb inhibited the production of IFN-gamma by 75%, but did not inhibit IL-10 production, while CTLA4IgG4 completely inhibited the production of both IFN-gamma and IL-10. In secondary allogeneic pig MLRs, CTLA4IgG4, but not anti-CD154 mAb, induced Ag-specific T cell anergy. CTLAIgG4 completely blocked the indirect pathway of allorecognition, while anti-CD154 mAb blocked the indirect response by approximately 50%. The generation of porcine CTLs was inhibited by CTLA4IgG4, but not by anti-CD154 mAb. Human anti-porcine xenogeneic MLRs were blocked by CTLA4IgG4, but only minimally by anti-CD154 mAb. Finally, CTLA4IgG4 prevented secondary xenogeneic human anti-porcine T cell responses. These data indicate that blockade of the B7-CD28 pathway was more effective than blockade of the CD40-CD154 pathway in inhibiting allogeneic pig T cell responses and xenogeneic human anti-pig T cell responses in vitro. These findings have implications for inhibiting cell-mediated immune responses in pig-to-human xenotransplantation.     

Xenogeneic mixed leucocyte response

Xenogeneic mixed leucocyte cultures composed of human, chimpanzee, baboon, goat, sheep, pig, and dog cells were set up with a variety of plasma culture supplements. The characteristics of the human leucocyte response to xenogeneic cells was similar to its response to allogeneic cells. Peak response to xenogeneic stimulation occurred on the same culture day as the peak response to allogeneic stimulation. Similar numbers of xenogeneic and allogeneic cells produced peak stimulation of cells from any one individual. There was, however, a wide variation in the response of human lymphocytes to both allogeneic and xenogeneic cells. A factor in the plasma supplement specific for responding or stimulating cells inhibited the mixed leucocyte response in some combinations and could be removed by absorption techniques.     

Influences of vascularization and osteogenic cells on heterotopic bone formation within a madreporic ceramic in rats

Research in biomaterials for bone reconstruction has led to elaborate osteogenic composites that combine porous ceramics with bone marrow stromal cells. The aim of this study was to evaluate the influence of direct vascularization of such composites on osteogenesis and the ability to produce a vascularized bone substitute transplant in an ectopic muscular site. Sixty-four coralline biomaterials were implanted in 32 Fisher rats under four conditions: (1) alone (reference group M, n = 16), (2) coated with bone marrow stromal cells (group MC, n = 16), (3) combined with a vascular pedicle (group MV, n = 16), or (4) coated with bone marrow stromal cells and combined with a vascular pedicle (MCV group, n = 16). The number of vessels in the pores (vessel-pore ratio) of the implants and the proportion of pores showing bone ingrowth (bone-pore ratio) were measured at 2, 4, 6, and 8 weeks on four implants of each group. Compared with the reference group, angiogenesis was higher when the biomaterial was combined with a vascular pedicle or was coated with osteoprogenitor cells. The association of both vascular pedicle and osteoprogenitor cells increased vascularization by 60 percent (p = 0.003) and osteogenesis by 62 percent (p < 0.001). A combination of both vascular pedicle and bone marrow osteoprogenitor cells in coralline implants enhances neovascularization and osteogenesis after implantation in ectopic intramuscular sites to a greater extent than either does alone.     

Human neural stem cells and astrocytes,but not neurons,suppress an allogeneic lymphocyte response

Transplantation of human neural stem cells (NSCs) and their derivatives is a promising future treatment for neurodegenerative disease and traumatic nervous system lesions. An important issue is what kind of immunological reaction the cellular transplant and host interaction will result in. Previously, we reported that human NSCs, despite expressing MHC class I and class II molecules, do not trigger an allogeneic T cell response. Here, the immunocompetence of human NSCs, as well as differentiated neural cells, was further studied. Astrocytes expressed both MHC class I and class II molecules to a degree equivalent to that of the NSCs, whereas neurons expressed only MHC class I molecules. Neither the NSCs nor the differentiated cells triggered an allogeneic lymphocyte response. Instead, these potential donor NSCs and astrocytes, but not the neurons, exhibited a suppressive effect on an allogeneic immune response. The suppressive effect mediated by NSCs most likely involves cell–cell interaction. When the immunogenicity of human NSCs was tested in an acute spinal cord injury model in rodent, a xenogeneic rejection response was triggered. Thus, human NSCs and their derived astrocytes do not initiate, but instead suppress, an allogeneic response, while they cannot block a graft rejection in a xenogeneic setting.     

Xenogeneic rejection among three botryllids (compound Ascidians)

Xenogeneic rejection was observed among colonies of three botryllids, Botryllus scalaris, Botryllus primigenus, and Botrylloides simodensis. Allogeneic recognition occurs in each of these species, but the manner of allogeneic rejection differs among them. We studied xenogeneic rejection reactions among these species under the following conditions: colony contact at natural growing edges, colony contact at artificially cut surfaces, and injection of xenogeneic blood plasma into a vascular vessel. In the first two cases, xenogeneic rejection occurred only in Botryllus primigenus and Botrylloides simodensis. The features of that xenogeneic rejection were similar to those of allogeneic rejection in each of these two botryllids. Injection of xenogeneic blood plasma induced responses similar to those of allogeneic rejection in all three botryllids. It is interesting to note that colonies of Botryllus scalaris never showed any response against injected blood plasma from allogeneic incompatible colonies, unlike the responses seen in colonies of the other two botryllids under the same conditions. On the basis of these results, the relationship between allogeneic and xenogeneic rejection in botryllids is discussed.     

