Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154+ Aspergillus fumigatus specific T-cell quantification.

Flow cytometric quantification of CD154+ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154+ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154+ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154+ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.

Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells.

CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification.

Evaluation of Aspergillus and Mucorales specific T-cells and peripheral blood mononuclear cell cytokine signatures as biomarkers of environmental mold exposure.

Mold specific T-cells have been described as a supportive biomarker to monitor invasive mycoses and mold exposure. This study comparatively evaluated frequencies and cytokine profiles of Aspergillus fumigatus and Mucorales reactive T-cells depending on environmental mold exposure. Peripheral blood mononuclear cells (PBMCs) obtained from 35 healthy donors were stimulated with mycelial lysates of A. fumigatus and three human pathogenic Mucorales species. CD154+ specific T-cells were quantified by flow cytometry. In a second cohort of 20 additional donors, flow cytometry was complemented by 13-plex cytokine assays. Mold exposure of the subjects was determined using a previously established questionnaire. Highly exposed subjects exhibited significantly greater CD154+A. fumigatus and Mucorales specific naïve and memory T-helper cell frequencies. Significant correlation (r = 0.48 - 0.79) was found between A. fumigatus and Mucorales specific T-cell numbers. Logistic regression analyses revealed that combined analysis of mold specific T-cell frequencies and selected cytokine markers (A. fumigatus: IL-5 and TNF-α, R. arrhizus: IL-17A and IL-13) significantly improves classification performance, resulting in 75-90 % predictive power using 10-fold cross-validation. In conclusion, mold specific T-cell frequencies and their cytokine signatures offer promising potential in the assessment of environmental mold exposure. The cytokines identified in this pilot study should be validated in the clinical setting, e. g. in patients with hypersensitivity pneumonitis.

Susceptibility of A. fumigatus-specific T-cell assays to pre-analytic blood storage and PBMC cryopreservation greatly depends on readout platform and analytes.

Mould-specific T cells detectable by flow cytometry or ELISPOT were proposed as a novel biomarker in invasive aspergillosis. To define protocols facilitating sample shipment and longitudinal analysis, this study evaluated the susceptibility of different functional assays for A. fumigatus-specific T-cell quantification and characterisation to pre-analytic delays. PBMCs from 6 healthy donors were analysed after immediate isolation, after 6 hours whole blood storage or after cryopreservation using 3 different common media. Functional responses to A. fumigatus lysate stimulation were comparatively assessed by flow cytometry, ELISPOT and 14-plex cytokine assay. After 6 hours pre-analytic storage, all functional assays showed reduced detection rates, higher coefficients of variation (CV) and widely varying individual recovery indices of specific T-cell response. While cryopreservation resulted in sufficient yields and largely unaltered cellular composition, outcomes of functional readouts significantly differed from freshly processed samples. For CD154-based flow cytometry, only cryopreservation in RPMI supplemented with autologous serum resulted in satisfactory detection rates and CVs. For ELISPOT and cytokine secretion assays, none of the cryopreservation protocols provided sufficient concordance with immediately processed samples. Even using the same readout platform, individual analytes widely varied in their susceptibility to cryopreservation, highlighting that distinct optimisation is required depending on the downstream assay.

Analysis of the in vitro activity of human neutrophils against Aspergillus fumigatus in presence of antifungal and immunosuppressive agents.

Neutrophils are essential in the first line defense against moulds. This in vitro study assessed different neutrophil effector mechanisms in the presence of clinically relevant antifungal and immunosuppressive agents. Therapeutic concentrations of liposomal amphotericin B led to reduced IL-8 and oxidative burst response to the synthetic stimulus PMA, whereas no major alterations of oxidative burst, phagocytosis, or cytokine response to germinated stages of Aspergillus fumigatus and no supra-additive effects of antifungal and immunosuppressive drugs were observed. Conventional and liposomal amphotericin B as well as voriconazole, however, led to reduced neutrophil extracellular trap formation in response to A. fumigatus germ tubes.

Comparative Analysis of Inflammatory Cytokine Release and Alveolar Epithelial Barrier Invasion in a Transwell® Bilayer Model of Mucormycosis.

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.

Intra- and inter-individual variability of Aspergillus fumigatus reactive T-cell frequencies in healthy volunteers in dependency of mould exposure in residential and working environment.

