Relevance of maintenance triple-drug immunosuppression to bridle the amplification of rat cytomegalovirus infection after experimental lung transplantation.

Immunosuppressive therapy required to treat rejection after lung transplantation (LTx) contributes significantly to the pathogenesis of cytomegalovirus (CMV) infection and disease. In a weak allogeneic left LTx model in the rat (Fisher 344 [F344] to Wistar Kyoto [WKY] rats) we analyzed the influence of acute CMV infection on postoperative day (POD) 3, with application of standard triple-drug immunosuppression (TD-IS) (cyclosporin A, azathioprine, prednisolone) on late outcome after LTx. Native right lungs and syngeneic grafts (WKY to WKY) served as controls. Rats were sacrificed on POD 15, 30, 60, and 100. TD-IS completely prevented acute and chronic rejection in non-infected rats. Allografts of CMV-infected rats treated with TD-IS showed only mild perivascular infiltrations in 6/10 rats (POD 15 and 30), which persisted up to POD 100 in 4/10 rats. In the long-term course, mild isolated interstitial and alveolar changes were found in 40% of these animals. In conclusion, rat CMV infection partially neutralized the immunosuppressive effect of TD-IS. However, an amplification of CMV infection under TD-IS can be controlled and does not result in fatal outcome.

Development of obliterative bronchiolitis after allogeneic rat lung transplantation: implication of acute rejection and the time point of treatment.

BACKGROUND: Chronic allograft failure represents the major cause of late morbidity and mortality after solid organ transplantation. Despite the pathological and clinical changes of this disease being well-described, the etiology and the causative factors are still under discussion. Several clinical, as well experimental studies, emphasize the significance of acute rejection. In rat model of left lung allo-transplantation (F344-to-WKY) the influence of acute rejection (AR) on the development of chronic rejection (CR) was studied. METHODS: In Group I (n = 25) no immunosuppression was used, while methylprednisolone (MP) (10 mg/kg) was applied in Group II (n = 20) in the early phase of AR on postoperative Days 9, 10, 11 and in Group III (n = 20) during AR on Day 14, Day 15, Day 16. The rats were sacrificed on Day 5, Day 15/20, Day 30, Day 60, Day 100 and following HE-staining the extend of AR as well CR was graded according to the working formulation of The International Society of Heart and Lung Transplantation. RESULTS: In Group I, AR was found at Day 15 and Day 30 which resolved spontaneously and resulted in CR on Day 60 and Day 100. In Group II, signs of AR were less evident on Day 20, while mild AR persisted on Day 30 and Day 60. On Day 100, normal lung structure was found in all rats. The recipients of Group III showed decreased signs of AR in the early course, however, severe CR was found on Day 60 and Day 100. Extensive airway inflammation with destruction of the subepithelial layer of the smaller airways resulted in severe early obliterative bronchiolitis. CONCLUSIONS: Untreated severe AR in the early course after lung transplantation results in CR in the F344-to-WKY model. Preventive treatment with MP during the early phase of AR clearly diminishes the degree of AR and the graft recovers completely without any evidence of CR. Late application of steroids during the zenith of AR is successful to control the extent of AR, however, it fails to prevent CR.

Allograft conduit wall calcification in a model of chronic arterial graft rejection.

Early allograft vascular wall degeneration has emerged as a major important complication in young patients. To explain this mechanism, we reviewed studies on explants of allograft valved conduits implanted heterotopically into the infrarenal aorta in inbred rats (LEW; RT1I and CAP-RT1C). The following strain combinations (isografts and allografts) were used: syngeneic, LEW- > LEW, strongly allogeneic, and CAP > LEW (RT1- and non-RT1-incompatible). Second-set skin grafting was performed 3 weeks after the heterotrophic implant to test for immunogenicity and presensitization. The animals (LEW) were sacrificed serially on days 20, 30, 50, and 100 for immunofluorescence and SEM studies. Endothelial disruption was observed on day 30, while valve leaflets appeared normal. Humoral allograft rejection was demonstrated and associated with production of antibodies (IgG) against the endothelial cells and around the smooth muscle cells, and in areas of smooth cell necrosis, through 100 days. Neointimal repopulation by host cells and migrated smooth muscle cells was also observed in both viable and allovital grafts. Allovital grafts demonstrated more disorganized collagen and elastic fibers, as well as calcific degeneration in the media and neointima on day 50; the viable conduits showed such structural changes on day 100. In conclusion, vascular walls of allovital conduits calcified earlier than the viable conduits without discernible calcification of the valves. There is therefore evidence to prove causative relationships between cellular viability, immune response, and fibroproliferative calcific degeneration in allograft vascular conduits.

