Magnetic resonance imaging as a noninvasive adjunct to conventional assessment of functional differences between kidneys in vivo and during ex vivo normothermic machine perfusion.

Normothermic machine perfusion (NMP) is increasingly considered for pretransplant kidney quality assessment. However, fundamental questions about differences between in vivo and ex vivo renal function, as well as the impact of ischemic injury on ex vivo physiology, remain unanswered. This study utilized magnetic resonance imaging (MRI), alongside conventional parameters to explore differences between in vivo and ex vivo renal function and the impact of warm ischemia on a kidney's behavior ex vivo. Renal MRI scans and samples were obtained from living pigs (n = 30) in vivo. Next, kidney pairs were procured and exposed to minimal, or 75 minutes of warm ischemia, followed by 6 hours of hypothermic machine perfusion. Both kidneys simultaneously underwent 6-hour ex vivo perfusion in MRI-compatible NMP circuits to obtain multiparametric MRI data. Ischemically injured ex vivo kidneys showed a significantly altered regional blood flow distribution compared to in vivo and minimally damaged organs. Both ex vivo groups showed diffusion restriction relative to in vivo. Our findings underscore the differences between in vivo and ex vivo MRI-based renal characteristics. Therefore, when assessing organ viability during NMP, it should be considered to incorporate parameters beyond the conventional functional markers that are common in vivo.

Thulium fiber laser vs. holmium laser enucleation of the prostate: results of a prospective randomized non-inferiority trial.

PURPOSE: Holmium laser enucleation of the prostate (HoLEP) represents the current standard procedure for size-independent surgical therapy of benign prostatic obstruction (BPO). With advent of the novel laser technology thulium fiber laser (TFL), we hypothesized that the functional outcome of TFL enucleation of the prostate (ThuFLEP) is non-inferior compared to HoLEP. METHODS: From October 2021 to October 2022, 150 patients with BPO were recruited for the prospective randomized trial in accordance with CONSORT. Stratified randomization into the arms ThuFLEP (n = 74) or HoLEP (n = 76) was carried out. The primary endpoint was non-inferior international prostate symptom score (IPSS) and quality of life (QoL) at three months after treatment. Secondary endpoints were rates of complications, peak flow, residual urine and operation times. RESULTS: Preoperative characteristics showed no significant differences. Overall IPSS and QoL improved from 21 to 8 and 4 to 1.5, respectively, after three months of follow-up. No statistically significant differences between ThuFLEP and HoLEP were observed regarding median postoperative IPSS (8.5 vs. 7, p > 0.9), QoL (1 vs. 2, p = 0.6), residual urine (48 vs. 30ml, p = 0.065) and peak flow (19 vs. 17ml/s, p > 0.9). Similarly, safety profile was comparable with no statistically significant differences regarding rate of major complications (5.3 vs. 5.4%, p = 0.5), laser hemostasis time (3 vs. 2min, p = 0.2), use of additive electric coagulation (74 vs. 87%, p = 0.06) or electric coagulation time (8 vs. 8min, p = 0.4). CONCLUSIONS: In this prospective, randomized trial ThuFLEP showed non-inferior results compared to HoLEP in terms of functional outcomes measured by IPSS and QoL as primary endpoint. TRIAL REGISTRATION NUMBER: DRKS00032699 (18.09.2023, retrospectively registered).

Ex-Vivo Kidney Perfusion With Hemoglobin-Based Oxygen Carriers, Red Blood Cells, or No Oxygen Carrier.

