Gut microbiota and fecal volatilome profile inspection in metabolically healthy and unhealthy obesity phenotypes.

BACKGROUND: People with metabolically healthy (MHO) and metabolically unhealthy obesity (MUO) differ for the presence or absence of cardio-metabolic complications, respectively. OBJECTIVE: Based on these differences, we are interested in deepening whether these obesity phenotypes could be linked to changes in microbiota and metabolome profiles. In this respect, the overt role of microbiota taxa composition and relative metabolic profiles is not completely understood. At this aim, biochemical and nutritional parameters, fecal microbiota, metabolome and SCFA compositions were inspected in patients with MHO and MUO under a restrictive diet regimen with a daily intake ranging from 800 to 1200 kcal. METHODS: Blood, fecal samples and food questionnaires were collected from healthy controls (HC), and an obese cohort composed of both MHO and MUO patients. Most impacting biochemical/anthropometric variables from an a priori sample stratification were detected by applying a robust statistics approach useful in lowering the background noise. Bacterial taxa and volatile metabolites were assessed by qPCR and gas chromatography coupled with mass spectrometry, respectively. A targeted GC-MS analyses on SCFAs was also performed. RESULTS: Instructed to follow a controlled and restricted daily calorie intake, MHO and MUO patients showed differences in metabolic, gut microbial and volatilome signatures. Our data revealed higher quantities of specific pro-inflammatory taxa (i.e., Desulfovibrio and Prevotella genera) and lower quantities of Clostridium coccoides group in MUO subset. Higher abundances in alkane, ketone, aldehyde, and indole VOC classes together with a lower amount of butanoic acid marked the faecal MUO metabolome. CONCLUSIONS: Compared to MHO, MUO subset symptom picture is featured by specific differences in gut pro-inflammatory taxa and metabolites that could have a role in the progression to metabolically unhealthy status and developing of obesity-related cardiometabolic diseases. The approach is suitable to better explain the crosstalk existing among dysmetabolism-related inflammation, nutrient intake, lifestyle, and gut dysbiosis.

Quasi-3D morphology and modulation of focal adhesions of human adult stem cells through combinatorial concave elastomeric surfaces with varied stiffness.

In vivo cell niches are complex architectures that provide a wide range of biochemical and mechanical stimuli to control cell behavior and fate. With the aim to provide in vitro microenvironments mimicking physiological niches, microstructured substrates have been exploited to support cell adhesion and to control cell shape as well as three dimensional morphology. At variance with previous methods, we propose a simple and rapid protein subtractive soft lithographic method to obtain microstructured polydimethylsiloxane substrates for studying stem cell adhesion and growth. The shape of adult renal stem cells and nuclei is found to depend predominantly on micropatterning of elastomeric surfaces and only weakly on the substrate mechanical properties. Differently, focal adhesions in their shape and density but not in their alignment mainly depend on the elastomer stiffness almost regardless of microscale topography. Local surface topography with concave microgeometry enhancing adhesion drives stem cells in a quasi-three dimensional configuration where stiffness might significantly steer mechanosensing as highlighted by focal adhesion properties.

Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

Klotho is an anti-aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down-regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1-inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF-kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down-regulation of Klotho in TEC in vitro that was NF-kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre-implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down-regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down-regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti-senescence and anti-fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF-associated chronic allograft dysfunction.

Acquired lecithin:cholesterol acyltransferase deficiency as a major factor in lowering plasma HDL levels in chronic kidney disease.

