After Intestinal Transplantation Kidney Function Is Impaired by Downregulation of Epithelial Ion Transporters in the Ileum.

BACKGROUND: Intestinal transplantation is a treatment option for intestinal failure. Although nephrotoxic medication after transplantation is a major cause for posttransplant renal insufficiency, it remains unclear why kidney dysfunction is particularly frequent after intestinal transplantation. METHODS: This study analyzed messenger RNA expression of NHE3, DRA, and CFTR in 404 biopsies obtained between day 2 and 1508 from the terminal ileum of 10 adult intestinal transplant recipients. RESULTS: The time courses of immunosuppression and glomerular filtration rate were correlated. In the first posttransplant year, expression of NHE3 and DRA, which mediate NaCl absorption, was diminished to a greater degree than that of CFTR, which mediates chloride secretion. Reduced NHE3 and DRA expression was associated with high tacrolimus trough levels. Titration of tacrolimus to low levels by year 2 was paralleled by partially restored NHE3 and DRA expression. In cell culture experiments, similar effects of tacrolimus on transporter expression were detected. In patients, both reduced tacrolimus levels and recovery of NHE3 and DRA expression were associated with stabilization of renal function. CONCLUSIONS: Our data strongly suggest that tacrolimus impairs absorption of NaCl and water from the transplanted ileum, leading to volume depletion and impaired renal function. This may be reversible by reduction of tacrolimus to lower levels without increased rates of rejection or chronic graft failure.

Reevaluating the concept of treating experimental tumors with a mixed bacterial vaccine: Coley's Toxin.

Several decades after Coley's initial work, we here systematically analyzed tumoricidal as well as immunostimulatory effects of the historical preparation Coley's Toxin (CT), a safe vaccine made of heat-inactivated S. pyogenes and S. marcescens. First, by performing in vitro analysis, established human pancreatic carcinoma cell lines responded with dose- and time-dependent growth inhibition. Effects were attributed to necrotic as well as apoptotic cell death as determined by increased Caspase 3/7 levels, raised numbers of cells with sub-G1-DNA, and induced p21(waf) expression, indicative for cell cycle arrest. Besides, CT effectively stimulated human peripheral blood leukocytes (huPBL) from healthy volunteers. Quantitative gene expression analysis revealed upregulated mRNA levels of selected Toll-like receptors. Flow cytometric phenotyping of CT-stimulated huPBLs identified raised numbers of CD25(+)-activated leukocytes. In vivo, repetitive, local CT application was well tolerated by animals and induced considerable delay of Panc02 tumors. However, systemic treatment failed to affect tumor growth. Antitumoral effects following local therapy were primarily accompanied by stimulation of innate immune mechanisms. Data presented herein prove that the historical approach of using killed bacteria as active immunotherapeutic agents still holds promise, and further careful preclinical analyses may pave the way back into clinical applications.

Up-regulation of ICAM-1 during cold ischemia triggers early neutrophil infiltration in human pancreas allograft reperfusion.

BACKGROUND: Graft pancreatitis is induced by ischemia/reperfusion injury in which neutrophil infiltration is believed to be a crucial early event. This observation suggests the presence of adhesion molecules already at the time of reperfusion. Therefore, this study was performed to evaluate the pattern of ICAM-1 and P-Selectin expression on human pancreas allografts following cold ischemia and reperfusion. PATIENTS AND METHODS: We performed an analysis of pancreas biopsy specimens taken from 13 patients undergoing pancreas transplantation compared with pancreas specimens from 10 patients following resection. Cryostat sections were stained with monoclonal antibodies against CD11b, a neutrophil marker, and the adhesion molecules ICAM-1 and P-Selectin. RESULTS: Extensive infiltration of CD11b-positive cells was detected in venules and capillaries of pancreas allografts after reperfusion (18.38 +/- 0.87) compared with controls (T1 4.22 +/- 0.55) or with tissue specimens at about 10 hours of cold ischemia (2.60 +/- 0.35; P < .001). Similarly, the pattern of P-Selectin showed a moderate expression before organ harvest (1.54 +/- 0.21) and in samples during cold ischemia (1.46 +/- 0.24) followed by a significantly greater number of P-Selectin-positive cells after reperfusion (2.54 +/- 0.18; P = .005). ICAM-1 was only weakly expressed on the surface of the venular endothelium in all controls (0.77 +/- 0.12). In contrast to P-Selectin, ICAM-1 showed prominent up-regulation during cold ischemia (2.23 +/- 0.23; P < .001) with no further increase after reperfusion (2.23 +/- 0.17). CONCLUSION: The data suggested that ICAM-1 was already up-regulated during cold ischemia, possibly representing the mechanism of early neutrophil infiltration observed in human pancreatic ischemia/reperfusion injury.

An in vitro system for the determination of individualized immunosuppression.

The risk of complications of immunosuppressive treatment in organ transplantation increases with the aggregate amount of immunosuppressive medication given to the patient. As the doses of immunosuppressive agents required to achieve comparable effects show considerable variability, methods to assess individual sensitivity toward immunosuppressive regimens are urgently needed. The aim of this study was to develop such an in vitro test system. As immunological model for allogeneic transplantation, individual pairs of recipient-derived lymphocytes and of donor-derived B lymphocytes mimicking HLA expression of cells in the transplanted organ were isolated and assessed in mixed-lymphocyte cultures (MLC). Alloreactivity was readily observed and MLC consisted of CD8(+) and CD4(+) T cells as well as CD56(+) natural killer cells. A proliferation assay to measure the response of individual MLC on the immunosuppression by cyclosporine (CsA) was developed. The concentrations of CsA leading to growth reductions by 50% (inhibitory concentration 50, IC(50)) were found between 110 and 220 ng/mL, which was near the trough whole blood levels for CsA. Accordingly, the IC(90) values (660 to 1760 ng/mL) were near the target values for peak whole blood levels. We believe that these data present a simple and potentially useful in vitro technology that allows for the prediction of individual responses to immunosuppressive therapeutic regimens.