INTRODUCTION: Normothermic machine perfusion (NMP) of donor kidneys provides the opportunity to assess and improve organ viability prior to transplantation. This study explored the necessity of an oxygen carrier during NMP and whether the hemoglobin-based oxygen carrier (HBOC-201) is a suitable alternative to red blood cells (RBCs). METHODS: Porcine kidneys were perfused with a perfusion solution containing either no-oxygen carrier, RBCs, or HBOC-201 for 360 min at 37°C. RESULTS: Renal flow and resistance did not differ significantly between groups. NMP without an oxygen carrier showed lower oxygen consumption with higher lactate and aspartate aminotransferase levels, indicating that the use of an oxygen carrier is necessary for NMP. Cumulative urine production and creatinine clearance in the RBC group were significantly higher than in the HBOC-201 group. Oxygen consumption, injury markers, and histology did not differ significantly between these two groups. However, methemoglobin levels increased to 45% after 360 min in the HBOC-201 group. CONCLUSIONS: We conclude that HBOC-201 could be used as an alternative for RBCs, but accumulating methemoglobin levels during our perfusions indicated that HBOC-201 is probably less suitable for prolonged NMP. Perfusion with RBCs, compared to HBOC-201, resulted in more favorable renal function during NMP.

KIDNEY-PAGER: analysis of circulating tumor DNA as a biomarker in renal cancer - an observational trial. Study protocol.

BACKGROUND: Management of localized renal cell carcinoma (RCC) is challenged by inaccurate methods to assess the risk of recurrence and deferred detection of relapse and residual disease after radical or partial nephrectomy. Circulating tumor DNA (ctDNA) has been proposed as a potential biomarker in RCC. PURPOSE: Conduction of an observational study to evaluate the validity of ctDNA as a biomarker of the risk of recurrence and subclinical residual disease to improve postoperative surveillance. MATERIAL AND METHODS: Urine and blood will be prospectively collected before and after surgery of the primary tumor from up to 500 patients until 5 years of follow-up. ctDNA analysis will be performed using shallow whole genome sequencing and cell-free methylated DNA immunoprecipitation sequencing. ctDNA levels in plasma and urine will be correlated to oncological outcomes. Residual blood and urine as well as tissue biopsies will be biobanked for future research. INTERPRETATION: Results will pave the way for future ctDNA-guided clinical trials aiming to improve RCC management.

Corticomedullary shunting after ischaemia and reperfusion in the porcine kidney?

BACKGROUND: Renal perfusion may redistribute from cortex to medulla during systemic hypovolaemia and after renal ischaemia for other reasons, but there is no consensus on this matter. We studied renal perfusion after renal ischaemia and reperfusion. METHODS: Renal perfusion distribution was examined by use of 153Gadolinium-labeled microspheres (MS) after 2 h (hrs) and 4 h ischaemia of the pig kidney followed by 4 h of reperfusion. Intra-arterial injected MS are trapped in the glomeruli in renal cortex, which means that MS are not present in the medulla under normal physiological conditions. RESULTS: Visual evaluation after reperfusion demonstrated that MS redistributed from the renal cortex to the medulla in 6 out of 16 pigs (38%) subjected to 4 h ischaemia and in one out of 18 pigs subjected to 2 h ischaemia. Central renal uptake of MS covering the medullary/total renal uptake was significantly higher in kidneys subjected to 4 h ischaemia compared with pigs subjected to 2 h ischaemia (69 ± 5% vs. 63 ± 1%, p < 0.001), and also significantly higher than in the contralateral kidney (69 ± 5% vs. 63 ± 2%, p < 0.001). Analysis of blood and urine demonstrated no presence of radioactivity. CONCLUSION: The study demonstrated the presence of MS in the renal medulla in response to renal ischaemia and reperfusion suggesting that severe ischaemia and reperfusion of the pig kidney leads to opening of functional shunts bypassing glomeruli.

Dosing Limitation for Intra-Renal Arterial Infusion of Mesenchymal Stromal Cells.

The immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) make MSC therapy a promising therapeutic strategy in kidney disease. A targeted MSC administration via the renal artery offers an efficient delivery method with limited spillover to other organs. Although local administration alleviates safety issues with MSCs in systemic circulation, it introduces new safety concerns in the kidneys. In a porcine model, we employed intra-renal arterial infusion of ten million allogenic adipose tissue-derived MSCs. In order to trigger any potential adverse events, a higher dose (hundred million MSCs) was also included. The kidney function was studied by magnetic resonance imaging after the MSC infusion and again at two weeks post-treatment. The kidneys were assessed by single kidney glomerular filtration rate (skGFR) measurements, histology and inflammation, and fibrosis-related gene expression. None of the measured parameters were affected immediately after the administration of ten million MSCs, but the administration of one hundred million MSCs induced severe adverse events. Renal perfusion was reduced immediately after MSC administration which coincided with the presence of microthrombi in the glomeruli and signs of an instant blood-mediated inflammatory reaction. At two weeks post-treatment, the kidneys that were treated with one hundred million MSCs showed reduced skGFR, signs of tissue inflammation, and glomerular and tubular damage. In conclusions, the intra-renal administration of ten million MSCs is well-tolerated by the porcine kidney. However, higher concentrations (one hundred million MSCs) caused severe kidney damage, implying that very high doses of intra-renally administered MSCs should be undertaken with caution.

Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study.

Normothermic machine perfusion (NMP) of injured kidneys offers the opportunity for interventions to metabolically active organs prior to transplantation. Mesenchymal stromal cells (MSCs) can exert regenerative and anti-inflammatory effects in ischemia-reperfusion injury. The aims of this study were to evaluate the safety and feasibility of MSC treatment of kidneys during NMP using a porcine autotransplantation model, and examine potential MSC treatment-associated kidney improvements up to 14 days posttransplant. After 75 min of kidney warm ischemia, four experimental groups of n = 7 underwent 14 h of oxygenated hypothermic machine perfusion. In three groups this was followed by 240 min of NMP with infusion of vehicle, 10 million porcine, or 10 million human adipose-derived MSCs. All kidneys were autotransplanted after contralateral nephrectomy. MSC treatment did not affect perfusion hemodynamics during NMP or cause adverse effects at reperfusion, with 100% animal survival. MSCs did not affect plasma creatinine, glomerular filtration rate, neutrophil gelatinase-associated lipocalin concentrations or kidney damage assessed by histology during the 14 days, and MSCs retention was demonstrated in renal cortex. Infusing MSCs during ex vivo NMP of porcine kidneys was safe and feasible. Within the short posttransplant follow-up period, no beneficial effects of ex vivo MSC therapy could be demonstrated.

Goal-Directed Fluid Therapy Does Not Improve Early Glomerular Filtration Rate in a Porcine Renal Transplantation Model.

BACKGROUND: Insufficient fluid administration intra- and postoperatively may lead to delayed renal graft function (DGF), while fluid overload increases the risk of heart failure, infection, and obstipation. Several different fluid protocols have been suggested to ensure optimal fluid state. However, there is a lack of evidence of the clinical impact of these regimens. This study aimed to determine whether individualized goal-directed fluid therapy (IGDT) positively affects the initial renal function compared to a high-volume fluid therapy (HVFT) and to examine the effects on renal endothelial glycocalyx, inflammatory and oxidative stress markers, and medullary tissue oxygenation. The hypothesis was that IGDT improves early glomerular filtration rate (GFR) in pigs subjected to renal transplantation. METHODS: This was an experimental randomized study. Using a porcine renal transplantation model, animals were randomly assigned to receive IGDT or HVFT during and until 1 hour after transplantation from brain-dead donors. The kidneys were exposed to 18 hours of cold ischemia. The recipients were observed until 10 hours after reperfusion, which included GFR measured as clearance of chrom-51-ethylendiamintetraacetat (Cr-EDTA), animal weight, and renal tissue oxygenation by fiber optic probes. The renal expression of inflammatory and oxidative stress markers as well as glomerular endothelial glycocalyx were analyzed in the graft using polymerase chain reaction (PCR) technique and immunofluorescence. RESULTS: Twenty-eight recipient pigs were included for analysis. We found no evidence that IGDT improved early GFR compared to HVFT (P = .45), while animal weight increased more in the HVFT group (a mean difference of 3.4 kg [1.96-4.90]; P < .0001). A better, however nonsignificant, preservation of glomerular glycocalyx (P = .098) and significantly lower levels of the inflammatory marker cyclooxygenase 2 (COX-2) was observed in the IGDT group when compared to HVFT. COX-2 was 1.94 (1.50-2.39; P = .012) times greater in the HVFT group when compared to the IGDT group. No differences were observed in outer medullary tissue oxygenation or oxidative stress markers. CONCLUSIONS: IGDT did not improve early GFR; however, it may reduce tissue inflammation and could possibly lead to preservation of the glycocalyx compared to HVFT.

