Ischemia-Reperfusion Injury in Kidney Transplantation: Mechanisms and Potential Therapeutic Targets.

Kidney transplantation offers a longer life expectancy and a better quality of life than dialysis to patients with end-stage kidney disease. Ischemia-reperfusion injury (IRI) is thought to be a cornerstone in delayed or reduced graft function and increases the risk of rejection by triggering the immunogenicity of the organ. IRI is an unavoidable event that happens when the blood supply is temporarily reduced and then restored to an organ. IRI is the result of several biological pathways, such as transcriptional reprogramming, apoptosis and necrosis, innate and adaptive immune responses, and endothelial dysfunction. Tubular cells mostly depend on fatty acid (FA) ß-oxidation for energy production since more ATP molecules are yielded per substrate molecule than glucose oxidation. Upon ischemia-reperfusion damage, the innate and adaptive immune system activates to achieve tissue clearance and repair. Several cells, cytokines, enzymes, receptors, and ligands are known to take part in these events. The complement cascade might start even before organ procurement in deceased donors. However, additional experimental and clinical data are required to better understand the pathogenic events that take place during this complex process.

Emerging Hallmarks of Metabolic Reprogramming in Prostate Cancer.

Prostate cancer (PCa) is the most common male malignancy and the fifth leading cause of cancer death in men worldwide. Prostate cancer cells are characterized by a hybrid glycolytic/oxidative phosphorylation phenotype determined by androgen receptor signaling. An increased lipogenesis and cholesterogenesis have been described in PCa cells. Many studies have shown that enzymes involved in these pathways are overexpressed in PCa. Glutamine becomes an essential amino acid for PCa cells, and its metabolism is thought to become an attractive therapeutic target. A crosstalk between cancer and stromal cells occurs in the tumor microenvironment because of the release of different cytokines and growth factors and due to changes in the extracellular matrix. A deeper insight into the metabolic changes may be obtained by a multi-omic approach integrating genomics, transcriptomics, metabolomics, lipidomics, and radiomics data.

ß3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

We previously reported the novel finding that ß3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of ß3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the ß3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Cancer Stem Cells in Renal Cell Carcinoma: Origins and Biomarkers.

The term "cancer stem cell" (CSC) refers to a cancer cell with the following features: clonogenic ability, the expression of stem cell markers, differentiation into cells of different lineages, growth in nonadhesive spheroids, and the in vivo ability to generate serially transplantable tumors that reflect the heterogeneity of primary cancers (tumorigenicity). According to this model, CSCs may arise from normal stem cells, progenitor cells, and/or differentiated cells because of striking genetic/epigenetic mutations or from the fusion of tissue-specific stem cells with circulating bone marrow stem cells (BMSCs). CSCs use signaling pathways similar to those controlling cell fate during early embryogenesis (Notch, Wnt, Hedgehog, bone morphogenetic proteins (BMPs), fibroblast growth factors, leukemia inhibitory factor, and transforming growth factor-ß). Recent studies identified a subpopulation of CD133+/CD24+ cells from ccRCC specimens that displayed self-renewal ability and clonogenic multipotency. The development of agents targeting CSC signaling-specific pathways and not only surface proteins may ultimately become of utmost importance for patients with RCC.

Complement System and the Kidney: Its Role in Renal Diseases, Kidney Transplantation and Renal Cell Carcinoma.

The crosstalk among the complement system, immune cells, and mediators of inflammation provides an efficient mechanism to protect the organism against infections and support the repair of damaged tissues. Alterations in this complex machinery play a role in the pathogenesis of different diseases. Core complement proteins C3 and C5, their activation fragments, their receptors, and their regulators have been shown to be active intracellularly as the complosome. The kidney is particularly vulnerable to complement-induced damage, and emerging findings have revealed the role of complement system dysregulation in a wide range of kidney disorders, including glomerulopathies and ischemia-reperfusion injury during kidney transplantation. Different studies have shown that activation of the complement system is an important component of tumorigenesis and its elements have been proved to be present in the TME of various human malignancies. The role of the complement system in renal cell carcinoma (RCC) has been recently explored. Clear cell and papillary RCC upregulate most of the complement genes relative to normal kidney tissue. The aim of this narrative review is to provide novel insights into the role of complement in kidney disorders.