Pancreatic cancer regression by intratumoural injection of live Streptococcus pyogenes in a syngeneic mouse model.

BACKGROUND: This study addressed the potential of bacteriolytic therapy using Streptococcus pyogenes in a syngeneic pancreatic carcinoma mouse model. METHODS: Panc02 tumours were either infected with S pyogenes or were treated with the equivalent volume of vehicle. In addition to assessment of tumour histology and immunohistochemistry, isolated splenocytes were analysed by flow cytometry. Interferon (IFN) gamma secretion as a reaction of splenocytes against tumour cells was shown through the ELISpot technique. A cytotoxic effect of lymphocytes against tumour targets was detected by lactate dehydrogenase (LDH) release. Cytokine levels in serum were measured. RESULTS: A single application of live bacteria into established Panc02 tumours resulted in complete tumour regression. This antitumoral effect was accompanied by massive leucocyte infiltration into the tumours as well as a significant and sustained elevation of systemic levels of the proinflammatory cytokines IFN gamma, tumour necrosis factor alpha and interleukin 6. Lymphocytes obtained from treated mice specifically recognised syngeneic tumour cells in IFN gamma-ELISpot, and most importantly in cellular cytotoxicity assays, indicating a tumour-specific immune response. CONCLUSIONS: We provide data that both the direct lytic activity of S pyogenes towards tumour cells and the infection-driven infiltration of tumours by cells of the innate immune system lead to damage of tumour cells followed by a dissemination of tumour components. This last outcome allows for the activation of tumour-specific effector cells, most probably in draining lymph nodes, promoted by the proinflammatory context. Taken together, these data indicate that the application of live S pyogenes may be a promising new treatment strategy for advanced pancreatic cancer patients that warrants further investigation.

High prevalence of Mycobacterium avium subspecies paratuberculosis IS900 DNA in gut tissues from individuals with Crohn's disease.

BACKGROUND AND AIMS: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in Crohn's disease (CD) tissues. Therefore, we examined IS900 in a large number of gut samples from patients with CD (n = 100) and ulcerative colitis (UC, n = 100), and in non-inflamed control tissues (nIBD, n = 100). We hypothesised that IS900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD. METHODS: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. RESULTS: IS900 PCR detection rate was significantly higher in CD tissue samples (52%) than in UC (2%) or nIBD (5%) specimens (p<0.0001). In CD patients, IS900 DNA was detected in samples from both diseased small bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS900 detection rates and clinical phenotypic characteristics in CD could be established. However, corticosteroid medication constituted a factor which tended to have a negative influence on IS900 DNA detection rates in CD (p<0.01). CONCLUSIONS: The presence of MAP specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn's disease.

Frameshift peptide-derived T-cell epitopes: a source of novel tumor-specific antigens.

Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C-terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono- and dinucleotide repeat-bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40-activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA-A2.1-restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (-1) frameshift mutation in the TGFbetaIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor-specific antigens. It is tempting to speculate that a frameshift peptide-directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic at-risk individuals in HNPCC families by a prophylactic vaccination strategy.

Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene.

The use of the cytosine deaminase (CD)/5-fluorocytosine suicide system as a cancer gene therapy approach enables selective killing of CD-modified cells as well as the ablation of non-modified tumor cells due to a bystander effect that has been suggested to involve the immune system in vivo. Using a stable CD transfectant of the tumorigenic rat adenocarcinoma cell line AS (AS/CD), an antitumoral response against the CD expressing cell line as well as the parental cell line could be induced by stepwise vaccinations in syngeneic animals. AS/CD tumor regression occurred independently of 5-fluorocytosine treatment and was sufficient to protect 37% of the animals against subsequent challenge with tumorigenic doses of the parental AS cell line. Immune rats contained lymphocytes able to specifically lyse CD modified as well as unmodified AS tumor cells in vitro, most likely contributing to the in vivo antitumoral reaction. Thus, the CD suicide system seems to be suitable not only for a local tumor gene therapy but also for the application as therapy of metastatic tumors and minimal residual disease.

Recombinant gp100 protein presented by dendritic cells elicits a T-helper-cell response in vitro and in vivo.

Induction of a helper T (TH)-cell response is a critical element in the generation of anti-tumor immunity. The majority of immunotherapeutic approaches have so far been concerned with the generation of cytotoxic T lymphocytes (CTLs). This also accounts for gp100, a melanoma-associated protein which induces a potent CTL response. Because of the high immunogenicity of gp100, we considered it of special interest to explore the feasibility of generating gp100-specific TH cells. Human dendritic cells (DCs) were loaded with recombinant gp100 protein, and the response of autologous TH cells was evaluated in vitro and in vivo. We have observed that gp100 peptides can be presented by DCs of certain MHC class II haplotypes, which led to proliferation and cytokine production of TH-1 cells in vitro. Furthermore, transfer of gp100 protein-loaded human DCs into SCID mice also induced proliferation of autologous, unprimed peripheral blood leukocytes (PBLs) and selective expansion of TH cells. When human T cells from the spleen of SCID mice were recovered and restimulated in vitro, they strongly proliferated in response to gp100-loaded DCs, while showing minimal proliferative activity in response to DCs loaded with a control antigen. Thus, it is possible to induce an efficient MHC class II-restricted TH response by in vitro stimulation or in vivo vaccination with DCs which have been loaded with a purified tumor-associated antigen.