OBJECTIVES: It has been suggested that a low plasma high-density lipoprotein cholesterol (HDL-C) level contributes to the high cardiovascular disease risk of patients with chronic kidney disease (CKD), especially those undergoing haemodialysis (HD). The present study was conducted to gain further understanding of the mechanism(s) responsible for the low HDL-C levels in patients with CKD and to separate the impact of HD from that of the underlying CKD. METHODS: Plasma lipids and lipoproteins, HDL subclasses and various cholesterol esterification parameters were measured in a total of 248 patients with CKD, 198 of whom were undergoing HD treatment and 40 healthy subjects. RESULTS: Chronic kidney disease was found to be associated with highly significant reductions in plasma HDL-C, unesterified cholesterol, apolipoprotein (apo)A-I, apoA-II and LpA-I:A-II levels in both CKD cohorts (with and without HD treatment). The cholesterol esterification process was markedly impaired, as indicated by reductions in plasma lecithin:cholesterol acyltransferase (LCAT) concentration and activity and cholesterol esterification rate, and by an increase in the plasma preß-HDL content. HD treatment was associated with a further lowering of HDL levels and impaired plasma cholesterol esterification. The plasma HDL-C level was highly significantly correlated with LCAT concentration (R = 0.438, P < 0.001), LCAT activity (R = 0.243, P < 0.001) and cholesterol esterification rate (R = 0.149, P = 0.031). Highly significant correlations were also found between plasma LCAT concentration and levels of apoA-I (R = 0.432, P < 0.001), apoA-II (R = 0.275, P < 0.001), LpA-I (R = 0.326, P < 0.001) and LpA-I:A-II (R = 0.346, P < 0.001). CONCLUSION: Acquired LCAT deficiency is a major cause of low plasma HDL levels in patients with CKD, thus LCAT is an attractive target for therapeutic intervention to reverse dyslipidaemia, and possibly lower the cardiovascular disease risk in these patients.

Rapamycin-induced hypophosphatemia and insulin resistance are associated with mTORC2 activation and Klotho expression.

Rapamycin, an immunosuppressive drug used to prevent rejection after kidney transplantation, influences phosphate homeostasis, induces insulin resistance and has been shown to prolong lifespan in animal models. Because Klotho is an aging-suppressor gene controlling phosphate metabolism and insulin sensitivity, we investigated the influence of rapamycin on Klotho expression. A total of 100 kidney transplant recipients, 50 chronically treated with rapamycin and 50 with calcineurin inhibitors, were enrolled; 20 healthy subjects were employed as control. In the rapamycin group, serum phosphate was lower than in the CNI group with an increase in phosphate excretion and a reduction in its reabsorption. In addition, rapamycin increased insulin resistance as shown by HOMA index. Rapamycin treatment of an immortalized proximal tubular cell line induced the expression of Klotho, the phosphorylation of AKT in Ser473, downstream target of mTORC2 and the expression of RICTOR, mTORC2 main component. AKT inhibition reduced the rapamycin-induced expression of Klotho. In vivo rapamycin treatment induced higher degree of RICTOR and AKT Ser(473) expression directly correlating with long-term rapamycin exposure, FE(PO4) and HOMA index. In conclusion, our data would suggest that rapamycin may influence phosphate homeostasis and insulin resistance modulating Klotho expression through mTORC2 activation.

IL-17 expression by tubular epithelial cells in renal transplant recipients with acute antibody-mediated rejection.

Acute rejection is still a common complication of kidney transplantation. IL-17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL-17 graft expression in renal transplant recipients with acute antibody-mediated rejection (ABMR), acute T-cell-mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin-inhibitor toxicity (CNI). In acute ABMR, tubular IL-17 protein expression was significantly increased compared to TCMR, where most of the IL-17⁺ cells were CD4⁺ graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL-17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL-17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL-17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a-induced IL-17 expression. The use of steroids and monoclonal antibodies reduced IL-17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL-17 in ABMR and this event might be mediated by the complement system activation featuring this condition.

Micro opioid receptor A118G polymorphism and post-operative pain: opioids' effects on heterozygous patients.