MUC1 Expression Affects the Immunoflogosis in Renal Cell Carcinoma Microenvironment through Complement System Activation and Immune Infiltrate Modulation.

Mucin1 (MUC1), a glycoprotein associated with an aggressive cancer phenotype and chemoresistance, is aberrantly overexpressed in a subset of clear cell renal cell carcinoma (ccRCC). Recent studies suggest that MUC1 plays a role in modulating cancer cell metabolism, but its role in regulating immunoflogosis in the tumor microenvironment remains poorly understood. In a previous study, we showed that pentraxin-3 (PTX3) can affect the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system (C1q) and releasing proangiogenic factors (C3a, C5a). In this scenario, we evaluated the PTX3 expression and analyzed the potential role of complement system activation on tumor site and immune microenvironment modulation, stratifying samples in tumors with high (MUC1H) versus tumors with low MUC1 expression (MUC1L). We found that PTX3 tissue expression was significantly higher in MUC1H ccRCC. In addition, C1q deposition and the expressions of CD59, C3aR, and C5aR were extensively present in MUC1H ccRCC tissue samples and colocalized with PTX3. Finally, MUC1 expression was associated with an increased number of infiltrating mast cells, M2-macrophage, and IDO1+ cells, and a reduced number of CD8+ T cells. Taken together, our results suggest that expression of MUC1 can modulate the immunoflogosis in the ccRCC microenvironment by activating the classical pathway of the complement system and regulating the immune infiltrate, promoting an immune-silent microenvironment.

MUC1 Tissue Expression and Its Soluble Form CA15-3 Identify a Clear Cell Renal Cell Carcinoma with Distinct Metabolic Profile and Poor Clinical Outcome.

An altered metabolism is involved in the development of clear cell renal carcinoma (ccRCC). MUC1 overexpression has been found to be associated with advanced disease and poor prognosis. In this study, we evaluated the metabolomic profile of human ccRCC, according to MUC1 expression, and integrated it with transcriptomic data. Moreover, we analyzed the role of MUC1 in sustaining ccRCC aggressiveness and the prognostic value of its soluble form CA15-3. Integrated metabolomic and transcriptomic analysis showed that MUC1-expressing ccRCC was characterized by metabolic reprogramming involving the glucose and lipid metabolism pathway. In addition, primary renal cancer cells treated with a small interfering RNA targeting MUC1 (siMUC1) migrated and proliferated at a slower rate than untreated cancer cells. After cisplatin treatment, the death rate of cancer cells treated with siMUC1 was significantly greater than that of untreated cells. Kaplan-Meier curves showed significant differences in CSS and PFS among groups of patients with high versus low levels of CA15-3. In a multivariate analysis, CA15-3 was an independent adverse prognostic factor for cancer-specific and progression-free survival. In conclusion, MUC1 expressing ccRCC is characterized by a particular metabolic reprogramming. The inhibition of MUC1 expression decreases cell motility and viability and improves cisplatin susceptibility, suggesting that this pathway can regulate de novo chemotherapy resistance in ccRCC.

Metabolomic Approaches for Detection and Identification of Biomarkers and Altered Pathways in Bladder Cancer.

Metabolomic analysis has proven to be a useful tool in biomarker discovery and the molecular classification of cancers. In order to find new biomarkers, and to better understand its pathological behavior, bladder cancer also has been studied using a metabolomics approach. In this article, we review the literature on metabolomic studies of bladder cancer, focusing on the different available samples (urine, blood, tissue samples) used to perform the studies and their relative findings. Moreover, the multi-omic approach in bladder cancer research has found novel insights into its metabolic behavior, providing excellent start-points for new diagnostic and therapeutic strategies. Metabolomics data analysis can lead to the discovery of a "signature pathway" associated with the progression of bladder cancer; this aspect could be potentially valuable in predictions of clinical outcomes and the introduction of new treatments. However, further studies are needed to give stronger evidence and to make these tools feasible for use in clinical practice.

