Molecular Profiling of Tissue Samples with Chronic Rejection from Patients with Chronic Lung Allograft Dysfunction: A Pilot Study in Cystic Fibrosis Patients.

Chronic rejection (CR) is the main culprit for reduced survival and quality of life in patients undergoing lung transplantation (Ltx). High-throughput approaches have been used to unveil the molecular pathways of CR, mainly in the blood and/or in bronchoalveolar lavage. We hypothesized that a distinct molecular signature characterizes the biopsies of recipients with clinically confirmed histological signs of CR. Eighteen cystic fibrosis patients were included in the study and RNA sequencing was performed in 35 scheduled transbronchial biopsies (TBBs): 5 with acute cellular rejection, 9 with CR, and 13 without any sign of post-LTx complication at the time of biopsy; 8 donor lung samples were used as controls. Three networks with 33, 26, and 36 differentially expressed genes (DEGs) were found in TBBs with CR. Among these, seven genes were common to the identified pathways and possibly linked to CR and five of them (LCN2, CCL11, CX3CL1, CXCL12, MUC4) were confirmed by real-time PCR. Immunohistochemistry was significant for LCN2 and MUC4. This study identified a typical gene expression pattern in TBBs with histological signs of CR and the LCN2 gene appeared to play a central role. Thus, it could be crucial in CR pathophysiology.

Post-hoc analysis of a tool to predict kidney failure in patients with IgA nephropathy.

BACKGROUND: Recently, a tool based on two different artificial neural networks has been developed. The first network predicts kidney failure (KF) development while the second predicts the time frame to reach this outcome. In this study, we conducted a post-hoc analysis to evaluate the discordant results obtained by the tool. METHODS: The tool performance was analyzed in a retrospective cohort of 1116 adult IgAN patients, as were the causes of discordance between the predicted and observed cases of KF. RESULTS: There was discordance between the predicted and observed KF in 216 IgAN patients (19.35%) all of whom were elderly, hypertensive, had high serum creatinine levels, reduced renal function and moderate or severe renal lesions. Many of these patients did not receive therapy or were non-responders to therapy. In other IgAN patients the tool predicted KF but the outcome was not reached because patients responded to therapy. Therefore, in the discordant group (prediction did not match the observed outcome) the proportion of patients having or not having KF was strongly associated with treatment (P < 0.0001). CONCLUSIONS: The post-hoc analysis shows that discordance in a low number of patients is not an error, but rather the effect of positive response to therapy. Thus, the tool could both help physicians to determine the prognosis of the disease and help patients to plan for their future.

Prediction of chronic kidney disease and its progression by artificial intelligence algorithms.

BACKGROUND AND OBJECTIVE: Aim of nephrologists is to delay the outcome and reduce the number of patients undergoing renal failure (RF) by applying prevention protocols and accurately monitoring chronic kidney disease (CKD) patients. General practitioners and nephrologists are involved in the first and in the late stages of the disease, respectively. Early diagnosis of CKD is an important step in preventing the progression of kidney damage. Our aim was to review publications on machine learning algorithms (MLAs) that can predict early CKD and its progression. METHODS: We conducted a systematic review and selected 55 articles on the application of MLAs in CKD. PubMed, Medline, Scopus, Web of Science and IEEE Xplore Digital Library of the Institute of Electrical and Electronics Engineers were searched. The search terms were chronic kidney disease, artificial intelligence, data mining and machine learning algorithms. RESULTS: MLAs use enormous numbers of predictors combining them in non-linear and highly interactive ways. This ability increases when new data is added. We observed some limitations in the publications: (i) databases were not accurately reviewed by physicians; (ii) databases did not report the ethnicity of the patients; (iii) some databases collected variables that were not important for the diagnosis and progression of CKD; (iv) no information was presented on the native kidney disease causing CKD; (v) no validation of the results in external independent cohorts was provided; and (vi) no insights were given on the MLAs that were used. Overall, there was limited collaboration among experts in electronics, computer science and physicians. CONCLUSIONS: The application of MLAs in kidney diseases may enhance the ability of clinicians to predict CKD and RF, thus improving diagnostic assistance and providing suitable therapeutic decisions. However, it is necessary to improve the development process of MLA tools.

The molecular mechanisms of inflammation and scarring in the kidneys of immunoglobulin A nephropathy : Gene involvement in the mechanisms of inflammation and scarring in kidney biopsy of IgAN patients.

