Y. Nakafusa

Intrathymic injection of donor alloantigens induces donor-specific vascularized allograft tolerance without immunosuppression.

The induction of donor-specific tolerance could prevent the side effects of immunosuppression while improving allograft survival. Male adult Buffalo (RT1b) rats underwent an intrathymic (IT), portal venous (PV), intrasplenic (IS), or subcutaneous (SQ) injection of 25 χ 106 major histocompatibility complex (MHC) mismatched Lewis (RT11), UV-B-irradiated Lewis (RT11), ACI (RT12), or syngeneic Buffalo (RT1b) splenocytes. At the completion of the donor alloantigen injection, 1 mL rabbit anti-rat lymphocyte serum (ALS) was administered intraperitoneally to the Buffalo recipients, and 21 days later a heterotopic Lewis or ACI heart was transplanted. Intrathymic injection of donor alloantigen induced a donor-specific tolerance that allowed the cardiac allograft to survive indefinitely (mean survival time [MST] > 140.7 days) in 84% of the recipients without further immunosuppression, whereas groups receiving antigen injections at other sites (PV, IS, and SQ) plus ALS rejected cardiac allografts in normal fashion (MST ≈ 8.0 days). Buffalo recipient rats with long-term surviving Lewis cardiac allografts after Lewis IT injection and ALS subsequently rejected a heterotopic third-party ACI cardiac allograft in normal fashion (MST ≈ 7 days), whereas a second Lewis cardiac allograft was not rejected (MST > 116 days). Microchimerism is unlikely because Lewis allograft survival was also prolonged (MST > 38.7 days) in rats receiving UV-B-irradiated splenocytes IT, which cannot proliferate. Survival of Lewis renal allografts was also prolonged, but not indefinitely, in Buffalo recipients possessing a long-term surviving Lewis cardiac allograft (MST ≈ 17.6 days versus 7 days for control). This model emphasizes the potential role of exposure of immature thymocytes to foreign donor alloantigens during maturation in the thymic environment for the development of unresponsiveness to an MHC-mismatched donor-specific vascularized allograft.

Induction of donor-specific tolerance to cardiac but not skin or renal allografts by intrathymic injection of splenocyte alloantigen.

We have recently found that donor-specific tolerance to a cardiac allograft can be achieved after the intrathymic (i.t.) injection of donor splenocytes and a single intraperitoneal injection of rabbit antirat lymphocyte serum. The present study evaluated whether the tolerance induced by splenocytes injected i.t. could also prevent the rejection of kidney and skin allografts. Male Buffalo (RT1b) rats were given 25 x 10(6) fully MHC-mismatched unfractionated Lewis (RT1l) splenocytes by i.t. injection plus 1 ml of ALS i.p. and 21 days later underwent a Lewis heterotopic cardiac, orthotopic renal, or skin transplant. Lewis i.t. injection induced a donor-specific tolerance with indefinite cardiac allograft survival (> 153.1 days) in 88% of the recipients without the need for further immunosuppression, while renal and skin allograft survival was prolonged (kidney 14.8 days vs. control 7.8 days; skin 11.6 days vs. control 9.2 days) but were still rejected. Buffalo recipients with a long-surviving Lewis cardiac allograft after Lewis i.t. injection were still able to reject a third-party heterotopic ACI (RT1a) cardiac allograft in normal time (7.0 days), but did not reject a second Lewis cardiac allograft (> 100.0 days). In contrast, however, Buffalo recipients with long-surviving Lewis cardiac allografts did reject a Lewis skin allograft in normal time (10.0 days) and a Lewis renal allograft in a prolonged manner (17.6 days) without causing the rejection of the Lewis cardiac allografts. These data support the important role tissue-specific non-MHC antigens may play in the rejection of kidney and skin allografts.