Expression of costimulatory ligand CD70 on steady-state dendritic cells breaks CD8+ T cell tolerance and permits effective immunity.

Steady-state dendritic cells (DCs) maintain peripheral T cell tolerance, whereas mature DCs generate immunity. CD70 is a costimulatory ligand acquired upon DC maturation. To determine its impact on T cell fate, we have generated mice that constitutively express CD70 in conventional DCs (cDCs). In these mice, naive CD4+ and CD8+ T cells spontaneously convert into effector cells. Administration of peptide without adjuvant, which is ordinarily tolerogenic, elicited tumor-eradicating CD8+ T cell responses and robust CD4+ T cell-independent memory. CD70 was also constitutively expressed in cDCs that inducibly present viral epitopes. In this case, tolerance induction was prevented as well. The antigen-presenting DCs generated protective immunity to virus infection and broke a pre-existing state of CD8+ T cell tolerance. Thus, the sole expression of CD70 by otherwise immature cDCs sufficed to convert CD8+ T cell tolerance into immunity, defining the importance of CD27-CD70 interactions at the interface between T cell and DC.

Identification of a dendritic cell receptor that couples sensing of necrosis to immunity.

Injury or impaired clearance of apoptotic cells leads to the pathological accumulation of necrotic corpses, which induce an inflammatory response that initiates tissue repair. In addition, antigens present in necrotic cells can sometimes provoke a specific immune response and it has been argued that necrosis could explain adaptive immunity in seemingly infection-free situations, such as after allograft transplantation or in spontaneous and therapy-induced tumour rejection. In the mouse, the CD8alpha+ subset of dendritic cells phagocytoses dead cell remnants and cross-primes CD8+ T cells against cell-associated antigens. Here we show that CD8alpha+ dendritic cells use CLEC9A (also known as DNGR-1), a recently-characterized C-type lectin, to recognize a preformed signal that is exposed on necrotic cells. Loss or blockade of CLEC9A does not impair the uptake of necrotic cell material by CD8+ dendritic cells, but specifically reduces cross-presentation of dead-cell-associated antigens in vitro and decreases the immunogenicity of necrotic cells in vivo. The function of CLEC9A requires a key tyrosine residue in its intracellular tail that allows the recruitment and activation of the tyrosine kinase SYK, which is also essential for cross-presentation of dead-cell-associated antigens. Thus, CLEC9A functions as a SYK-coupled C-type lectin receptor to mediate sensing of necrosis by the principal dendritic-cell subset involved in regulating cross-priming to cell-associated antigens.

Metabolic MRI With Hyperpolarized 13 C-Pyruvate for Early Detection of Fibrogenic Kidney Metabolism.