Short-course cyclosporin A therapy for definite allograft valve survival immunosuppression in allograft valve operations.

This study was designed to determine the effect of short-course cyclosporin A therapy (10 mg/kg daily for 14 days) on allograft valve survival across the histocompatibility barriers in the following rat models; (1) syngeneic Lewis to Lewis (herein referred to as autografts), (2) weakly allogeneic AS to Lewis (RT1 compatible, non-RT1-incompatible), and (3) strongly allogeneic CAP to Lewis (RT1 and non-RT1-incompatible). Cyclosporin A-treated and untreated recipient animals (Lewis) received allovital and antibiotic-treated viable allografts implanted into the infrarenal aorta. Second-set skin grafting was performed 3 weeks after heterotopic valve implantation to test for immunogenicity and presensitization. The animals (Lewis) were sacrificed serially on days 20, 50, 100, and 150 for immunofluorescence study using mouse monoclonal antibodies (OX6) directed at class II endothelial surface antigens. The allografts in weakly allogenic strains showed no humoral response under a short course of cyclosporin A. The cyclosporin A-untreated allovital grafts and the viable (antibiotic-treated) valves demonstrated fibrocalcification on the 100th and 150th postoperative days, respectively. In conclusion, it seems that a short course of nontoxic immunosuppression could arrest allograft rejection and thus prevent early degeneration of allografts. Furthermore, antibiotic-treated viable allografts seemed to be more durable than allovital grafts.

Blood monocytes and spleen macrophages differentiate into microglia-like cells on monolayers of astrocytes: morphology.

Several morphological and functional properties of microglial cells, the resident immunoeffector cells of the central nervous system (CNS), differ from those of monocytes/macrophages in other tissues. Microglia are assumed to derive from myelonocytic lineage, possibly as a distinct subpopulation that diverges from a common cell line early in ontogeny, invades the CNS, proliferates, and differentiates into ameboid and then ramified microglia. We tested the hypothesis that some morphological and functional properties of microglia are induced in myelomonocytic cells by nervous tissue, specifically astrocytes. In the present in vitro studies we compared the differentiation of microglia, blood monocytes, and spleen macrophages on acellular substrates and on monolayers of astrocytes and fibroblasts. On acellular substrates, microglial cells at first acquire an ameboid morphology; later they show a few short, unbranched processes. On monolayers of pure astrocytes, microglial cells at first also differentiate into ameboid cells, but after 5 to 7 days they start to develop processes with large lamellopodial tips. These lengthen and branch continuously during the next 2 weeks in vitro, demarcating a round to oval territory around the small ellipsoid cell body. By contrast, on monolayers of fibroblasts the microglial cells develop an ameboid morphology, but do not grow the typical long branched processes of the ramified form. Blood monocytes and spleen macrophages behave indistinguishably from microglia both on acellular and cellular substrates, i.e., on astroglia they develop the ramified form, while on fibroblasts they retain the ameboid shape. When microglia, macrophages, or monocytes are cultured on coverslips on top of astrocytic monolayers, i.e., physically separated from the astroglia, but exposed to the medium conditioned by astrocytes, a significant proportion of them also develop the ramified shape. These findings indicate that the ramified shape of microglia is induced by astrocytes. Since this morphology can also be induced in blood monocytes and macrophages, we take this to be further evidence for the proposition that microglial cells are derived from the myelomonocytic lineage, and, moreover, that properties of resident macrophages are largely determined by tissue components of their host organ.

Injection of v-mos-transformed and irradiated macrophages leads to longlasting specific acceptance of MHC-allogeneic heart grafts and specific prolongation of skin graft survival in mice.