Intrathymic injection of donor alloantigens induces specific tolerance to cardiac allografts

The induction of donor-specific tolerance would eliminate the risk of long-term immunosuppression while ensuring allograft function and survival. Male Buffalo (RT1b) rats were exposed to donor alloantigen by an intrathymic, intrasplenic, s.c., or i.v. injection of 25 ± 106 syngeneic Buffalo (RT1b) or MHC fully mismatched Lewis (RT11), ACI (RT1a), or UV-B irradiated Lewis (RT11) splenocytes. The Buffalo recipients were given 1 cc of rabbit antirat antilymphocyte serum (ALS) i.p. at the time of the donor antigen injection, and 21 days later received a heterotopic Lewis or ACI heart transplant. Only intrathymic alloantigen injection induced a donor-specific tolerance which allowed the cardiac allograft to survive indefinitely (mean survival time [MST] > 176.8 days) in >86% of the recipients without the need for further immunosuppression, whereas groups receiving antigen injections at other sites rejected cardiac allografts in control time (MST ± 7.0 days). Histologic examination of long-term tolerated Lewis cardiac allografts revealed the presence of healthy cardiac myocytes without mononuclear infiltration. Buffalo rats with a long-term surviving Lewis cardiac allograft did not reject a second Lewis cardiac allograft (MST > 100.0 days), but rejected a heterotopic ACI cardiac allograft in normal time (MST ± 7.0 days). By limiting dilution analysis (LDA), maturation of donor-specific CTLs (pCTL) from long-term recipient splenocytes was markedly diminished, whereas third party pCTL was not altered, and T helper precursors were moderately decreased without alteration in the peripheral CD4+ and CD8+ phenotype frequencies. MLC responses of recipients with long-term surviving cardiac allografts to donor-specific and third party stimulation were not significantly different from naive controls. Microchimerism is unlikely because Lewis allograft survival was also prolonged (MST > 96.0 days) in rats receiving UV-B irradiated Lewis splenocytes which cannot proliferate. The absence of increased allograft survival after transfer of long-term recipient splenocytes into naive animals suggests that donor-specific suppressor cells are not present. Additionally, in vitro lymphocyte proliferative responses to mitogenic or allogeneic stimulation in MLC was not diminished by the addition of these longterm recipient splenocytes. This model emphasizes the importance of exposure of T cell precursors to foreign donor alloantigen in the thymic environment for the development of unresponsiveness to a donor-specific vascularized allograft.

Induction of Tolerance to Islet Xenografts in a Concordant Rat-to-Mouse Model

Induction of tolerance to concordant rat islet xenografts (150 Wistar-Furth [WF] islets) in streptozocin-induced (STZ) diabetic mice (C57BU/6) was determined at three different sites for islet implantation (thymus, kidney capsule, and liver). Islets transplanted into the thymus or kidney capsule were either fresh or cultured at 24°C for 7 days, and the mice received a single injection of either anti-mouse lymphocyte serum (MALS) alone or anti-rat lymphocyte serum (RALS) and MALS. Islets transplanted into the liver via the portal vein were cultured at 24°C for 7 days, and the mice received a single injection of MALS and RALS. To document the induction of tolerance, recipients with islet xenografts surviving >100 days were made diabetic again by STZ (thymus and liver) or nephrectomy (kidney capsule) and received a second transplant of 150 fresh WF islets in the kidney capsule. Kidney capsule placement of fresh or cultured islets with MALS alone or MALS and RALS did not induce tolerance in a significant number of recipients. The intrathymic transplantation of fresh or cultured islets with MALS alone resulted in prolonged WF islet xenog raft survival (mean survival time of 39.7 ± 7.9 days) but did not result in tolerance, whereas the administration of MALS and RALS with the intrathymic placement of fresh or cultured islets induced tolerance in ∼50% of the mice. Intrahepatic transplantation of cultured islets with MALS and RALS resulted in tolerance to donor islets in 90% of the recipients. Donor specificity was evaluated by a third major histocompatability complex–disparate fresh Lewis islet xenograft. Approximately 50% of these xenografts were not rejected, suggesting that the tolerance may not be haplotype-specific. The mechanisms involved in inducing tolerance to islet xenografts are not clearly established; however, this study clearly demonstrates that tolerance to xenogeneic islets can be achieved by using a brief period of immunosuppression in the intrathymic or intrahepatic transplantation site, with intrahepatic implantation achieving tolerance in a higher percentage of recipients than the intrathymic site.