Novel Insights into Autophagy and Prostate Cancer: A Comprehensive Review.

Autophagy is a complex process involved in several cell activities, including tissue growth, differentiation, metabolic modulation, and cancer development. In prostate cancer, autophagy has a pivotal role in the regulation of apoptosis and disease progression. Several molecular pathways are involved, including PI3K/AKT/mTOR. However, depending on the cellular context, autophagy may play either a detrimental or a protective role in prostate cancer. For this purpose, current evidence has investigated how autophagy interacts within these complex interactions. In this article, we discuss novel findings about autophagic machinery in order to better understand the therapeutic response and the chemotherapy resistance of prostate cancer. Autophagic-modulation drugs have been employed in clinical trials to regulate autophagy, aiming to improve the response to chemotherapy or to anti-cancer treatments. Furthermore, the genetic signature of autophagy has been found to have a potential means to stratify prostate cancer aggressiveness. Unfortunately, stronger evidence is needed to better understand this field, and the application of these findings in clinical practice still remains poorly feasible.

Renal Cell Carcinoma as a Metabolic Disease: An Update on Main Pathways, Potential Biomarkers, and Therapeutic Targets.

Clear cell renal cell carcinoma (ccRCC) is the most frequent histological kidney cancer subtype. Over the last decade, significant progress has been made in identifying the genetic and metabolic alterations driving ccRCC development. In particular, an integrated approach using transcriptomics, metabolomics, and lipidomics has led to a better understanding of ccRCC as a metabolic disease. The metabolic profiling of this cancer could help define and predict its behavior in terms of aggressiveness, prognosis, and therapeutic responsiveness, and would be an innovative strategy for choosing the optimal therapy for a specific patient. This review article describes the current state-of-the-art in research on ccRCC metabolic pathways and potential therapeutic applications. In addition, the clinical implication of pharmacometabolomic intervention is analyzed, which represents a new field for novel stage-related and patient-tailored strategies according to the specific susceptibility to new classes of drugs.

Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

Endothelial dysfunction is a hallmark of LPS-induced acute kidney injury (AKI). Endothelial cells (ECs) acquired a fibroblast-like phenotype and contributed to myofibroblast generation through the endothelial-to-mesenchymal transition (EndMT) process. Of note, human adult renal stem/progenitor cells (ARPCs) enhance the tubular regenerative mechanism during AKI but little is known about their effects on ECs. Following LPS exposure, ECs proliferated, decreased EC markers CD31 and vascular endothelial cadherin, and up-regulated myofibroblast markers, collagen I, and vimentin. The coculture with ARPCs normalized the EC proliferation rate and abrogated the LPS-induced EndMT. The gene expression analysis showed that most of the genes modulated in LPS-stimulated ARPCs belong to cell activation and defense response pathways. We showed that the ARPC-specific antifibrotic effect is exerted by the secretion of CXCL6, SAA4, and BPIFA2 produced after the anaphylatoxin stimulation. Next, we investigated the molecular signaling that underlies the ARPC protective mechanism and found that renal progenitors diverge from differentiated tubular cells and ECs in myeloid differentiation primary response 88-independent pathway activation. Finally, in a swine model of LPS-induced AKI, we observed that activated ARPCs secreted CXCL6, SAA4, and BPIFA2 as a defense response. These data open new perspectives on the treatment of both sepsis- and endotoxemia-induced AKI, suggesting an underestimated role of ARPCs in preventing endothelial dysfunction and novel strategies to protect the endothelial compartment and promote kidney repair.-Sallustio, F., Stasi, A., Curci, C., Divella, C., Picerno, A., Franzin, R., De Palma, G., Rutigliano, M., Lucarelli, G., Battaglia, M., Staffieri, F., Crovace, A., Pertosa, G. B., Castellano, G., Gallone, A., Gesualdo, L. Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.

CTR2 identifies a population of cancer cells with stem cell-like features in patients with clear cell renal cell carcinoma.