The single-nucleotide-polymorphism (SNP) 118A>G in the micro-1 opioid receptor gene (OPRM1) is associated with a decrease in the analgesic effects of opioids. The aim of this study is to assess whether 118A >G polymorphism could influence the analgesic response to opioid-based postoperative pain (POP) therapy. The study consisted of two parts: section alpha, observational, included 199 subjects undergoing scheduled surgical procedures with pain management standardized on surgery invasiveness and on expected level of postoperative pain; section beta, randomized, included 41 women undergoing scheduled caesarean delivery with continuous intra-operative epidural anesthesia and post-operative analgesia (CEA). In both sections, POP was measured over 48 h (T6h-T24h-T48h) by the visual analogue scale (VAS). In section beta we also tested the responsiveness of hypothalamic-pituitary-adrenal axis (HPA) expressed by cortisol levels. In section alpha, with cluster analysis, subjects were analyzed according to their genotype: a group (no. 1) of 34 patients reporting VAS score >3 at every time lapse was identified and included only A118G carriers, while wild-type (A118A - absence of 118A>G polymorphism) patients were unevenly distributed between those with cluster no. 2 (VAS score <3 at every study steps) and those with cluster no. 3 (VAS score progressively reducing from T6h). In section beta, A118G carriers receiving epidural sufentanil had the lowest VAS scores at T24h; also in these patients, cortisol levels remained more stable, with a mild decrease at T6h. This study shows that the OPRM1 118A>G polymorphism affects postoperative pain response in heterozygous patients: they have a different postoperative pain response than patients with wild-type genes, which may affect the efficacy of the analgesic therapy.

Ozone eliminates novel coronavirus Sars-CoV-2 in mucosal samples.

Recent investigations have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is able to resist on the surfaces and that the diffusion occurs through droplets that can remain suspended in the air as an aerosol. The ozone generated in situ from oxygen is an active ingredient with a 'biocidal' action, but little is known about its capacity to inactivate specifically SARS-CoV-2. Here we show, for the first time, the efficiency of the ozone treatment to neutralize the SARS-CoV-2 present in nasopharynx secretion samples with high viral load. Our data show that ozone is effective in SARS-CoV-2 elimination.

Effective and safe off-label use of caplacizumab treatment in a middle-aged obese male.

This study shows clinical efficacy and safety profile of an off-label use of caplacizumab for the treatment of immune-mediated thrombotic thrombocytopenic purpura in a middle-aged obese male patient manifesting aphasia, weakness and unconsciousness. Routine blood tests revealed haemolytic anaemia, severe thrombocytopenia (platelet count=20×109/L) and moderate creatinine increase. Diagnosis was based on the clinical judgement and laboratory determinations (undetectable ADAMTS13 activity and presence of anti-ADAMTS13 antibodies). The patient underwent plasma-exchange and an adjunctive treatment with prednisone (1mg/Kg/day), but the occurrence of a refractory and exacerbated form of disease suggested also using rituximab (375mg/m2 weekly for 4 weeks) and caplacizumab as salvage treatments. The caplacizumab was given at 10mg/day subcutaneously without the first intravenous bolus. Because von Willebrand factor inhibition, platelet count recovery and remission of symptoms were achieved, use of caplacizumab with this scheme appeared to be as effective as the approved one. Although this is an off-label use, this case highlights the potential of this new treatment, in terms of drug's efficacy and safety.