OBJECTIVES: Fibrosis is the final common pathway for chronic kidney disease and the best predictor for disease progression. Besides invasive biopsies, biomarkers for its detection are lacking. To address this, we used hyperpolarized 13 C-pyruvate MRI to detect the metabolic changes associated with fibrogenic activity of myofibroblasts. MATERIALS AND METHODS: Hyperpolarized 13 C-pyruvate MRI was performed in 2 pig models of kidney fibrosis (unilateral ureteral obstruction and ischemia-reperfusion injury). The imaging data were correlated with histology, biochemical, and genetic measures of metabolism and fibrosis. The porcine experiments were supplemented with cell-line experiments to inform the origins of metabolic changes in fibrogenesis. Lastly, healthy and fibrotic human kidneys were analyzed for the metabolic alterations accessible with hyperpolarized 13 C-pyruvate MRI. RESULTS: In the 2 large animal models of kidney fibrosis, metabolic imaging revealed alterations in amino acid metabolism and glycolysis. Conversion from hyperpolarized 13 C-pyruvate to 13 C-alanine decreased, whereas conversion to 13 C-lactate increased. These changes were shown to reflect profibrotic activity in cultured epithelial cells, macrophages, and fibroblasts, which are important precursors of myofibroblasts. Importantly, metabolic MRI using hyperpolarized 13 C-pyruvate was able to detect these changes earlier than fibrosis-sensitive structural imaging. Lastly, we found that the same metabolic profile is present in fibrotic tissue from human kidneys. This affirms the translational potential of metabolic MRI as an early indicator of fibrogenesis associated metabolism. CONCLUSIONS: Our findings demonstrate the promise of hyperpolarized 13 C-pyruvate MRI for noninvasive detection of fibrosis development, which could enable earlier diagnosis and intervention for patients at risk of kidney fibrosis.

Multimodal imaging in rats reveals impaired neurovascular coupling in sustained hypertension.

BACKGROUND AND PURPOSE: Arterial hypertension is an important risk factor for cerebrovascular diseases, such as transient ischemic attacks or stroke, and represents a major global health issue. The effects of hypertension on cerebral blood flow, particularly at the microvascular level, remain unknown. METHODS: Using the spontaneously hypertensive rat (SHR) model, we examined cortical hemodynamic responses on whisker stimulation applying a multimodal imaging approach (multiwavelength spectroscopy, laser speckle imaging, and 2-photon microscopy). We assessed the effects of hypertension in 10-, 20-, and 40-week-old male SHRs and age-matched male Wistar Kyoto rats (CTRL) on hemodynamic responses, histology, and biochemical parameters. In 40-week-old animals, losartan or verapamil was administered for 10 weeks to test the reversibility of hypertension-induced impairments. RESULTS: Increased arterial blood pressure was associated with a progressive impairment in functional hyperemia in 20- and 40-week-old SHRs; baseline capillary red blood cell velocity was increased in 40-week-old SHRs compared with age-matched CTRLs. Antihypertensive treatment reduced baseline capillary cerebral blood flow almost to CTRL values, whereas functional hyperemic signals did not improve after 10 weeks of drug therapy. Structural analyses of the microvascular network revealed no differences between normo- and hypertensive animals, whereas expression analyses of cerebral lysates showed signs of increased oxidative stress and signs of impaired endothelial homeostasis upon early hypertension. CONCLUSIONS: Impaired neurovascular coupling in the SHR evolves upon sustained hypertension. Antihypertensive monotherapy using verapamil or losartan is not sufficient to abolish this functional impairment. These deficits in neurovascular coupling in response to sustained hypertension might contribute to accelerate progression of neurodegenerative diseases in chronic hypertension.

Pulmonary embolisms and infections after renal trauma.

INTRODUCTION: The objective of this study was to describe and evaluate the management of patients with renal trauma and their complications at the Department of Urology at Aarhus University Hospital (AUH), Denmark. METHODS: All patients diagnosed with renal injury due to trauma and with contact to the Department of Urology at the AUH, Denmark, between March 2016 and March 2021 were included. Patients were identified by the International Classification of Diseases, Tenth version, code and data obtained from electronic patient records. RESULTS: A total of 58 patients were identified. The median age was 33 years (7-95 years) and the median length of hospitalisation was five days (range: 0-52 days). All patients were evaluated with a multiphase computed tomography upon admission. Injuries to the kidney were graded using the American Association for the Surgery of Trauma kidney injury scale. Twelve percent had grade I injury, 26% had grade II injury, 26% had grade III injury, 36% had grade IV injury and 3% had grade V injury. In the acute phase, all patients were managed non-operatively. Early complications were found in 24% of patients. Pulmonary embolism was diagnosed in 7%. Furthermore, 7% had an infection as a late complication and all of these patients had also had an early infection. A total of 60% were followed up with a renal-scintigraphy three months after their renal trauma. This examination had no consequence for any of the patients. CONCLUSIONS: No patients died due to the renal trauma. However, many experienced complications in terms of infections and pulmonary embolisms. These data support earlier findings and suggest that a renal scintigraphy after renal traumas may be obsolete. FUNDING: None. TRIAL REGISTRATION: Not relevant.