Graft-versus-host reactivity of mouse thymocytes: effect of in vitro treatment with anti-TL serum.

The lymph ducts efferent from prefemoral nodes of sheep were cannulated and the lymph flow monitored during immune responses to injected allogeneic lymphocytes or xenogeneic murine P815 mastocytoma cells. Changes in the lymph began 5–6 days after injection of allogeneic cells but at 3–4 days after injection of xenogeneic cells, in both systems the number of large cells in the lymph increased to reach peak values of up to 40% of the total. The in vitro cytotoxic activity of lymph cells, cell supernatants, or cell free lymph was determined by measuring the release of ⁵¹Cr from prelabeled target lymphocytes or P815 cells. The cytotoxic mechanisms that were detected in the allogeneic and xenogeneic systems were similar; in both cases the lymph cells were cytotoxic only during the large cell response, and when the immunoblast numbers had returned to normal levels in the lymph no further cell-mediated cytotoxic effects were detected. During the blast response lymphocytes alone caused some target cell damage but their cytotoxic effector function was greatly increased in cultures containing complement or normal blood white cells. It was concluded that the lymph immunoblasts caused some target cell damage by direct action, but the majority of their cytotoxic activity was associated with synthesis and secretion of complement-dependent antibody (C.D.A.) and leukocyte-dependent antibody (L.D.A.).     

Immunogenicity of allogeneic mesenchymal stem cells

Mesenchymal stem cells (MSCs) inhibit proliferation of allogeneic T cells and express low levels of major histocompatibility complex class I (MHCI), MHCII and vascular adhesion molecule-1 (VCAM-1). We investigated whether their immunosuppressive properties and low immunophenotype protect allogeneic rat MSCs against cytotoxic lysis in vitro and result in a reduced immune response in vivo. Rat MSCs were partially protected against alloantigen-specific cytotoxic T cells in vitro. However, after treatment with IFN-γ and IL-1β, MSCs upregulated MHCI, MHCII and VCAM-1, and cytotoxic lysis was significantly increased. In vivo, allogeneic T cells but not allogeneic MSCs induced upregulation of the activation markers CD25 and CD71 as well as downregulation of CD62L on CD4(+) T cells from recipient rats. However, intravenous injection of allo-MSCs in rats led to the formation of alloantibodies with the capacity to facilitate complement-mediated lysis, although IgM levels were markedly decreased compared with animals that received T cells. The allo-MSC induced immune response was sufficient to lead to significantly reduced survival of subsequently injected allo-MSCs. Interestingly, no increased immunogenicity of IFN-γ stimulated allo-MSCs was observed in vivo. Both the loss of protection against cytotoxic lysis under inflammatory conditions and the induction of complement-activating antibodies will likely impact the utility of allogeneic MSCs for therapeutic applications.     

Chemoimmunotherapeutic effect of cyclophosphamide on the highly metastatic MAT 13762 tumor

Summary We have observed that cyclophosphamide (CY) treatment of 13762 mammary adenocarcinoma tumor-bearing rats was found to cause tumor regression. Tumor-bearing animals cured with three low doses of CY were partially immune against IV and SC challenge with a high dose of 13762 cells. This immune protection mechanism in CY-cured animals appears to be a T (Ig^–) cell-mediated response. Irradiated rats reconstituted with CY-cured animal spleen cells were also partially protected against IV and SC challenge with 13762 cells, whereas irradiated rats reconstituted with CY-control animal spleen cells were not. In vitro primary and secondary cell-mediated cytotoxic activity of CY-cured spleen cells against target 13762 cells was low. The possible relevance of this tumor-model study is in the understanding of CY-induced tumor immune response and its role in preventing metastases or perhaps recurrent tumor growth.     

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

Contrasting effects of alloantiserum and xenoantiserum on cell-mediated lysis of tumor cells

The effect of anti-EL-4 serum on antibody-dependent cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC) was studied in allogeneic and xenogeneic systems. Inbred strains of BALB/c mice and Lewis rats were immunized with EL-4 tumor cells. Using microcytotoxic assays of ⁵¹Cr release from labeled EL-4 cells, complement-dependent cytolysis, ADCC, and CMC were determined. Complement-dependent cytolysis was observed in both systems. Although ADCC was demonstrated in both systems, the kinetics of cytolysis were different. Xenoantisera and alloantisera had opposite effects on CMC. Incubation of EL-4 target cells with BALB/c anti-EL-4 serum resulted in inhibition of CMC by immune BALB/c spleen cells. In contrast, treatment of EL-4 target cells with Lewis anti-EL-4 serum potentiated the CMC of immune Lewis spleen cells. It is thought that differences in the strength of response, antibody characteristics, and effector cells may determine the degree of inhibition or potentiation observed in these systems.     

