Methoxyethyl-modified intercellular adhesion molecule-1 antisense phosphorothiateoligonucleotides inhibit allograft rejection, ischemic-reperfusion injury, and cyclosporine-induced nephrotoxicity.

BACKGROUND: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity. METHODS/RESULTS: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. CONCLUSIONS: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

Selection of lowly immunogenic and highly tolerogenic donor and recipient allochimeric class I major histocompatibility complex proteins.

BACKGROUND: Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1. An rat alloantigen. We examined the potential of alpha1h-RT1. An versus alpha2h-RT1. An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts. METHODS: The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1. An AAs. Allochimeric and wild-type (WT)-RT1. An proteins were generated in an Escherichia coli expression system. RESULTS: A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1. An protein; alpha2h148-179n-RT1.Ac protein was ineffective. However, subcutaneous injection of 100 mug WT-RT1. An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation. CONCLUSIONS: The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

Fast hematopoietic recovery after bone marrow engraftment needs physiological proximity of stromal and stem cells.

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT1(1)) rats were either injected IV with syngeneic sex-mismatched 80 x 10(6) I-BM or transplanted with 80 x 10(6) V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.

Microenvironment elements involved in the development of pancreatic cancer tumor.

Introduction. In spite of intensive research during many years, pancreatic adenocarcinoma remains one of the deadliest cancers. The surgical intervention remains main possibility of treatment because chemotherapy and radiotherapy has a minimal impact on long-term survival. We are still looking for the weak points of this devastating disease. Materials and Methods. Pancreatic tumor tissue samples were collected from 36 patients. Immunohistochemistry staining was used to evaluate expression of growth factors and immune infiltrates. Activity of MMP2 and MMP9 was assessed by gelatin zymography on 7.5% SDS-PAGE gel with 0.1% gelatin. Results. All growth factors were strongly expressed in pancreatic tumor tissue. We found that level of expression of c-Met receptor was higher for G3 tumors than for G2 tumors. Also we found that active MMP2 was present at all stages of tumor while active MMP9 just at more advanced tumors. Abundant immune cells infiltration was distinctive for tumor tissue, especially macrophages were infiltrating tumor tissue. We found that amount of macrophages was associated with lymph nodes metastases. Conclusion. In our research we demonstrated that among many factors influencing tumor microenvironment c-Met receptor, infiltrating macrophages and MMP2 have significant influence on development and invasion of pancreatic cancer.

Analysis of pancreatic cancer microenvironment: role of macrophage infiltrates and growth factors expression.

BACKGROUND: Research over the last twenty years has yielded much insight into pancreatic cancer biology, but it has neither improved diagnostics methods nor the way of treatment. The question remains as to what the critical deciding factor is in making pancreatic cancer such an aggressive disease. METHODS: Pancreatic tumor tissue came from 36 patients. To assess lymphatic vessels color lymphangiography and immunohistochemistry were used. Activity of matrix metalloproteinases was studied with gel and in situ zymography. Expression of growth factors and infiltrating immune cells were investigated using immunohistochemistry. RESULTS: Our study revealed that the structures that correspond to lymphatic vessels were not observed in tumor center but only at the edge of the tumor. All studied growth factors were present in tumor tissue. We found that the difference in expression between G2 and G3 stage was statistically relevant in cases of c-Met receptor. Inflammatory cells were present around neoplastic glands and also strongly around nerves infiltrated by cancer cells. The number of infiltrating macrophages in tumor tissue was significantly higher in group with metastases to lymph nodes. CONCLUSION: We showed two factors that influence pancreatic cancer progression and invasion: c-Met receptors and macrophages infiltrating tumor tissue. Based on our analysis, this indicates that epithelial-mesenchymal transition might be crucial in the progression of pancreatic cancer.

Allograft rejection requires STAT5a/b-regulated antiapoptotic activity in T cells but not B cells.

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.