In vitro studies have revealed that the v-mos-transformed clone mos2 (mos2) of the murine macrophage cell line P388D1 (D1) (H-2d) is capable of inducing a state of specific unresponsiveness in MHC-allogeneic unprimed T cells. Here, we present data on the in vivo relevance of these findings. Male C57bl/6 mice (H-2b) were injected i.p. 6 times with 10(7) of the following irradiated cell types: D1, mos3, mos2, DBA/2-(H-2d) or C3H-(H-2k) spleen macrophages. DBA/2 and C3H skin or heart grafts were performed 10 days after the last injection. The normal rejection time for allogeneic skin was 7.5 days and for allogeneic hearts, was 12.8 days. After injection of D1 or mos3, DBA/2 skin grafts were rejected after 4.5 and 6.5 days, respectively, and the hearts, after 15.4 and 18.6 days, respectively. Third-partly C3H grafts were rejected normally (7.0 days). In contrast, injection of mos2 led to prolongation of DBA skin graft survival to 12.3 days. DBA/2 hearts were accepted for more than 160 days as revealed by heart beating. Again, C3H grafts were rejected normally (11.0 days). DBA/2 skin grafts on day 102 after heart grafting survived for 30 days, indicating hyporesponsiveness against these grafts. These results confirmed the in vitro findings. The mos2 cells obviously induced a state of specific unresponsiveness in otherwise unmanipulated recipients. However, the duration of this unresponsiveness induced by the injection of irradiated cells was dependent on the organ type.

Oncogene transformation can induce tolerogenicity in murine macrophages after down-regulation of immunogenicity without altering major histocompatibility complex antigen expression.

In vitro studies on cell lines may allow analyses of the mechanisms of immunogenicity and tolerogenicity in cells. We used a model of oncogenic transformation of an established murine macrophage cell line and report here that one v-mos-transformed clone expressing unaltered high amounts of MHC class I and II antigens does not induce proliferation of unprimed T cells in primary mixed lymphocyte reactions, in sharp contrast to its non-transformed parental cells. Interestingly, this clone induces specific unresponsiveness, as revealed by the lack of responsiveness of MHC-specific T cells when subsequently exposed to the pertinent MHC alloantigens in immunogenic form but unaltered MHC-third party responsiveness of the naive spleen T cells. Furthermore, the accessory function of this clone is strongly reduced. These functional defects could be overcome by the addition of exogenous interleukin-1 alpha (IL-1 alpha). Analysis of mRNA expression showed a significant and selective reduction of IL-1 alpha mRNA levels when compared with parental cells.

Biocompatibility of silicone oil and high-density liquids. An immunologic study.

Silicone oil and high-density liquids can be useful tools in the treatment of complicated retinal detachments. To test whether these substances induce specific and/or unspecific inflammatory responses, we performed immunoassays on 60 rats of two different inbred strains. After sensitization, each rat was injected in the right foot pad with either silicone oil, fluorosilicone oil, perfluorooctane, or perfluoropolyether. Depending on the inbred strain, the volume of the foot pads increased up to the fifth day, indicating an injection-induced inflammatory reaction. Histopathological examination of rats injected with silicone oil showed infiltration by mononuclear cells and occasional giant cells. In rats receiving fluorosilicone oil, additional eosinophilic reactions were observed. The most pronounced reactions occurred after injection of the two perfluorocarbons, which induced a greater number of giant cells and a slight granulomatous reaction. The degree of tissue response was independent of the type of sensitization; the majority of the draining popliteal lymph nodes showed no enlargement. Our findings demonstrate that all substances tested induced inflammatory though unspecific responses.

[Immunological reactions against preserved tracheal transplants? Studies on inbred rat strains]. / Immunologische Reaktion gegen konservierte Trachealtransplantate? Untersuchungen bei Ratteninzuchtstämmen.

To determine whether chemical (cialit, merthiolate, alcohol, formaldehyde solution) preserved tracheal segments maintain immunogenicity we performed systematic transplantation-immunological investigations using in vivo-, in vitro- and immunohistological tests. In contrast to other authors no indication of residual antigenicity was found. The survival rates after orthotopic grafting of preserved tracheal segments were shortened. This seems due to the loss of the mucociliary clearance, caused by chemical induced damages in the tracheal mucosa.

High-dose cytostatic agents in allogeneic bone marrow transplantation: comparison of the engraftment promoting potential.