Generation and characterization of three induced pluripotent stem cells lines from an 86-year old female individual diagnosed with an invasive lobular mammary carcinoma.

Invasive lobular carcinoma (ILC) is a distinct type of breast cancer and is accounting up to 10-15 % of all mammary carcinomas showing a pronounced increase in incidence rates over the last two decades. We generated three induced pluripotent stem cell (iPSC) lines from CD34+ progenitor cells isolated from a mammary carcinoma patient diagnosed with ILC. Here, we describe the characterization of the iPSCs by array-based comparative genomic hybridization (array CGH), immunocytochemistry, flow cytometry, reverse transcriptase polymerase chain reaction and directed in vitro differentiation. The iPSC lines will find application in the field of breast cancer research.

Multiplex immunofluorescence staining of coverslip-mounted paraffin-embedded tissue sections.

Animal and human tissues are used extensively in physiological and pathophysiological research. Due to both ethical considerations and low availability, it is essential to maximize the use of these tissues. Therefore, the aim was to develop a new method allowing for multiplex immunofluorescence (IF) staining of kidney sections in order to reuse the same tissue section multiple times. The paraffin-embedded kidney sections were placed onto coated coverslips and multiplex IF staining was performed. Five rounds of staining were performed where each round consisted of indirect antibody labelling, imaging on a widefield epifluorescence microscope, removal of the antibodies using a stripping buffer, and then re-staining. In the final round, the tissue was stained with hematoxylin/eosin. Using this method, tubular segments in the nephron, blood vessels, and interstitial cells were labeled. Furthermore, by placing the tissue on coverslips, confocal-like resolution was obtained using a conventional widefield epifluorescence microscope and a 60x oil objective. Thus, using standard reagents and equipment, paraffin-embedded tissue was used for multiplex IF staining with increased Z-resolution. In summary, this method offers time-saving multiplex IF staining and allows for the retrieval of both quantitative and spatial expressional information of multiple proteins and subsequently for an assessment of the tissue morphology. Due to the simplicity and integrated effectivity of this multiplex IF protocol, it holds the potential to supplement standard IF staining protocols and maximize use of tissue.

Regulation of synaptic connectivity in schizophrenia spectrum by mutual neuron-microglia interaction.

The examination of post-mortem brain tissue suggests synaptic loss as a central pathological hallmark of schizophrenia spectrum (SCZ), which is potentially related to activated microglia and increased inflammation. Induced pluripotent stem cells serve as a source for neurons and microglia-like cells to address neuron-microglia interactions. Here, we present a co-culture model of neurons and microglia, both of human origin, to show increased susceptibility of neurons to microglia-like cells derived from SCZ patients. Analysis of IBA-1 expression, NFκB signaling, transcription of inflammasome-related genes, and caspase-1 activation shows that enhanced, intrinsic inflammasome activation in patient-derived microglia exacerbates neuronal deficits such as synaptic loss in SCZ. Anti-inflammatory pretreatment of microglia with minocycline specifically rescued aberrant synapse loss in SCZ and reduced microglial activation. These findings open up possibilities for further research in larger cohorts, focused clinical work and longitudinal studies that could facilitate earlier therapeutic intervention.

Breast cancer patient-derived microtumors resemble tumor heterogeneity and enable protein-based stratification and functional validation of individualized drug treatment.

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

H2S-Enriched Flush out Does Not Increase Donor Organ Quality in a Porcine Kidney Perfusion Model.