Severe acute respiratory syndrome coronavirus 2 may exploit human transcription factors involved in retinoic acid and interferon-mediated response: a hypothesis supported by an in silico analysis.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19), resulting in acute respiratory disease, is a worldwide emergency. Because recently it has been found that SARS-CoV is dependent on host transcription factors (TF) to express the viral genes, efforts are required to understand the molecular interplay between virus and host response. By bioinformatic analysis, we investigated human TF that can bind the SARS-CoV-2 sequence and can be involved in viral transcription. In particular, we analysed the key role of TF involved in interferon (IFN) response. We found that several TF could be induced by the IFN antiviral response, specifically some induced by IFN-stimulated gene factor 3 (ISGF3) and by unphosphorylated ISGF3, which were found to promote the transcription of several viral open reading frame. Moreover, we found 22 TF binding sites present only in the sequence of virus infecting humans but not bat coronavirus RaTG13. The 22 TF are involved in IFN, retinoic acid signalling and regulation of transcription by RNA polymerase II, thus facilitating its own replication cycle. This mechanism, by competition, may steal the human TF involved in these processes, explaining SARS-CoV-2's disruption of IFN-I signalling in host cells and the mechanism of the SARS retinoic acid depletion syndrome leading to the cytokine storm. We identified three TF binding sites present exclusively in the Brazilian SARS-CoV-2 P.1 variant that may explain the higher severity of the respiratory syndrome. These data shed light on SARS-CoV-2 dependence from the host transcription machinery associated with IFN response and strengthen our knowledge of the virus's transcription and replicative activity, thus paving the way for new targets for drug design and therapeutic approaches.

Dysfunctional DC subsets in RCC patients: ex vivo correction to yield an effective anti-cancer vaccine.

Dendritic cells (DCs) are potent antigen-presenting cells responsible for the activation and functional polarization of specific T cells. In patients with renal cell carcinoma (RCC) and other cancers, coordinate DC and T cell defects have been reported. In particular, DC and T cell functional subsets that are not conducive to tumor clearance are hypothesized to predominate in patients with advanced-stage disease. Two major peripheral blood DC subsets have been identified in humans: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) that are believed to mediate contrasting effects on cancer immunity. Given the lack of information regarding DC subsets in patients with RCC, in the present study we have investigated the comparative frequencies and activation states of mDC and pDC in peripheral blood, cancer tissues and lymph nodes of patients with RCC using flow cytometry and immunohistochemistry. Three monoclonal antibodies (mAbs) reactive against specific DC subsets (BDCA-2 or BDCA-4 for pDC and BDCA-1 and BDCA-3 which represent two distinct subsets of mDC, mDC1 and mDC2, respectively) were employed. We observed a significant reduction of both DC subsets in the peripheral blood of patients as compared to normal donors. Similarly, both mDC and pDC were recruited in large numbers into RCC tumor tissues, where they displayed an immature phenotype (DC-LAMP(-)) and appeared unable to differentiate into mature DC (CD83(+)) that were competent to migrate to draining lymph nodes. However, we were readily able to generate ex vivo mDC from RCC patients. These DC stimulated robust anti-tumor CTL in vitro and would be envisioned for use in DC-based vaccines applied in patients with RCC whose existing immune system is judged dysfunctional, anergic or prone to undergo apoptosis.

Hyperuricemia and Risk of Cardiovascular Outcomes: The Experience of the URRAH (Uric Acid Right for Heart Health) Project.

The latest European Guidelines of Arterial Hypertension have officially introduced uric acid evaluation among the cardiovascular risk factors that should be evaluated in order to stratify patient's risk. In fact, it has been extensively evaluated and demonstrated to be an independent predictor not only of all-cause and cardiovascular mortality, but also of myocardial infraction, stroke and heart failure. Despite the large number of studies on this topic, an important open question that still need to be answered is the identification of a cardiovascular uric acid cut-off value. The actual hyperuricemia cut-off (> 6 mg/dL in women and 7 mg/dL in men) is principally based on the saturation point of uric acid but previous evidence suggests that the negative impact of cardiovascular system could occur also at lower levels. In this context, the Working Group on uric acid and CV risk of the Italian Society of Hypertension has designed the Uric acid Right for heArt Health project. The primary objective of this project is to define the level of uricemia above which the independent risk of CV disease may increase in a significantly manner. In this review we will summarize the first results obtained and describe the further planned analysis.

ID2-VEGF-related pathways in the pathogenesis of Kaposi's sarcoma: a link disrupted by rapamycin.