We investigated the potential of various cytostatic agents for preventing graft rejection following allogeneic bone marrow transplantation. LEW rats received a lethal dose (35 mg/kg) of busulfan followed by injection of 1 x 10(8) F1(CAP x LEW) marrow cells, which are unable to induce a graft-versus-host reaction in LEW recipients. Rejection of the marrow graft was assessed by monitoring haematocrit and granulocyte counts. Due to its weak immunosuppressive activity, busulfan by itself is unable to allow engraftment of allogeneic marrow. Therefore, agents administered in addition to busulfan can be tested for their capacity to prevent marrow graft rejection. 120 mg/kg of cyclophosphamide, 20 mg/kg of ACNU and 240 mg/kg of ifosfamide completely prevented rejection of the allogeneic marrow. Maximum doses of BCNU applicable in conjunction with busulfan reduced the rejection rate to 12% (30 mg/kg) and 17% (40 mg/kg), whereas the antitumour agents thiotepa, melphalan, and carboplatin exhibited a very limited engraftment-promoting potential in this experimental setting. Thus, BCNU (carmustine), ACNU (nimustine), and ifosfamide might be suitable candidates for conditioning of allogeneic bone marrow graft recipients.

Effect of post-transplant methotrexate, cyclosporin A and prednisolone on graft rejection after allogeneic bone marrow transplantation.

Methotrexate, cyclosporin A and prednisolone have been shown to improve graft survival rates in solid organ transplantation. However, little is known concerning their capacity to promote lasting engraftment of allogeneic bone marrow. Therefore, we tested these agents in LEW rats receiving MHC-mismatched marrow after pretreatment with a myeloablative dose of busulfan plus different doses of total body irradiation (TBI). To avoid mortality due to graft-versus-host reaction (GHVR), F1(CAP x LEW) marrow was transferred. Hematological parameters were determined twice weekly to monitor engraftment and rejection. The pretreatment was lethal but not sufficiently immunosuppressive to ensure lasting engraftment in all animals. Thus, post-transplant immunosuppressive protocols could be evaluated for their capacity to improve engraftment rates. Standard clinical doses of methotrexate (0.25 mg/kg i.p. day 1, 3, 6, 11, 18, 25), cyclosporin A (10 mg/kg orally day 0-28) and prednisolone (1 mg/kg i.p. day 0-28) were administered and proved to be of nearly equivalent toxicity in our system. All three agents failed to allow engraftment after busulfan alone. After additional conditioning with 1.5 Gy of TBI, methotrexate and cyclosporin A reduced the rejection rate from 100% to 59% and 70%, respectively. When 3 Gy of TBI were added to busulfan, cyclosporin A and prednisolone were able to reduce the rejection rate from 67% to 33% and 39%, respectively, whereas 0.12 and 0.25 mg/kg methotrexate completely prevented graft rejections. After cessation of cyclosporin A therapy, late secondary rejections were frequently observed. These results demonstrate that postgrafting immunosuppression with protocols conventionally used for the prophylaxis of GVHR is able to facilitate lasting engraftment of MHC-mismatched bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenicity testing of food proteins: in vitro and in vivo trials in rats.

The immunogenicity of an industrially produced bacterial food protein (single cell protein, SCP) was analyzed in a comparative study. SCP, casein and ovalbumin were injected into or fed to rats. The systemic response was tested in vitro with a lymphocyte transformation test and in vivo with a footpad swelling assay. Feeding of casein induced cell-mediated reactivity against casein, while feeding of SCP or ovalbumin had no effect. One injection of casein led to sensitization compared with three injections of SCP. Prior feeding of SCP did not abrogate the response to injected SCP. The footpad swelling test is suggested as a particularly effective model for helping to determine the immunological risk of foodstuffs to man.

Assessment of different preservation and storage conditions on aortic valves: introduction of a quantitative measuring method of the integrity of endothelial cells.

The fate of human allogeneic aortic valves depends mainly on their histological and immunological condition at the time of transplantation. A screening test making novel use of Alcian Blue was used to determine the integrity of endothelial cells as a prerequisite to their function. The dye uptake into the nucleus was measured quantitatively. The test was used to compare the effect of different storage mediums and temperatures (+4 degrees C, -30 degrees C, -80 degrees C, DMSO, FCS, RPMI, antibiotic solution) on aortic valves of rats. The cell integrity decreased with increasing storage time and higher storage temperature. The cryoprotective agent DMSO had no essential effect on the maintenance of cell integrity.