A new bicornuate model of rat uterus transplantation.

INTRODUCTION: Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child. MATERIAL AND METHODS: We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. RESULTS: Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. CONCLUSIONS: Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and, therefore, comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

Moderate LMWH Anticoagulation Improves Success Rate of Hind Limb Allotransplantation in Mice.

Background: The mouse hind limb model represents a powerful research tool in vascularized composite tissue allotransplantation, but its applicability is limited due to poor graft survival (62%-83%). Vascular thrombosis and massive hemorrhage are the major causes for these drop-outs. We hypothesize that because of better anticoagulation effect and lower risk of thrombocytopenia, application of low molecular weight heparin (LMWH) will minimize vascular complications and enhance graft and animal survival. Methods: Fifty allogeneic hind limb transplantations were performed (C57BL/6 to DBA/2 mice) using five different anticoagulation protocols. Bleeding and thromboembolic events were recorded macroscopically by postoperative hemorrhage and livid discoloration of the graft, respectively. Graft perfusion and survival were monitored daily by capillary-refill-time of graft toes within 2-3 seconds. Vascular congestion and tissue necrosis were examined by histological evaluation of hematoxylin-eosin-stained tissue sections. Results: All transplantations were technically successful. Increase in thromboembolic events and a concomitant decrease in bleeding events were observed with the decreasing concentration of heparin in the perfusion solution. Although treatment of donor and recipient with low dose of LMWH could not reduce thromboembolic events, moderate dose effectively reduced these events. Compared with the poor outcome of graft perfusion with heparin alone, additional treatment of donor and recipient with low dose of LMWH improved graft and animal survival by 18%. Interestingly, animals treated with moderate dose of LMWH demonstrated 100% graft and animal survival. Conclusions: Treatment of donor and recipient mice with a moderate dose of LMWH prevents vascular complications and improves the outcome of murine hind limb transplants.

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism.

Aims: Expanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation. Methods and results: First, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach. Conclusion: Uremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Renal inflamm-aging provokes intra-graft inflammation following experimental kidney transplantation.

Donor age is a major risk factor for allograft outcome in kidney transplantation. The underlying cellular mechanisms and the recipient's immune response within an aged allograft have yet not been analyzed. A comprehensive immunophenotyping of naïve and transplanted young versus aged kidneys revealed that naïve aged murine kidneys harbor significantly higher frequencies of effector/memory T cells, whereas regulatory T cells were reduced. Aged kidney-derived CD8+ T cells produced more IFNγ than their young counterparts. Senescent renal CD8+ T and NK cells upregulated the cytotoxicity receptor NKG2D and the enrichment of memory-like CD49a+ CXCR6+ NK cells was documented in aged naïve kidneys. In the C57BL/6 to BALB/c kidney transplantation model, recipient-derived T cells infiltrating an aged graft produced significantly more IFNγ, granzyme B and perforin on day 7 post-transplantation, indicating an enhanced inflammatory, cytotoxic response towards the graft. Pre-treatment of aged kidney donors with the senolytic drug ABT-263 changed the recipient-derived effector molecule profile to significantly reduced levels of IFNγ and IL-10 compared to controls. Graft function after ABT-263 pre-treatment was significantly improved 28 days post kidney transplantation. In conclusion, renal senescence also occurs at the immunological level (inflamm-aging) and aged organs provoke an altered recipient-dominated immune response in the graft.