MHC Class II Presenting Cells Are Necessary for the Induction of Intrathymic Tolerance

OBJECTIVE This study determined the form of cellular donor MHC alloantigen necessary for the induction of intrathymic tolerance. BACKGROUND The authors have achieved indefinite donor-specific tolerance, to a fully MHC-disparate rat heterotopic cardiac allograft, after the pretransplant intrathymic injection of unfractionated donor splenocytes and a single injection of rabbit anti-rat lymphocyte serum (ALS), without subsequent immunosuppression. METHODS Male 4-12-week-old Buffalo (RT1b) rats underwent an intrathymic injection of either fractionated Lewis (RT1(1)) red blood cells (purified by Ficoll gradient) or T lymphocytes (purified by nylon wool column and plastic adherence), both of which express only MHC class I alloantigens, or B lymphocytes, macrophages, and dendritic cells (purified by plastic adherence) which express both MHC class I and class II alloantigens. At the completion of alloantigen injection the Buffalo recipient rats were given 1 ml of ALS intraperitoneally. Twenty-one days later a heterotopic Lewis heart was transplanted. RESULTS The intrathymic injection of the fractions of Lewis MHC class I and class II expressing B lymphocytes, macrophages, and dendritic cells induced a donor-specific tolerance that resulted in indefinite Lewis cardiac allograft survival (MST > 125 days) in all recipients without further immunosuppression, whereas groups receiving MHC class I expressing red blood cell or T lymphocyte injections plus ALS rejected Lewis cardiac allografts with a MST of 7.3 and 16.5 days, respectively, thus indicating that the MHC class II expressing cell is necessary for the induction of intrathymic tolerance. Buffalo recipients with a long-term surviving Lewis cardiac allograft, after Lewis MHC class II expressing cells were still able to reject a third-party heterotopic ACI (RT1a) cardiac allograft in normal time (MST = 7.0 days), but did not reject a second Lewis cardiac allograft (MST > 100 days). Additionally, the intrathymic injection of MHC class II expressing cells resulted in decreased interleukin-2 (IL-2) production and an 80% decrease in in vitro donor-specific cell mediated cytotoxicity, whereas the cytolytic response to a third party was unaltered. CONCLUSION Donor MHC class II, and not class I, expressing cells are the cells in donor splenocytes, injected intrathymically, responsible for the development of donor-specific allograft tolerance.

Gadolinium chloride inhibits lipopolysaccharide-induced mortality and in vivo prostaglandin E2 release By splenic macrophages.

The monocytic phagocytic system, consisting primarily of tissue macrophages of the liver and spleen, produces prostaglandin E2 (PGE2), a modulator of the septic response. Macrophages are known to internalize gadolinium chloride (GD), a lanthanide metal, which inhibits phagocytic function. Thus we studied the effect of in vivo GD on lipopolysacchride (LPS)-induced mortality and on LPS-stimuIated PGE2 release by cultured splenic macrophages. GD (7 mg/kg intravenously) given on the two days prior to LPS challenge (30 mg/kg intravenously) completely prevented the uniform mortality in rats. This protective effect was transient since rechallenge with LPS 10 days later was uniformly lethal. Previous work in this laboratory has established a critical role of arginine concentration on macrophage behavior in vitro. Therefore, to establish culture conditions reflective of the milieu within the portal venous system, alanine and arginine levels were measured in the portal and hepatic veins of normal and endotoxemic (LPS, 10 mg/kg intraperitoneally) rats. In contrast to alanine levels, which were not altered by endotoxemia, there was a reduction of arginine concentrations from a range of 50 to 250 μmol/L in normal rats to a range of 10 to 50 μmol/L after LPS challenge. Consequently subsequent in vitro assays of splenic macrophage secretory behavior were performed in concentrations of 1200 μmol/L arginine (in standard RPMI-1640), as well as in concentrations reflective of physiologic arginine levels (10 and 100 μmol/L in modified RPMI-1640). Rat splenic macrophages harvested after two consecutive days of either in vivo saline or GD injection (7 mg/kg intravenously) were stimulated with LPS (0.025 to 2.5 μg/ml). At 72 hours of culture, the release of PGE2 by splenic macrophages from GD-treated rats was significantly (P<0.0001) reduced at all LPS concentrations. Increased PGE2 production was not present when the splenic macrophages were cultured in the supraphysiologic arginine (1200 μmol/L) concentration. The results demonstrate the relevance of physiologic arginine concentrations in cell culture studies and suggest that the protection conferred by GD against septic mortality may be related to downregulation of the release of immunosuppressive PGE2 by the monocytic phagocytic system.