Invasive aspergillosis remains a deadly disease in immunocompromised patients, whereas the combination of an exaggerated immune response and continuous exposure lead to various hyperinflammatory diseases. This pilot study aimed to gain an overview of the intra- and inter-individual variability in Aspergillus fumigatus reactive T-helper cells in healthy adults and the correlation with environmental mould exposure. In this flow cytometric study, the frequencies of CD154+ A. fumigatus reactive T cells were evaluated in 70 healthy volunteers. All subjects completed a standardised questionnaire addressing their mould exposure. Subjects with intensive mould exposure in their professional or residential surrounding demonstrated considerably higher mean frequencies of A. fumigatus reactive T-helper and T-memory cells. Comparative evaluation of multiple measurements over time demonstrated relatively conserved reactive T-cell frequencies in the absence of major changes to the exposure profile, whereas those frequently exposed in professional environment or with changes to their risk score demonstrated a marked dependency of antigen reactive T-cell frequencies on recent mould exposure. This pilot study was the first to provide data on the intra-individual variability in A. fumigatus reactive T-cell frequencies and its linkage to mould encounter. Fungus reactive T cells are to be considered a valued tool for the assessment of environmental mould exposure.

Preoperative serum h-FABP concentration is associated with postoperative incidence of acute kidney injury in patients undergoing cardiac surgery.

BACKGROUND: Fatty acid binding protein (FABP) is an intracellular transport protein associated with myocardial damage size in patients undergoing cardiac surgery. Furthermore, elevated FABP serum concentrations are related to a number of common comorbidities, such as heart failure, chronic kidney disease, diabetes mellitus, and metabolic syndrome, which represent important risk factors for postoperative acute kidney injury (AKI). Data are lacking on the association between preoperative FABP serum level and postoperative incidence of AKI. METHODS: This prospective cohort study investigated the association between preoperative h-FABP serum concentrations and postoperative incidence of AKI, hospitalization time and length of ICU treatment. Blood samples were collected according to a predefined schedule. The AKI Network definition of AKI was used as primary endpoint. All associations were analysed using descriptive and univariate analyses. RESULTS: Between 05/2009 and 09/2009, 70 patients undergoing cardiac surgery were investigated. AKI was observed in 45 patients (64%). Preoperative median (IQR) h-FABP differed between the AKI group (2.9 [1.7-4.1] ng/ml) and patients without AKI (1.7 [1.1-3.3] ng/ml; p = 0.04), respectively. Patients with AKI were significantly older. No statistically significant differences were found for gender, type of surgery, operation duration, CPB-, or X-Clamp time, preoperative cardiac enzymes, HbA1c, or CRP between the two groups. Preoperative h-FABP was also correlated with the length of ICU stay (rs = 0.32, p = 0.007). CONCLUSIONS: We found a correlation between preoperative serum h-FABP and the postoperative incidence of AKI. Our results suggest a potential role for h-FABP as a biomarker for AKI in cardiac surgery.

Hyperthermic intraperitoneal chemotherapy in patients with peritoneal carcinomatosis: role of heat shock proteins and dissecting effects of hyperthermia.

BACKGROUND: In patients with isolated peritoneal carcinomatosis (PC) of gastrointestinal cancer, hyperthermic intraperitoneal chemotherapy (HIPEC) represents a promising treatment option integrated into multimodal concepts. Heat shock proteins (HSP) seem to play a major role in cellular stress during HIPEC therapy. We analyzed differentially hyperthermic conditions and HSPs responsible for cell stress-mediated repair mechanisms in tumor tissues from patients who underwent HIPEC therapy and in an in vitro hyperthermic model. METHODS: Tumor tissues from our patient cohort with isolated PC were selected for further analysis when representative material was available before and after HIPEC therapy. To further dissect the role of HSPs under conditions of hyperthermia, gene and protein expression was additionally determined, together with cellular apoptosis and proliferation in human HT-29 colon cancer cells. RESULTS: Differently up-regulated HSP70/72 and HSP90 gene and protein expression was found in all investigated patient tumors. In vitro studies confirmed observations from clinical tumor analysis as underlying HSP-mediated cell stress mechanisms. Moreover, results from proliferation and apoptosis assays combined with differentiated HSP expression analysis demonstrated the relevance of preselecting specific target temperatures to achieve optimal toxic effects on remaining tumor cells in vivo. CONCLUSIONS: Therapeutic approaches like HIPEC to achieve antiproliferative and apoptosis-inducing cellular effects in patients with PC are negatively influenced by highly conserved HSP mechanisms in tumor cells. This study shows for the first time that specific hyperthermic conditions are necessary to be established to achieve optimal toxic effects on tumor cells during HIPEC therapy, a finding that opens potentially new therapeutic strategies.

Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer.

BACKGROUND: Regulatory T cells (Treg) expressing the transcription factor forkhead-box protein P3 (Foxp3) have been identified to counteract anti-tumor immune responses during tumor progression. Besides, Foxp3 presentation by cancer cells itself may also allow them to evade from effector T-cell responses, resulting in a survival benefit of the tumor. For colorectal cancer (CRC) the clinical relevance of Foxp3 has not been evaluated in detail. Therefore the aim of this study was to study its impact in colorectal cancer (CRC). METHODS AND FINDINGS: Gene and protein analysis of tumor tissues from patients with CRC was performed to quantify the expression of Foxp3 in tumor infiltrating Treg and colon cancer cells. The results were correlated with clinicopathological parameters and patients overall survival. Serial morphological analysis demonstrated Foxp3 to be expressed in cancer cells. High Foxp3 expression of the cancer cells was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in overall patient survival. CONCLUSIONS: Our findings strongly suggest that Foxp3 expression mediated by cancer cells rather than by Treg cells contribute to disease progression.

Allergic predisposition, histamine and histamine receptor expression (H1R, H2R) are associated with complicated courses of sigmoid diverticulitis.

BACKGROUND: We aimed to evaluate our hypothesis that allergic predisposition and expression of histamine receptors might contribute to complicated courses of sigmoid diverticulitis. METHODS: Expression of histamine and histamine receptors (H1R, H2R) was analysed on protein level (immunohistochemistry/immunofluorescence (IF)) as well as mRNA level (reverse transcription-PCR (RT-PCR) in surgical specimen of patients (n = 101) having undergone resection for sigmoid diverticulits (n = 57 complicated diverticulitis/n = 44 non-complicated diverticulitis). RESULTS: The mean number of comorbid diseases per patient was 1.76 ± 1.25. Thirty-nine of 101 patients (38.6%) exhibited allergic predisposition (grass poll, food, drug, pets, etc.). Comorbid diseases were significantly associated with complicated diverticulitis (p = 0.027). Complicated sigmoid diverticulitis was significantly associated with high H1R and H2R expression (p < 0.001). Furthermore, an association of complicated diverticulitis with allergic predisposition was found (odds ratio = 3.2, p = 0.0097). IF double-labelling experiments showed a strong correlation of increased histamine expression with expression of H1R and H2R on intestinal enterocytes (histamine/H1R, rho = 0.841, p < 0.0001 and histamine/H2R, rho = 0.806, p < 0.0001). The results of increased H1R and H2R expression in complicated sigmoid diverticulitis were also detected on mRNA level in a subset of patients (RT-PCR, p = 0.009). CONCLUSIONS: Our findings suggest that allergic predisposition might be another important risk factor for complicated courses of acute sigmoid diverticulitis and linked with histamine receptor expression. Supportive therapies with antihistaminic drugs might become an option. Allergic predisposition might be worth considering when indicating surgery for sigmoid diverticulitis.

LgR5 expression and cancer stem cell hypothesis: clue to define the true origin of esophageal adenocarcinomas with and without Barrett's esophagus?

BACKGROUND: Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett's Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis. MATERIALS AND METHODS: Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunohistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates. RESULTS: LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis. CONCLUSION: The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.

Inhibition of nuclear translocation of calcineurin suppresses T-cell activation and prevents acute rejection of donor hearts.

BACKGROUND: Inhibition of calcineurin (CnA) activity by cyclosporine A (CsA) is the mainstay in immunosuppressive therapy. CsA inhibits the phosphatase activity of the cytosolic phosphatase CnA and, therefore, prevents the dephosphorylation and subsequently nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). However, CsA has multiple other targets within the cell and is, therefore, not specific. We developed a new approach to inhibit CnA/NFAT signaling. This synthetic peptide prevented CnA nuclear translocation in vitro. The purpose of this study was to demonstrate that this novel approach could potentially inhibit T-cell function in vitro and in vivo. METHODS: T-cell activation (Jurkat T cells, naïve rat T cells, and peripheral human T cells) was assessed by protein synthesis, interleukin (IL)-2 promoter activity, and IL-2 levels after T-cell activation. Immunohistological stainings for CnA were performed to investigate nuclear localization of CnA. The immunosuppressive effects in vivo of the synthetic peptide were investigated in rats with heterotopic transplanted hearts. RESULTS: The nuclear localization signal peptide significantly decreased alloantigen-specific T-lymphocyte proliferation, IL-2 promoter activity, and IL-2 production (338% ± 27% vs. 149% ± 11%, n=8, P<0.05) in cultured T cells by inhibition of CnA nuclear translocation. The synthetic peptide also significantly decreased the number of graft infiltrating CD8 T lymphocytes. Moreover, treatment with the synthetic inhibitory inhibited acute graft rejection (5 ± 0.6 days vs. 12 ± 2 days, n=10, P<0.05). CONCLUSIONS: Inhibition of nuclear translocation of CnA is a novel approach to inhibit the activation of the CnA/NFAT signaling cascade. Further studies have to demonstrate the long-term use of this principle in vivo.