Neutrophil Gelatinase-Associated Lipocalin Protects from ANCA-Induced GN by Inhibiting TH17 Immunity.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a diagnostic marker of intrinsic kidney injury produced by damaged renal cells and by neutrophils. ANCA-associated vasculitis features necrotizing crescentic GN (NCGN), and ANCA-activated neutrophils contribute to NCGN. Whether NGAL plays a mechanistic role in ANCA-associated vasculitis is unknown. METHODS: We measured NGAL in patients with ANCA-associated vasculitis and mice with anti-myeloperoxidase (anti-MPO) antibody-induced NCGN. We compared kidney histology, neutrophil functions, T cell proliferation and polarization, renal infiltrating cells, and cytokines in wild-type and NGAL-deficient chimeric mice with anti-MPO antibody-induced NCGN. To assess the role of TH17 immunity, we transplanted irradiated MPO-immunized MPO-deficient mice with bone marrow from either wild-type or NGAL-deficient mice; we also transplanted irradiated MPO-immunized MPO/IL-17A double-deficient mice with bone marrow from either IL-17A-deficient or NGAL/IL-17A double-deficient mice. RESULTS: Mice and patients with active ANCA-associated vasculitis demonstrated strongly increased serum and urinary NGAL levels. ANCA-stimulated neutrophils released NGAL. Mice with NGAL-deficient bone marrow developed worsened MPO-ANCA-induced NCGN. Intrinsic neutrophil functions were similar in NGAL-deficient and wild-type neutrophils, whereas T cell immunity was increased in chimeric mice with NGAL-deficient neutrophils with more renal infiltrating TH17 cells. NGAL-expressing neutrophils and CD3+ T cells were in close proximity in kidney and spleen. CD4+ T cells showed no intrinsic difference in proliferation and polarization in vitro, whereas iron siderophore-loaded NGAL suppressed TH17 polarization. We found significantly attenuated NCGN in IL-17A-deficient chimeras compared with MPO-deficient mice receiving wild-type bone marrow, as well as in NGAL/IL-17A-deficient chimeras compared with NGAL-deficient chimeras. CONCLUSIONS: Our findings support that bone marrow-derived, presumably neutrophil, NGAL protects from ANCA-induced NCGN by downregulating TH17 immunity.

Natural Killer Cells Promote Kidney Graft Rejection Independently of Cyclosporine A Therapy.

Natural Killer (NK) cells have recently been recognized as key players in antibody-mediated chronic allograft failure, thus requiring a comprehensive understanding whether NK cells can escape conventional immunosuppressive regimens. Influence of cyclosporine A (CyA) on NK cell function was studied in a mouse model of allogeneic kidney transplantation (KTX, BALB/c to C57BL/6). Recipients were treated daily with CyA (10 mg/kg) for seven or 14 days for long term survival (day 56). Administration of CyA in recipients resulted in significantly reduced frequencies of intragraft and splenic CD8+ T cells, whereas the latter illustrated reduced IFNγ production. In contrast, intragraft and splenic NK cell frequencies remained unaffected in CyA recipients and IFNγ production and degranulation of NK cells were not reduced as compared with controls. Depletion of NK cells in combination with CyA resulted in an improvement in kidney function until day 7 and prolonged graft survival until day 56 as compared to untreated controls. Surviving animals demonstrated higher intragraft frequencies of proliferating CD4+FoxP3+Ki67+ regulatory T (TREG) cells as well as higher frequencies of CD8+CD122+ TREG. We here demonstrate that NK cell depletion combined with CyA synergistically improves graft function and prolongs graft survival, suggesting that NK cell targeting constitutes a novel approach for improving KTX outcomes.

Autofluorescence: A potential pitfall in immunofluorescence-based inflammation grading.

Immunofluorescence (IF) staining of paraffin-embedded tissues is a frequently used method to answer research questions or even detect the abundance of a certain protein for diagnostic use. However, the signal originating from specific antibody-staining might be distorted by autofluorescence (AF) of the assessed tissue. Although the AF phenomenon is well known, its presence is often neglected by insufficient staining controls. In this study, we describe the existence of cellular AF in paraffin-embedded healthy and inflamed human and murine colonic tissues and present ways to reduce AF. The AF signal is detectable at emission spectra from 425 nm-738 nm, upon excitation from 403.6-638.7 nm and appears more pronounced in inflamed tissues. Most signals are located subepithelially in the tissue and in blood vessels. Previous studies have shown that the AF signals are caused by lipofuscin, which accumulates in lamina propria immune cells. In murine small intestine AF signals are present in granules in the Paneth cell zone. For alleviation of the AF signal, sudan black b (SBB) or copper sulfate was used. Incubation of the tissue slices with either one of the substances reduced AF. In conclusion, AF appears as an intrinsic biomarker for colonic inflammation. The dominant existence of AF in immune cells of IBD tissue elucidates the importance of negative controls and the limitation of IF staining for potential diagnostic purposes.

Prediction of delayed graft function and long-term graft survival by serum and urinary neutrophil gelatinase-associated lipocalin during the early postoperative phase after kidney transplantation.