In vitro analysis of gadolinium chloride abrogation of the systemic tolerance induced by portal venous administration of ultraviolet B-irradiated donor cells.

BACKGROUND Normally, a Buffalo (BUF) recipient (RT1b) rejects a heterotopically transplanted Lewis (LEW) heart (RT1l) drained into the portal vein (PV) within 14 days. However, the addition of PV administration of 25x10(6) ultraviolet B (UVB)-treated LEW spleen cells (SC) to BUF recipients 7 days before cardiac transplantation results in 70% long-term allograft survival. METHODS In this study, we used gadolinium chloride (GdCl3) (7 mg/kg/day) to selectively block the phagocytosis of recipient hepatic Kupffer cells before PV injection of UVB-treated donor SC to examine the mechanism of tolerance induction, as measured by in vitro analysis of mixed lymphocyte culture (MLC), T cell cytotoxicity, T helper cell precursors (pTH), and cytotoxic T cell precursors (pCTL) by limiting dilution analysis. RESULTS A BUF recipient that received untreated or gamma-irradiated LEW SC intraportally reacted to in vitro stimulation by LEW alloantigen with increased MLC proliferation, T cell cytotoxicity, pTH and pCTL frequencies, and interleukin-2 production. In contrast, SC from BUF that received UVB-treated LEW SC were hyporesponsive on MLC stimulation by donor LEW alloantigen and exhibited markedly reduced cytotoxicity, pTH and pCTL frequency, and interleukin-2 production. However, normal in vitro responsiveness resulted with stimulation by third-party Brown-Norway (RT1n) SC, thus indicating that the systemic hyporesponsiveness was specific for the UVB donor alloantigen given PV. On the other hand, GdCl3 given by intravenous injection daily for 3 days before PV alloantigen blocked the induction of in vitro hyporesponsiveness. CONCLUSION Therefore, prevention of alloantigen sequestration by GdCl3 inhibition of hepatic Kupffer cell phagocytosis was pivotal in preventing the development of portal venous tolerance.

Advantages of Donor-Specific Blood Transfusion in the Rat Via the Portal Vein for Renal Transplantation

It is well known that blood transfusion can promote immune unresponsiveness to a renal allograft in both animals and humans. The present study examined the differences between either portal venous (PV) or intravenous (IV) donor-specific transfusion (DST) alone on the survival of rat renal allografts without other immunosuppression. Seven days before LEW (RT1(1)) renal transplantation, recipient WKA rats (RT1u) were inoculated with graded doses (1.0 to 0.0001 mL) of LEW whole blood through either the portal vein or the tail vein. A dose of 0.001 mL whole blood PV resulted in significantly greater prolongation of renal allograft survival than occurred with IV preimmunization (38.5 +/- 10.6 days v 12.5 +/- 2.7 days). Although recipient survival was not significantly different for the two routes for a lower dose (0.1 mL) of whole blood preimmunization 7 days before transplantation, azotemia and rejection were prevented when the PV route, but not the IV route, was used. However, when 0.1 mL of whole blood was administered 5 days before transplantation, survival was prolonged only in animals preimmunized by the PV route (54.7 +/- 14.0 days v 14.3 +/- 4.0 days). In the stronger reciprocal combination of WKA to LEW, there was an advantage of PV with 0.1 mL whole blood, but not with 1 mL. These results indicate that the beneficial effects of DST on renal allograft survival can be obtained when a smaller volume of blood is given via the PV rather than the IV route and a shorter interval elapses between preimmunization and transplantation.

Asymptomatic adenocarcinoma arising from a gastric duplication cyst: A case report

Highlights • Adenocarcinoma arising from a gastric duplication cyst is extremely rare.• This is the 2nd asymptomatic case in the English literature.• During our close observation of 4 years, malignant transformation had occurred from a gastric duplication cyst.• When morphological change appears, we strongly recommend surgical treatment without delay.