Kidney biopsy is the cornerstone for the diagnosis of immunoglobulin A nephropathy (IgAN). The immunofluorescence technique evidences the IgA deposits in the glomeruli; the routine histology shows degree of active and chronic renal lesions. The spectrum of renal lesions is highly variable, ranging from minor or no detectable lesions to diffuse proliferative or crescentic lesions. Over the past three decades, renal transcriptomic studies have been performed on fresh or frozen renal tissue, and formalin-fixed paraffin-embedded kidney tissue specimens obtained from archival histological repositories. This paper aims to describe (1) the transcriptomic profiles of the kidney biopsy and (2) the potential urinary biomarkers that can be used to monitor the follow-up of IgAN patients. The use of quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), microarrays and RNA-sequencing (RNA-seq) techniques on renal tissue and separated compartments of the nephron such as glomeruli and tubule-interstitium has clarified many aspects of the renal damage in IgAN. Recently, the introduction of the single-cell RNA-seq techniques has overcome the limitations of the previous methods, making that it is possible to study the whole renal tissue without the dissection of the nephron segments; it also allows better analysis of the cell-specific gene expression involved in cell differentiation. These gene products could represent effective candidates for urinary biomarkers for clinical decision making. Finally, some of these molecules may be the targets of old drugs, such as corticosteroids, renin-angiotensin-aldosterone blockers, and new drugs such as monoclonal antibodies. In the era of personalized medicine and precision therapy, high-throughput technologies may better characterize different renal patterns of IgAN and deliver targeted treatments to individual patients.

TRIM8 Blunts the Pro-proliferative Action of ΔNp63α in a p53 Wild-Type Background.

The p53 gene family network plays a pivotal role in the control of many biological processes and therefore the right balance between the pro-apoptotic and pro-survival isoforms is key to maintain cellular homeostasis. The stability of the p53 tumor suppressor protein and that of oncogenic ΔNp63α, is crucial to control cell proliferation. The aberrant expression of p53 tumor suppressor protein and oncogenic ΔNp63α contributes to tumorigenesis and significantly affects anticancer drug response. Recently, we demonstrated that TRIM8 increases p53 stability, potentiating its tumor suppressor activity. In this paper, we show that TRIM8 simultaneously reduces the level of the pro-proliferative ΔNp63α protein, in both a proteasomal and caspase-1 dependent way, thereby playing a critical role in the cellular response to DNA damaging agents. Moreover, we provided evidence that ΔNp63α in turn, suppresses TRIM8 gene expression by preventing p53-mediated transactivation of TRIM8, therefore suggesting the existence of a negative feedback loop. These findings indicate that TRIM8 exerts its anticancer power through a joint action that provides on one hand, the activation of the p53 tumor suppressor role, and on the other the quenching of the oncogenic ΔNp63α protein activity. The enhancement of TRIM8 activity may offer therapeutic benefits and improve the management of chemoresistant tumors.

Social Dysfunction in Older Age and Relationships with Cognition, Depression, and Apathy: The GreatAGE Study.

BACKGROUND: Most studies focused on only one measure of social dysfunction in older age, without proper validation and distinction across different dimensions including subjectivity, structural, and functional aspects. OBJECTIVE: We sought to validate the Social Dysfunction Rating Scale (SDRS) and its factorial structure, also determining the association of SDRS with cognitive functions, global psychopathology, and social deprivation. METHODS: The SDRS was administered to 484 Italian community-dwelling elderly, recruited in the GreatAGE study, a population-based study on aging conducted in Castellana Grotte, Bari, Southern Italy. We determined objective and subjective psychometric properties of SDRS against the gold standard evaluation of social dysfunction according to the Semi-structured Clinical Diagnostic Interview for DSM-IV-TR Axis I Disorders (SCID-I) criterion. RESULTS: The SDRS showed a moderate accuracy with an optimal cut-off of 26 maximized with higher sensitivity (0.74,95% CI:0.63-0.84) than specificity (0.57,95% CI:0.50-0.64). A five-factor structure was carried out and five dimensions of SDRS were identified (loneliness; social isolation; feeling of contribution/uselessness; lack of leisure activities; anxiety for the health). Education and global cognitive functions were inversely correlated to SDRS, while a direct association with global psychopathology, depression, and apathy was found. The prevalence of higher SDRS scores was major in subjects with current psychiatric disorders versus other subjects.∥Conclusion: The SDRS could be a valid instrument to capture both size and quality of social dysfunction, both in subjects with psychiatric disorders and in normal subjects. Several categories of social dysfunction differed only in the degree of health deprivation, not in social or material deprivation.

Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway-Effect of SH reagents.

Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours.

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase.

Valinomycin induced energy-dependent mitochondrial swelling, cytochrome c release, cytosolic NADH/cytochrome c oxidation and apoptosis.

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that "respiratory contact sites" are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program.

Ceramide-induced activation of cytosolic NADH/cytochrome c electron transport pathway: An additional source of energy for apoptosis.

We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.