Chemokines involved in tumor promotion and dissemination in patients with renal cell cancer.

BACKGROUND: Chemokines play a critical role in tumor initiation, progression, and metastasis and have been associated with poor prognosis in diverse malignancies. The prognostic impact of chemokines for renal cell cancer (RCC) remains to be defined. METHODS: Patients diagnosed with RCC and operated between 07/07 and 05/11 were differentially assessed for expression profiles of a series of chemokines and their receptors by RT-qPCR and Western Blot analysis (tumor and adjacent normal tissue, n=37) and by Luminex for corresponding serum expression levels. Results were statistically correlated with clinicopathologic parameters. RESULTS: Gene expression of CCL2, CCR7, CXCL12, CXCR3, CXCR5 and CX3CL1 chemokines was significantly down-regulated in tumor compared to normal tissue. The gene profile for CCR6 was positively correlated with tumor size and stage. A positive linear correlation was found between CXCL12 and tumor stage as well as between CX3CR1 and C-reactive protein. In contrast to clear cell RCCs those of a chromophobe type showed a significantly down-regulated gene expression for CCR6, CCL20, and CXCL12. The CXCR7 serum level was significantly increased in patients with tumor-related mortality during postoperative follow-up. CONCLUSIONS: Chemokines may serve as novel diagnostic and prognostic biomarkers for RCC. Studies on larger collectives are required for further assessment of potential clinical application.

MMP-1 is a (pre-)invasive factor in Barrett-associated esophageal adenocarcinomas and is associated with positive lymph node status.

BACKGROUND: Esophageal adenocarcinomas (EACs) arise due to gastroesophageal reflux, with Barrett's esophagus (BE) regarded as precancerous lesion. Matrix metalloproteinases (MMPs) might play a role during the multistep carcinogenetic process. METHODS: Expression of MMP-1 and -13 was analyzed in esophageal cancer (n = 41 EAC with BE, n = 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC), furthermore in BE without intraepithelial neoplasia (IN) (n = 18), and the cell line OE-33. MMP-1 was co-labelled with Ki-67 (proliferation), Cdx-2 (marker for intestinal metaplasia, BE) and analyzed on mRNA level. MMP-1 staining results were correlated with clinicopathological parameters. RESULTS: On protein level, MMP-1 expression was found in 39 of 41 (95%) EAC with BE, in 19 of 19 (100%) EAC without BE, in 6 of 10 (60%) ESCC, and in 10 of 18 (56%) BE without IN. No expression of MMP-13 was found in these specimens. Quantification showed 48% MMP-1 positive cells in EAC with BE, compared to 35% in adjacent BE (p < 0.05), 44% in EAC without BE, 32% in ESCC, and 4% in BE without IN. Immunofluorescence double staining experiments revealed increased MMP-1 expressing in proliferating cells (MMP-1+/Ki-67+) (r = 0.943 for BE and r = 0.811 for EAC). On mRNA-level, expression of MMP-1 was significantly higher in EAC compared to BE (p = 0.01) and confirmed immunohistochemical staining results. High MMP-1 levels were associated with lymph node metastases but not with poorer survival (p = 0.307). CONCLUSIONS: Our findings suggest that MMP-1 plays a role as preinvasive factor in BE-associated EAC. Expression of MMP-1 in proliferating BE and EAC cells suggest malignant proliferation following the clonal expansion model.

Heterotopic rat heart transplantation (Lewis to F344): early ICAM-1 expression after 8 hours of cold ischemia.

BACKGROUND: Primary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function. METHODS: In a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours. RESULTS: Using immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion. CONCLUSIONS: We hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.