PURPOSE: In clear cell renal cell carcinoma tissue samples we identified and characterized a population of renal cell carcinoma derived CD133+/CD24+ cancer cells. We studied differences between these cells and their nonneoplastic counterpart, tubular adult renal progenitor cells. MATERIALS AND METHODS: CD133+/CD24+ renal cell carcinoma derived cells were isolated from 40 patients. The mesenchymal phenotype and stemness proteomic profile of these renal cell carcinoma derived cells were characterized. Colony forming efficiency and self-renewal ability were tested by limiting dilution. Tumorigenic properties were evaluated in vitro by soft agar assay. The angiogenic response was evaluated in vivo by the chorioallantoic membrane angiogenic assay. Microarray analysis was performed on 6 tubular adult renal progenitor cell and 6 renal cell carcinoma derived cell clones. Membrane protein expression was evaluated by flow cytometry and immunofluorescence staining. RESULTS: CD133+/CD24+ cells were isolated from normal and tumor kidney tissue. Fluorescence activated cell sorting revealed that renal cell carcinoma derived cells did not express mesenchymal stem cell markers. CD133+/CD24+ tumor cells were more undifferentiated than tubular adult renal progenitor cells. Renal cell carcinoma derived cells were clonigenic and could differentiate into adipocytes, epithelial and osteogenic cells. They could also regenerate tumor cells in vitro and induce angiogenesis in vivo. Gene expression profile identified CTR2 as a membrane marker for this neoplastic population. CTR2 was involved in renal cell carcinoma derived cell cisplatin resistance. CONCLUSIONS: Our results indicate the presence of a CD133+/CD24+/CTR2+ cancer cell population in clear cell renal cell carcinoma. These cells have some stem cell-like features, including in vitro self-maintenance and differentiating capabilities, and they can induce an angiogenic response in vivo.

The Role of MUC1 in Renal Cell Carcinoma.

Mucins are a family of high-molecular-weight glycoproteins. MUC1 is widely studied for its role in distinct types of cancers. In many human epithelial malignancies, MUC1 is frequently overexpressed, and its intracellular activities are crucial for cell biology. MUC1 overexpression can enhance cancer cell proliferation by modulating cell metabolism. When epithelial cells lose their tight connections, due to the loss of polarity, the mucins become dispersed on both sides of the epithelial membrane, leading to an abnormal mucin interactome with the membrane. Tumor-related MUC1 exhibits certain features, such as loss of apical localization and aberrant glycosylation that might cause the formation of tumor-related antigen epitopes. Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies and it is the most common kidney cancer. The exact role of MUC1 in this tumor is unknown. Evidence suggests that it may play a role in several oncogenic pathways, including proliferation, metabolic reprogramming, chemoresistance, and angiogenesis. The purpose of this review is to explore the role of MUC1 and the meaning of its overexpression in epithelial tumors and in particular in RCC.

Spondin-2, a secreted extracellular matrix protein, is a novel diagnostic biomarker for prostate cancer.

PURPOSE: SPON2 belongs to the F-spondin family of secreted extracellular matrix proteins. It is deregulated in some tumors, including prostate cancer. In this prospective study we assessed the role of serum SPON2 as a biomarker for prostate cancer diagnosis as well as any association between SPON2 levels and clinicopathological features. We also compared the diagnostic performance of this biomarker to that of serum sarcosine, and percent free-to-total and total prostate specific antigen. MATERIALS AND METHODS: SPON2 was measured using a sandwich enzyme linked immunosorbent assay in serum samples from 286 patients with prostate cancer and 68 with no evidence of malignancy, as confirmed by 10 to 12-core ultrasound guided prostate biopsy. Nonparametric statistical tests and ROC analysis were done to assess the diagnostic performance of SPON2 vs the other biomarkers. RESULTS: Median serum SPON2 was significantly higher in patients with prostate cancer than in those with no evidence of malignancy (77.5 vs 23.6 ng/ml, p<0.0001). ROC analysis showed a higher predictive value of SPON2 (AUC 0.952) than of serum sarcosine (AUC 0.674), percent free-to-total prostate specific antigen (AUC 0.806) and total prostate specific antigen (AUC 0.561). Moreover, patients with low grade prostate cancer had higher median SPON2 levels (p=0.001). Spearman rank correlation confirmed a negative association with Gleason score (rs=-0.29, p=0.0005). CONCLUSIONS: We found evidence that SPON2 levels were significantly higher in patients with prostate cancer than in healthy individuals. Moreover, this biomarker had better diagnostic performance than serum sarcosine, and percent free-to-total and total prostate specific antigen. This greater accuracy was also present in a subset of patients with normal prostate specific antigen.

