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Background & Aims: Adult stem cells provide a promising alternative for the treatment of diabetes mellitus and end-stage liver diseases. We evaluated the differentiation potential of human peripheral blood monocytes into hepatocyte-like and pancreatic islet-like cells. Methods: Monocytes were treated with macrophage colony-stimulating factor and interleukin 3 for 6 days, followed by incubation with hepatocyte and pancreatic islet-specific differentiation media. Cells were characterized by flow cytometry, gene-expression analysis, metabolic assays, and transplantation for their state of differentiation and tissue-specific functions. Results: In response to macrophage colony-stimulating factor and interleukin 3, monocytes resumed cell division in a CD115-dependent fashion, which was associated with a down-regulation of the PRDM1 and ICSBP genes. These programmable cells of monocytic origin were capable of differentiating into neohepatocytes, which closely resemble primary human hepatocytes with respect to morphology, expression of hepatocyte markers, and specific metabolic functions. After transplantation into the liver of severe combined immunodeficiency disease/nonobese diabetic mice, neohepatocytes integrated well into the liver tissue and showed a morphology and albumin expression similar to that of primary human hepatocytes transplanted under identical conditions. Programmable cells of monocytic origin-derived pancreatic neoislets expressed β cell-specific transcription factors, secreted insulin and C peptide in a glucose-dependent manner, and normalized blood glucose levels when xenotransplanted into immunocompetent, streptozotocin-treated diabetic mice. Programmable cells of monocytic origin retained monocytic characteristics, notably CD14 expression, a monocyte-specific methylation pattern of the CD115 gene, and expression of the transcription factor PU.1. Conclusions: The ability to reprogram, expand, and differentiate peripheral blood monocytes in large quantities opens the real possibility of the clinical application of programmable cells of monocytic origin in tissue repair and organ regeneration. [Copyright &y& Elsevier]

Lymphocytes with activated β7 integrins (αEβ7 and α4β7) contribute to inflammatory reactions in the small bowel. Since the selective recruitment of lymphocytes to the lymphoid compartments of the small bowel is controlled by distinct adhesion molecule interactions, a compartment-dependent use of β7 integrins may influence the rejection response within intestinal transplants. To further delineate the nature of β7 integrin-mediated graft infiltration, we analysed their expression on T lymphocytes in the heterotopically transplanted small bowel of BALB/c and C57BL/6 mice- Lymphocytes isolated from the epithelium, lamina propria (LP). Peyer's patches (PP). and mesenteric lymph nodes (MLN) were analysed by three-color fluorescence flow cytometry using monoclonal antibodies (mAb) to integrin the subunits. lymphocyte markers, and MHC I of the donor and recipient strains. On postoperative day 5 (POD) after allogeneic small bowel transplantation (SBT), 43% of intraepithelial lymphocytes (IEL) and 63% of LP. 93% of MLN. and 93% of PP lymphocytes were of host origin. In the MLN and PP of allografts, a major infiltrating lymphocyte population consisted of CD8+ cells with increased expression of α4β7 and decreased expression of L-selectin. an adhesion molecule profile characteristic of intestinal effector cell phenotypes. An increase in α4β7 levels was also found on CD8+ host lymphocytes in the LP. The integrin profile of a number of host IEL suggests an ongoing transition from the phenotype of graft infiltrating lymphocytes with high levels of α4β7 and low levels of αEβ7 to that of resident IEL with high levels of αEβ7 and low levels of α4β7. The importance of β7-mediated lymphocyte trafficking to the graft is attested by the significant reduction in the host lymphocyte population in the LP. PP. and epithelium following the administration of a (β7-blocking mAb to allograft recipients. In conclusion, while the inllliration patterns of lymphocytes may vary between the lymphoid compartment.s of intestinal allografts. host CD8+ lymphocytes with high levels of α4β7 constitute a major effector cell population that affects the entire graft. [ABSTRACT FROM AUTHOR]

Increasing evidence supports the existence of regulatory T cells that may inhibit the allogeneic immune response after transplantation by secreting regulatory cytokines. To determine whether rat liver tolerance is associated with intrahepatic regulatory T cells secreting a characteristic cytokine profile, we analyzed the cytokine production of freshly isolated intragraft CD4+ T cells at different times postoperatively by semiquantitative reverse transcription–polymerase chain reaction and by enzyme-linked immunosorbent assay before and after in vitro stimulation. Orthotopic arterialized liver transplantation was performed in two allogeneic rat strain combinations, one with fatal acute rejection (DA-to-LEW) and one with long-lasting tolerance (LEW-to-DA) without immunosuppression despite a complete major histocompatibility complex mismatch (spontaneous liver tolerance). Liver allografts of both groups showed continuously increasing cellular infiltration between day 3 and day 7 after transplantation. In this inflammatory situation, very low levels of interleukin-13 were detectable directly after cell isolation, as well as after in vitro stimulation. However, after 30 days, intrahepatic CD4+T cells in the tolerance group were then able to express elevated messenger RNA levels of the anti-inflammatory cytokine interleukin-13 in response to stimulation. This result indicates the presence of an intragraft Th2-like CD4+ T cell population, which may have a regulatory function in the induction of liver tolerance. ( J Gastrointest Surg 2002;6:455–463.) [ABSTRACT FROM AUTHOR]