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as an early marker protein for kidney dysfunction in various clinical settings. In this prospective study we evaluated serial changes of serum and urinary NGAL within the first 7 days after kidney transplantation in 170 consecutive recipients. The main focus of this study was to assess the performance of serum and urinary NGAL in the prediction of delayed graft function (DGF) and two-year graft and patient survival. Serum and urine samples of 170 patients undergoing primary kidney transplantation from October 2010 to December 2012 were prospectively collected from day 0 to 7. NGAL was analyzed by ELISA. Multivariate regression models, receiver-operating characteristics (ROC), and areas under ROC curves (AUC) were used to identify predictors of DGF. DGF occurred in 52 patients (30.6%). Serum (AUC = 0.869) and urinary NGAL (AUC = 0.872) on postoperative day (POD) 2 could accurately predict DGF compared to serum creatinine (AUC = 0.619). Multivariate analyses revealed donor age, serum and urinary NGAL significantly associated with DGF (p<0.001). Recipient age was the only significant factor in a cox regression model influencing two-year graft and patient survival. In conclusion, serum and urinary NGAL are early predictors of DGF after kidney transplantation.

Disturbances in iron homeostasis result in accelerated rejection after experimental heart transplantation.

BACKGROUND: Clinical data suggest that iron disturbances deleteriously affect graft survival after heart transplantation (HTx), but immunological mechanisms underlying this phenomenon have not yet been elucidated. METHODS: To identify the mechanistic influence of iron in a murine model of HTx, fully allogeneic BALB/c donor organs were transplanted into iron-overloaded or iron-deficient C57BL/6 mice, and recipients were analyzed for functional and immunological parameters. RESULTS: After HTx, iron overload accelerated acute rejection as observed by shortened graft survival (HTx vs HTx + iron; p = 0.01), elevated rejection score (p < 0.01), and induction of troponin T (p < 0.01). Compared with controls, allografts and recipient spleens derived from iron-overloaded recipients were characterized by a pronounced graft infiltration of CD4+ T cells (p < 0.01), CD3-NKp46+ natural killer cells (p < 0.05), and reduced frequencies of regulatory T cells (p < 0.01). This was accompanied by lower mRNA expression levels of anti-inflammatory cytokines, including interleukin-10, transforming graft factor-ß, and Foxp3. Cardiac allograft survival was further tested under co-stimulation blockade (CTLA4-Ig) showing that naïve grafts transplanted into iron-overloaded recipients illustrated restricted graft outcome compared with wild types (p = 0.0051), which was rescued after treatment with the iron chelator deferoxamine. Iron deficiency (ID) also resulted in enhanced intragraft infiltration of inflammatory cells and accelerated rejection in the acute setting (HTx vs HTx + ID; p = 0.02) and after co-stimulation blockade (p = 0.0059). CONCLUSIONS: We provide novel insights into the understanding of disturbances in iron homeostasis and their consequences after HTX, allowing novel insights regarding improvements in personalized immunosuppression to prolong allograft survival.

Cell-penetrating self-nanoemulsifying drug delivery systems (SNEDDS) for oral gene delivery.

OBJECTIVE: The aim of study was to investigate whether cell-penetrating peptides could amplify cellular uptake of plasmid DNA (pDNA) loaded self-nanoemulsifying drug delivery systems (SNEDDS) by mucosal epithelial cells, thereby enhancing transfection efficiency. METHODS: HIV-1 Tat peptide-oleoyl conjugate (TAT-OL) was synthesized through amide bond formation between HIV-1 Tat-protein 49-57 (TAT) and oleoyl-chloride (OL). SNEDDS formulation contained 29.7% each of Cremophor EL, Capmul MCM and Crodamol, 9.9% propylene glycol and 1% TAT-OL. SNEDDS with OL instead of TAT-OL served as control. RESULTS: Fluorescent-microscopy demonstrated 0.5% (m/v) nanoemulsions were suitable for subsequent studies. Mucus diffusion of nanoemulsion loaded with fluorescein diacetate (FDA) was 1.5-fold increased by incorporation of TAT-OL. Confocal microscopy confirmed that droplets of nanoemulsions were successfully internalized. Furthermore, quantitative analysis showed that addition of TAT-OL increases uptake of nanoemulsions by 2.3- and 2.6-folds after 2 and 4 hours of incubation, respectively. Cellular internalization pathways were found with substantial decrease in uptake in presence of indomethacin and chlorpromazine. Transfection efficiency investigated on HEK-293-cells was found to be 1.7- and 1.8-fold higher for SNEDDS loaded with TAT-OL compared to Lipofectin and control, respectively. CONCLUSION: In comparison to prevailing lipid and polymer-based delivery systems, these novel cell-penetrating SNEDDS likely represent most effective, simplistic and expedite dosage form for mucosal gene delivery.