Kidney extraction time has a detrimental effect on post-transplantation outcome. This study aims to improve the flush-out and potentially decrease ischemic injury by the addition of hydrogen sulphide (H2S) to the flush medium. Porcine kidneys (n = 22) were extracted during organ recovery surgery. Pigs underwent brain death induction or a Sham operation, resulting in four groups: donation after brain death (DBD) control, DBD H2S, non-DBD control, and non-DBD H2S. Directly after the abdominal flush, kidneys were extracted and flushed with or without H2S and stored for 13 h via static cold storage (SCS) +/- H2S before reperfusion on normothermic machine perfusion. Pro-inflammatory cytokines IL-1b and IL-8 were significantly lower in H2S treated DBD kidneys during NMP (p = 0.03). The non-DBD kidneys show superiority in renal function (creatinine clearance and FENa) compared to the DBD control group (p = 0.03 and p = 0.004). No differences were seen in perfusion parameters, injury markers and histological appearance. We found an overall trend of better renal function in the non-DBD kidneys compared to the DBD kidneys. The addition of H2S during the flush out and SCS resulted in a reduction in pro-inflammatory cytokines without affecting renal function or injury markers.

Vascularization of cytochrome oxidase-rich blobs in the primary visual cortex of squirrel and macaque monkeys.

The close correlation between energy supply by blood vessels and energy consumption by cellular processes in the brain is the basis of blood flow-related functional imaging techniques. Regional differences in vascular density can be detected using high-resolution functional magnetic resonance imaging. Therefore, inhomogeneities in vascularization might help to identify anatomically distinct areas noninvasively in vivo. It was reported previously that cytochrome oxidase-rich blobs in the striate cortex of squirrel monkeys are characterized by a notably higher vascular density (42% higher than interblob regions). However, blobs have so far never been identified in vivo on the basis of their vascular density. Here, we analyzed blobs of the primary visual cortex of squirrel monkeys and macaques with respect to the relationship between vascularization and cytochrome oxidase activity. By double staining with cytochrome oxidase enzyme histochemistry to define the blobs and collagen type IV immunohistochemistry to quantify the blood vessels, a close correlation between oxidative metabolism and vascularization was confirmed and quantified in detail. The vascular length density in cytochrome oxidase blobs was on average 4.5% higher than in the interblob regions, a difference almost one order of magnitude smaller than previously reported. Thus, the vascular density that is closely associated with local average metabolic activity is a structural equivalent of cerebral metabolism and blood flow. However, the quantitative differences in vascularization between blob and interblob regions are small and below the detectability threshold of the noninvasive hemodynamic imaging methods of today.

The invariant chain transports TNF family member CD70 to MHC class II compartments in dendritic cells.

CD70 is a TNF-related transmembrane molecule expressed by mature dendritic cells (DCs), which present antigens to T cells via major histocompatibility complex (MHC) molecules. In DCs, CD70 localizes with MHC class II molecules in late endosomal vesicles, known as MHC class II compartments (MIICs). MIICs are transported to the immune synapse when a DC contacts an antigen-specific CD4(+) T cell. Consequently, MHC class II and CD70 are simultaneously exposed to the T cell. Thereby, T-cell activation via the antigen receptor and CD70-mediated co-stimulation are synchronized, apparently to optimize the proliferative response. We report here that the invariant chain (Ii), a chaperone known to transport MHC class II to MIICs, performs a similar function for CD70. CD70 was found to travel by default to the plasma membrane, whereas Ii coexpression directed it to late endosomes and/or lysosomes. In cells containing the MHC class II presentation pathway, CD70 localized to MIICs. This localization relied on Ii, since transport of CD70 from the Golgi to MIICs was impeded in Ii-deficient DCs. Biophysical and biochemical studies revealed that CD70 and Ii participate in an MHC-class-II-independent complex. Thus, Ii supports transport of both MHC class II and CD70 to MIICs and thereby coordinates their delivery to CD4(+) T cells.