The indirect alloimmune response seems to be restricted to a few dominant major histocompatibility complex (MHC)-derived peptides responsible for T-cell activation in allograft rejection. The molecular mechanisms of indirect T-cell activation have been studied using peptide analogues derived from the dominant allopeptide in vitro, whereas the in vivo effects of peptide analogues have not been well characterized yet. In the present study, we generated allochimeric peptide analogues by replacing the three allogeneic amino acids 5L, 9L, and 10T in the sequence of the dominant MHC class I allopeptide P1. These allochimeric peptide analogues were used to define the allogeneic amino acids critical for the MHC binding and TCR recognition. We found that position 5 (5L) of the dominant allopeptide acts as an MHC-binding residue, while the other two allogeneic positions, 9 and 10, are important for the T-cell receptor (TCR) recognition. A peptide containing the MHC-binding residue 5L, as the only different amino acid between donor (RT1.Au) and recipient (RT1.Al) sequences, did not induce proliferation of lymph node cells primed with the dominant peptide and prevented dominant peptide-induced acceleration of allograft rejection. Identification of MHC and TCR contact residues should facilitate the development of antigen-specific therapies to inhibit or regulate the indirect alloimmune response. [Copyright &y& Elsevier]

The recognition of major histocompatibility complex (MHC) allopeptides by recipient MHC class II-restricted CD4+ T cells via indirect pathway is a prerequisite for the generation of an immune response to the allograft. We tested 13-mer to 24-mer peptides from the MHC class I molecule for their possible immunogenicity in a fully MHC-mismatched rat strain combination. Our results confirm the hierarchical distribution of the immunogenicity of donor MHC class I peptides in the T cell alloactivation via indirect pathway. In addition, we show that allopeptide-induced immune response is critical for acute rejection of heart allografts. Among the seven allopeptides tested, peptide P1 was identified as immunodominant; it induced the greatest T cell proliferation and cytokine production in vitro as well as a significant reduction in allograft survival time. The TCR repertoire of T cells involved in the in vitro and in vivo responses induced by the dominant allopeptide P1 was found to be limited to the Vβ10 and Vβ 19 gene families. The identification of dominant allopeptides should greatly facilitate characterization of the specific T cell population responsible for allograft rejection and may be used to modulate the alloimmune response through antigen-specific therapy. [Copyright &y& Elsevier]

Background: During acute rejection of organ or tissue allografts T cells and macrophages are dominant infiltrating cells. CD4-positive T cells are important for the induction of allograft rejection and macrophages are important effector cells mediating cytotoxicity via production of nitric oxide (NO) by the inducible NO-synthase (iNOS). In the present study we analysed whether the destruction of primarily nonvascularised parathyroid allografts is also mediated by iNOS-positive macrophages. Methods: Hypocalcaemic Lewis rats received parathyroid isografts (from Lewis donors) and allografts (from Wistar Furth donors), respectively, under the kidney capsule. Levels of serum calcium above 2 mmol/L correlated with normal parathyroid function and below 2 mmol/L with parathyroid rejection. Accelerated parathyroid allograft rejection was induced by immunisation of Lewis recipients with the allogeneic peptide P1. Results: Determination of serum calcium levels is a useful parameter to control parathyroid graft function, and therefore to determine allograft rejection. Macrophages positive for both major histocompatibility complex (MHC) class II molecules and costimulatory molecules accumulated in iso- and allografts. but iNOS-positive macrophages were only detectable in allografts in the presence of activated CD4-positive I cells. These results confirm a cooperation between activated T cells and intragraft macrophages to induce macrophage iNOS expression. Recipients immunised with the allogeneic peptide P1 demonstrated accelerated rejection of allografts (mean ± SD: 92 ± 0.9 days) in contrast to nonimmunised animals (mean ± SD: 15.8 ± 1.8 days). Allografts of P1- immunised animals were infiltrated faster by activated CD4-positive T cells and, in addition, the infiltrates of iNOS- positive macrophages were stronger than those in allografts of nonimmunised animals. Conclusions: Intragraft iNOS-positive macrophages seem to be able to produce cytotoxic NO involved in the killing of allogeneic cells during the alloimmune response against primarily nonvascularised parathyroid organ grafts. Infiltrates of iNOS-negative macrophages found in parathyroid isografts were caused by antigen-independent inflammation triggered by surgically induced injury. The absence of activated T cells in isografts and their presence in allografts underlines their importance in inducing macrophage iNOS expression. [ABSTRACT FROM AUTHOR]

It has been extensively documented the role of the indirect pathway of allorecognition in allograft rejection. However, recent data demonstrate that the manipulation of this pathway could be also sufficient to promote prolongation of allograft survival. In the present study we evaluated the effect of preoperative immunization with the WF-specific MHC class II peptides RT1.D2 and RT1.B2 in combination with low-dose CsA from days 0 to 7 (5 mg/kg/day) and from days 8 to 30 (1 mg/kg/day) after WF small bowel transplantation. Seven days before and on the day of transplantation, LEW recipients were immunized with the two WF MHC class II peptides RT1.B2 and RT1.D2. The CsA monotherapy induced an allograft survival of 49.3 ± 6.1 days. MHC class II peptide immunization had a limited effect on allograft survival for RT1.D2 (47.1 ± 3.8 days) and induced prolongation of allograft survival for RT1.B2 (73.6 ± 34.6 days). This effect seems to be based on the absence or silence of RT1.B2-reactive T cells and rejection seems to be correlated with the presence of RT1.B2-specific T cells in the late phase. Therefore, the combination of RT1.B2 with low-dose CsA shifts the immunological response and protects small bowel allograft rejection. [Copyright &y& Elsevier]