Donor modification leads to prolonged survival of limb allografts

Chronic immunosuppression is essential for maintaining human hand transplant survival because composite tissue allografts are as susceptible to rejection as visceral organ allografts. Limb allografts comprise different types of tissues with varying antigenicities, and the immunosuppressive doses required for these allografts are as high or higher than those required for solid organ allotransplantation. In particular, bone marrow is an early target of the host immune response. This study reports on donor limb modification of the marrow compartment leading to prolonged survival of limb allografts.Chimeric limb allografts comprising a Lewis rat vascularized allograft and Brown Norway rat bone marrow were created. These chimeric limbs were transplanted into three recipients: (1) Buffalo rats (n = 12), where the chimeric limb was allogeneic for both vascular graft and bone marrow; (2) Lewis rats (n = 12), where the limb was allogeneic for marrow alone; and (3) Brown Norway rats (n = 12), where the limb was allogeneic for graft alone.This study found that Brown Norway recipients elicited significantly reduced cell-mediated and humoral immune responses in comparison with the Buffalo and Lewis recipients (p < 0.001 and p < 0.01, respectively). The Buffalo and Lewis recipients both elicited high cell-mediated and humoral responses. The Brown Norway recipients also had prolonged survival of limb tissue allograft in comparison with the other experimental groups.     

Studies of the immunological capacity of germ-free mouse radiation chimeras: I. Chimerism and humoral immune response

The genetic origin of both the functional lymphoid cell and progenitor cell populations of germ-free mouse radiation chimeras was assessed by indirect immunofluorescence (IIF), anti-H-2 cytotoxicity, and survival of lethally x-irradiated secondary recipients of chimera cell populations. These studies demonstrated that germ free C3H/He mice given 1000 R and 10⁷ DBA/2 bone marrow cells express H-2 antigens on their lymphoid and bone marrow cell populations characteristic of the DBA/2 donor. These cells persist for at least 14 months postirradiation and bone marrow transplantation. However, these allogeneic mouse radiation chimeras have a reduced humoral immune response to sheep erythrocytes (SRBC). This decreased humoral immune capacity as assessed by kinetic studies of the spleen plaque-forming cell (PFC) response is present throughout the life span of the chimera. The γ₁ PFC response shows extreme depression. The reduced humoral immune responsiveness to the thymusdependent SRBC antigen is considered to be due to the absence or malfunctioning of a thymocyte population.     

Bone Marrow Transplantation Concurrently Reconstitutes Donor Liver and Immune System across Host Species Barrier in Mice

Liver immunopathologic mechanisms during hepatotropic infection, malignant transformation, and autoimmunity are still unclear. Establishing a chimeric mouse with a reconstituted liver and immune system derived from a single donor across species is critical to study regional donor immune responses in recipient liver. Using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah ^-/-) and bone marrow transplantation (BMT), we reconstituted the donor''s hepatocytes and immune cells across host species barrier. Syngeneic, allogeneic or even xenogeneic rat BMT rescued most recipient fah^-/- mice against liver failure by donor BM-derived FAH⁺ hepatocytes. Importantly, immune system developed normally in chimeras, and the immune cells together with organ architecture were intact and functional. Thus, donor BM can across host species barrier and concurrently reconstitutes MHC-identical response between immune cells and hepatocytes, giving rise to a new simple and convenient small animal model to study donor''s liver immune response in mice.     

Innate and adaptive immune responses to nonvascular xenografts: evidence that macrophages are direct effectors of xenograft rejection

Nonvascularized xenograft rejection is T cell mediated, but is dependent on initial macrophage (Mphi) infiltration. We developed an i.p. transplant model to define the roles of Mphi and T cells in xenograft rejection. Nonobese diabetic or BALB/c mice were injected i.p. with xenogeneic, allogeneic, or syngeneic cells, and the responding cells in subsequent lavages were assessed by flow cytometry and adoptive transfer. Neutrophils and monocytes/elicited Mphi were rapidly recruited in response to xenogeneic pig (PK15 or spleen) cells and, to a significantly lesser extent, allogeneic cells. These innate responses preceded T cell infiltration and occurred in their absence in SCID mice. Syngeneic cells induced negligible neutrophil or Mphi responses. Neutrophils and Mphi induced by xenogeneic cells in SCID mice stimulated T cell recruitment after transfer to immunocompetent mice. T cells in turn were required for Mphi activation and xenogeneic cell rejection. Thus, Mphi harvested from immunocompetent but not SCID mice injected with xenogeneic cells expressed activation markers and rejected xenogeneic cells when transferred into SCID mice. These findings demonstrate the interdependent roles of Mphi and T cells in xenograft rejection. The requirement for Mphi reflects their ability to mount a rapid, local innate response that stimulates T cell recruitment and, having received T cell help, to act as direct effectors of rejection.     

Recognition of cellular implants by the brain's innate immune system

Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.