Donor brain death leads to differential immune activation in solid organs but does not accelerate ischaemia-reperfusion injury.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx.

cJun N-terminal kinase (JNK) phosphorylation of serine 36 is critical for p66Shc activation.

p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.

Biopsychronology: A Method Using Live Tissue Staining to Image Cell Function in the Kidney.

Methods to monitor the status of a graft prior to transplantation are highly desirable to avoid unnecessary surgical interventions and follow-up treatments and to optimize the clinical outcome as delayed graft function may lead to costly and lengthy follow-up treatments or even organ loss. As a promising step in this direction we present a method which combines the use of fine needle biopsies, the staining of living cells with dyes suitable to monitor mitochondrial status/cellular integrity, and live confocal real-time analysis.This approach provides information about the functional and structural intactness of an organ within a few minutes. To confirm the feasibility of this approach, we recently published a pilot study using rodent kidneys. The results demonstrated that this method is suitable to monitor organ damage caused by ischemia or short periods of reperfusion. This procedure required minimal time for sample preparation and data acquisition and is suitable for recording damage resulting from unphysiological stress to the organ.

Biopsychronology: live confocal imaging of biopsies to assess organ function.

Prolonged ischemia (I) times caused by organ procurement and transport are main contributors to a decrease in organ function, which is further enhanced during reperfusion (R). This combined damage, referred to as ischemia-reperfusion injury (IRI), is a main contributor to delayed graft function, which leads to costly and lengthy follow-up treatments or even organ loss. Methods to monitor the status of a graft prior to transplantation are therefore highly desirable to optimize the clinical outcome. Here, we propose the use of fine needle biopsies, which are analyzed by real-time live confocal microscopy. Such a combination provides information about the functional and structural integrity of an organ within a few minutes. To confirm the feasibility of this approach, we obtained fine needle biopsies from rodent kidneys and exposed them to various stress conditions. Following the addition of a range of live stains, biopsies were monitored for mitochondrial function, cell viability, and tissue integrity using confocal live cell imaging. Our data demonstrate that this procedure requires minimal time for sample preparation and data acquisition and is well suitable to record organ damage resulting from unphysiological stress.

A p38MAPK/MK2 signaling pathway leading to redox stress, cell death and ischemia/reperfusion injury.

BACKGROUND: Many diseases and pathological conditions are characterized by transient or constitutive overproduction of reactive oxygen species (ROS). ROS are causal for ischemia/reperfusion (IR)-associated tissue injury (IRI), a major contributor to organ dysfunction or failure. Preventing IRI with antioxidants failed in the clinic, most likely due to the difficulty to timely and efficiently target them to the site of ROS production and action. IR is also characterized by changes in the activity of intracellular signaling molecules including the stress kinase p38MAPK. While ROS can cause the activation of p38MAPK, we recently obtained in vitro evidence that p38MAPK activation is responsible for elevated mitochondrial ROS levels, thus suggesting a role for p38MAPK upstream of ROS and their damaging effects. RESULTS: Here we identified p38MAPKα as the predominantly expressed isoform in HL-1 cardiomyocytes and siRNA-mediated knockdown demonstrated the pro-oxidant role of p38MAPKα signaling. Moreover, the knockout of the p38MAPK effector MAPKAP kinase 2 (MK2) reproduced the effect of inhibiting or knocking down p38MAPK. To translate these findings into a setting closer to the clinic a stringent kidney clamping model was used. p38MAPK activity increased upon reperfusion and p38MAPK inhibition by the inhibitor BIRB796 almost completely prevented severe functional impairment caused by IR. Histological and molecular analyses showed that protection resulted from decreased redox stress and apoptotic cell death. CONCLUSIONS: These data highlight a novel and important mechanism for p38MAPK to cause IRI and suggest it as a potential therapeutic target for prevention of tissue injury.

Isolation of a novel thioflavin S-derived compound that inhibits BAG-1-mediated protein interactions and targets BRAF inhibitor-resistant cell lines.

Protein-protein interactions mediated through the C-terminal Bcl-2-associated athanogene (BAG) domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948)-a mixture of compounds resulting from the methylation and sulfonation of primulin base-has been shown to dose-dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70, or CRAF and decreases proliferation and viability. Here, we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines, but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells, Thio-2 interfered with intracellular signaling at the level of RAF, but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies.

RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins.

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.