Serum sarcosine is a risk factor for progression and survival in patients with metastatic castration-resistant prostate cancer.

AIM: Sarcosine has been identified as a differential metabolite that is greatly increased during progression from normal tissue to prostate cancer and metastatic disease. In this study we assessed the role of serum sarcosine in metastatic castration-resistant prostate cancer (mCRPC) patients. PATIENTS & METHODS: Data from 52 mCRPC patients treated with docetaxel-based chemotherapy were retrospectively analyzed. Receiver operating characteristic curves, and Kaplan-Meier and Cox multivariate analyses were performed. RESULTS: Median sarcosine values were significantly higher in mCRPC versus non-mCRPC patients (0.81 vs 0.52 nmol/µl; p < 0.0001). A significant correlation resulted between serum sarcosine levels and the duration of hormone sensitivity (Spearman's correlation coefficient: -0.51; p = 0.001). At multivariate analysis sarcosine was an independent prognostic factor of outcome in terms of overall and progression-free survival. CONCLUSION: Serum sarcosine values were significantly increased in patients with metastatic disease. Moreover, this biomarker is a risk factor for progression and survival in chemotherapy-treated mCRPC patients.

Cellular and Molecular Players in the Tumor Microenvironment of Renal Cell Carcinoma.

Globally, clear-cell renal cell carcinoma (ccRCC) represents the most prevalent type of kidney cancer. Surgery plays a key role in the treatment of this cancer, although one third of patients are diagnosed with metastatic ccRCC and about 25% of patients will develop a recurrence after nephrectomy with curative intent. Molecular-target-based agents, such as tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs), are recommended for advanced cancers. In addition to cancer cells, the tumor microenvironment (TME) includes non-malignant cell types embedded in an altered extracellular matrix (ECM). The evidence confirms that interactions among cancer cells and TME elements exist and are thought to play crucial roles in the development of cancer, making them promising therapeutic targets. In the TME, an unfavorable pH, waste product accumulation, and competition for nutrients between cancer and immune cells may be regarded as further possible mechanisms of immune escape. To enhance immunotherapies and reduce resistance, it is crucial first to understand how the immune cells work and interact with cancer and other cancer-associated cells in such a complex tumor microenvironment.

The dark side of lipid metabolism in prostate and renal carcinoma: novel insights into molecular diagnostic and biomarker discovery.

INTRODUCTION: Lipidomics focuses on the in-depth analysis of lipids, which are crucial macromolecules involved in a wide range of metabolic pathways. The increased intracellular accumulation of different classes of lipids in renal cell carcinoma (RCC) and prostate cancer (PCa) cells may be caused by elevated absorption or by increased de novo lipogenesis as a consequence of lipid metabolism reprogramming. The involvement of cholesterol metabolism in cancer's aberrant pathways has also been demonstrated. AREAS COVERED: This review provides an update on the most important lipidomics studies and applications in RCC and PCa, with a particular focus on how knowledge of aberrant lipid pathways may be used to identify biomarkers and novel therapeutic targets. In addition, the application of this methodologies have led to novel cancer subtypes identification and patient's risk stratification. Tracking tumor progression using specific biofluid metabolite profiles offers a huge translational opportunity for urological malignancies. EXPERT OPINION: Lipidomics is a promising branch of 'omics' approach and should include in next decade new standardized analysis methods and randomized clinical trials in order to reach the aim to use this high-throughput technique in patient-tailored therapy perspective.

Immune Checkpoint Inhibitors in Renal Cell Carcinoma: Molecular Basis and Rationale for Their Use in Clinical Practice.