The Id-proteins are a family of four related proteins implicated in the control of differentiation and cell-cycle progression. Down-regulation of Id-gene expression is essential for the differentiation of several cell types. In addition, deregulated Id2 activity inhibits the Rb tumor suppressor pathway and promotes the expression of vascular endothelial growth factor (VEGF). Several members of VEGF family could be involved in Kaposi's sarcoma (KS) development and progression. Lymphatic vascular endothelial hyaluronan receptor-1 (LYVE-1) is the first marker of lymphatic endothelial competence during development in the mature vasculature, and is also expressed on KS spindle cells. Rapamycin (RAPA), an immunosuppressive drug, has been shown to reverse KS growth and to reduce tumor angiogenesis. We evaluate, in transplantation-associated KS and in cultured KS-cells the RAPA effect on Id2 and on de novo lymphangiogenesis. Markers of lymphatic-endothelial-cells (VEGFR-3, LYVE-1) and Id2, expressed at low levels within the normal skin, were up-regulated in KS and returned to normal levels after RAPA introduction. The association between Id2 and lymphangiogenesis is suggested by co-localization of Id2, VEGFR-3 and LYVE-1. RAPA inhibition on Id2 expression was confirmed in vitro in KS-cells, both in basal conditions and upon stimulation with VEGF. In conclusion, our data would suggest a novel molecular mechanism for the antineoplastic effects of RAPA in posttransplant KS.

Waist-to-height ratio is the best anthropometric index in association with adverse cardiorenal outcomes in type 2 diabetes mellitus patients.

BACKGROUND/AIMS: Visceral obesity is a potent risk factor for both chronic kidney disease (CKD) and myocardial infarction (MI) in type 2 diabetes mellitus patients (T2DM). Short stature is also associated with higher risk for either coronary or kidney diseases. Thus, the aim of our study was to investigate the association of the frequency of cardiorenal complications with waist-to-height index (W/Ht) in T2DM. METHODS: This was a cross-sectional study where 958 T2DM patients were studied. Subjects with cardiorenal disease (CRD) were defined as those with both kidney dysfunction (KD) and MI. RESULTS: We found a significant excess of MI in patients with KD as compared to those without KD (28 vs. 14%, p < 0.0001). Interestingly, among the commonly used indices of obesity, only W/Ht and BMI were significantly associated with CRD risk. Moreover, only the W/Ht index (but neither BMI nor WC) was significantly associated with the risks for every component of CRD. Lastly, in the multivariate logistic regression analysis, W/Ht proved superior to the other traditional factors associated with risk for CRD. CONCLUSIONS: Our study in a large cohort of subjects demonstrated that a higher W/Ht index is the best anthropometric measure associated with adverse CRD outcomes of T2DM patients.

Proteinuria in the prognosis of IgA nephropathy.

IgA Nephropathy (IgAN) is the most common lesion causing primary glomerulonephritis in the world. The main clinical predictors of progression are: elevated blood pressure, high histological score and proteinuria. Although elevated serum creatinine concentration at diagnosis, increased excretion of cytochines, age at onset, obesity and genetic factors may all influence clinical outcome, it is quite clear that proteinuria is the hallmark of renal damage in IgAN. Patients with IgAN and little or no proteinuria (<500 mg/day) have low risk of progression in the short term, while the rate of decline in renal function is 25-fold faster in those with sustained proteinuria >3 g/day. The product of duration (years) and urinary protein excretion (g/day) at the time of renal biopsy is more significantly correlated with progression. So, this so called proteinuria index may be a useful predictor for glomerular and interstitial histopathological changes and the fate of renal function in IgAN. The progression of IgAN may be slowed by antihypertensive and antiproteinuric therapy, such as angiotensin converting enzyme inhibitors and/or angiotensin II receptor blockers, that can minimize secondary glomerular injury. Proteinuria has been shown to be an adverse prognostic factor in IgAN, with a strong relationship between proteinuria and prognosis and established importance of remission. Consequently, targeting proteinuria may be a valid surrogate for individualized kidney protective therapy.