Renal cell carcinoma (RCC) is the seventh most common cancer in men and the ninth most common cancer in women worldwide. There is plenty of evidence about the role of the immune system in surveillance against tumors. Thanks to a better understanding of immunosurveillance mechanisms, immunotherapy has been introduced as a promising cancer treatment in recent years. Renal cell carcinoma (RCC) has long been thought chemoresistant but highly immunogenic. Considering that up to 30% of the patients present metastatic disease at diagnosis, and around 20-30% of patients undergoing surgery will suffer recurrence, we need to identify novel therapeutic targets. The introduction of immune checkpoint inhibitors (ICIs) in the clinical management of RCC has revolutionized the therapeutic approach against this tumor. Several clinical trials have shown that therapy with ICIs in combination or ICIs and the tyrosine kinase inhibitor has a very good response rate. In this review article we summarize the mechanisms of immunity modulation and immune checkpoints in RCC and discuss the potential therapeutic strategies in renal cancer treatment.

Serum sarcosine increases the accuracy of prostate cancer detection in patients with total serum PSA less than 4.0 ng/ml.

BACKGROUND: Sarcosine is reported to be a differential metabolite that is greatly increased during prostate cancer (PCa) progression. In this study, we assessed the role of serum sarcosine as a biomarker for PCa, as well as any association between sarcosine levels and clinical-pathological parameters. METHODS: Sarcosine was measured by fluorometric assay in serum samples from 290 PCa patients and 312 patients with no evidence of malignancy (NEM), confirmed by 8-12 core prostate biopsies. Nonparametric statistical tests and receiver operating characteristics (ROC) analyses were performed to assess the diagnostic performance of sarcosine in different (prostate-specific antigen) PSA ranges. RESULTS: ROC analyses in subjects with PSA < 4 ng/ml showed a higher predictive value of sarcosine (AUC = 0.668) versus total PSA (AUC = 0.535) (P = 0.03), whereas for the other two PSA ranges (4-10 ng/ml and >10 ng/ml), percent ratio of free to total PSA (%fPSA) showed a predictive superiority over sarcosine. Moreover, in patients with a PSA < 4 ng/ml, the percentage of low/intermediate-grade cancers was positively associated with sarcosine levels (P = 0.005). The specificities for serum sarcosine, %fPSA, PSA, and the logistic regression model at 95% sensitivity were 24.4, 3.41, 2.22, and 28.4%, respectively. CONCLUSIONS: We provide evidence that serum sarcosine has a higher predictive value than tPSA and %fPSA in patients with PSA < 4 ng/ml. Moreover, sarcosine levels were significantly different in low grade versus high grade cancers in this subset of patients, suggesting that this marker may be a further tool not only for diagnosing PCa in normal PSA and abnormal DRE/TRUS patients but also for selecting candidates for non-aggressive therapies and active surveillance.

Cryopreserved penile tunica albuginea for allotransplantation: a morphological and ultrastructural investigation.

INTRODUCTION: Peyronie's disease, a connective tissue disorder of penile tunica albuginea (TA) associated with penile deformity, curvature, pain, and erectile dysfunction, is best managed surgically, but suitable graft biomaterials are not available. AIM: To establish whether cryopreservation affects human TA in view of its use in allotransplants. METHODS: The effects on TA samples of the two most widely used tissue cryopreservation methods were investigated using an ad hoc panel of histochemical, immunohistochemical, and ultrastructural tests. Apoptotic cells were evaluated using the terminal deoxynucleotidyl transferase method of end labeling (TUNEL) assay. MAIN OUTCOME MEASURES: Assessment of tissue integrity and arrangement of collagen and elastic fibers in thawed TA. RESULTS: Both cryofixation methods provided TA tissue suitable for use as graft material. Significant ultrastructural changes, namely, a greater diameter of collagen fibrils, were detected in sections preserved in liquid nitrogen; nonetheless, such increase never exceeded the normal range. The comprehensive panel of assays used proved suitable to characterize the thawed tissue. CONCLUSION: Human TA is suitable for cryopreservation; freezing at -80 °C provides better results than preservation in liquid nitrogen.