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A greater number of screened conditions is often considered to equate to better screening, whereas it may be due to conditions being counted differently. This manuscript describes a harmonised Australasian approach to listing target conditions found on bloodspot screening panels. Operational definitions for target disorders and incidental findings were developed and applied to disorder lists. A gap analysis was performed between five, state-based Australian newborn screening programme disorder lists and the single national New Zealand and state-level Californian versions. Screening panels were found to be broadly similar. Gap analysis with Californian data reflected differences in jurisdictional approval (for example, haemoglobinopathies and lysosomal disorders not being recommended in Australasia). Differences amongst Australasian panels reflected varied the timeframes recommended in order to implement newly approved disorders, as well as decisions to remove previously screened disorders. A harmonised approach to disorder counting is essential to performing valid comparisons of newborn bloodspot screening panels.

OBJECTIVES: Since its implementation 50 years ago in Quebec, Canada, newborn screening for congenital hypothyroidism has become one of the most successful public health measures worldwide. Screening programmes across Australia and New Zealand are characterised by significant commonalities in screening algorithms, and a high degree of regional cooperation in harmonisation efforts. We aimed to conduct a comprehensive survey of current performance and practices related to the total testing process for congenital hypothyroidism screening and provide recommendations for harmonisation priorities within our region. METHODS: A survey was conducted involving the six newborn screening laboratories which provide complete geographic coverage across Australasia. Approximately 360,000 newborns are screened annually. Survey questions incorporated pre-analytical, analytical, and post-analytical aspects of the screening programmes and an extensive 5-year (2016-2020) retrospective analysis of individual programme performance data. Responses from individual screening programmes were collated. RESULTS: The uptake of newborn screening was over 98% for the six major jurisdictions. All programmes have adopted a single-tier thyroid stimulating hormone (TSH) strategy using the Perkin Elmer GSP instrument. Significant similarities exist between programmes for recommended age of collection and recollection protocols for low birthweight newborns. The process for the determination of TSH cutoffs varies between programmes. TSH lower cut-offs for borderline-positive and positive notifications between 12-15 and 12-25 mIU/L blood, respectively. Recall rates vary between 0.08 and 0.20%. The case definition for congenital hypothyroidism generally includes biochemical and radiological parameters in addition to the commencement of thyroxine. All programmes reported collecting biochemical and clinical data on infants with positive screening tests, and positive predictive values vary between 23.6 and 77.

Pentraxins are a family of evolutionarily conserved pattern recognition molecules with pivotal roles in innate immunity and inflammation, such as opsonization of pathogens during bacterial and viral infections. In particular, the long Pentraxin 3 (PTX3) has been shown to regulate several aspects of vascular and tissue inflammation during solid organ transplantation. Our study investigated the role of PTX3 as possible modulator of Complement activation in a swine model of renal ischemia/reperfusion (I/R) injury. We demonstrated that I/R injury induced early PTX3 deposits at peritubular and glomerular capillary levels. Confocal laser scanning microscopy revealed PTX3 deposits co-localizing with CD31+ endothelial cells. In addition, PTX3 was associated with infiltrating macrophages (CD163), dendritic cells (SWC3a) and myofibroblasts (FSP1). In particular, we demonstrated a significant PTX3-mediated activation of classical (C1q-mediated) and lectin (MBL-mediated) pathways of Complement. Interestingly, PTX3 deposits co-localized with activation of the terminal Complement complex (C5b-9) on endothelial cells, indicating that PTX3-mediated Complement activation occurred mainly at the renal vascular level. In conclusion, these data indicate that PTX3 might be a potential therapeutic target to prevent Complement-induced I/R injury.

CVD remains the major cause of mortality with graft functioning in Kidney transplant recipients (KTRs), with an estimated risk of CV events about 50-fold higher than in the general population. Many strategies have been considered to reduce the CV risk such as the use of mTOR inhibitors. We evaluate whether chronic mTOR inhibition might influence CV aging in KTRs studying the molecular mechanisms involved in this effect. We retrospectively analyzed 210 KTRs with stable graft function on therapy with CNI and mycophenolic acid (Group A, 105 pts.), or with CNI and mTORi (Everolimus, Group B, 105 pts.). The presence of mTOR inhibitor in immunosuppressive therapy was associated to increase serum levels of Klotho with concomitant reduction in FGF-23, with a significant decrease in left ventricular mass. In addition, KTRs with mTORi improved mitochondrial function/biogenesis in PBMC with more efficient oxidative phosphorylation, antioxidant capacity and glutathione peroxidase activity. Finally, group B KTRs presented reduced levels of inflammaging markers such as reduced serum pentraxin-3 and p21ink expression in PBMC. In conclusion, we demonstrated that mTOR inhibition in immunosuppressive protocols prevents the occurrence and signs of CV aging in KTRs.

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12–36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell i

Background and objectives Active antibody-mediated rejection is the main cause of kidney transplant loss, sharing with SLE the alloimmune response and the systemic activation of the IFN-a pathway. IgE-mediated immune response plays a key role in the development of SLE nephritis and is associated with IFN-a secretion. The aim of our study was to investigate IgE-mediated immune response in antibody-mediated rejection. Design, setting, participants, & measurements This was a cross-sectional study of 56 biopsy-proven antibodymediated rejection study participants, 80 recipients with normal graft function/histology (control), 16 study participants with interstitial fibrosis/tubular atrophy, and six participants with SLE. We evaluated graft IgE deposition, tryptase (a mast cell marker), and CD203 (a specific marker of activated basophils) by immunofluorescence/confocal microscopy. In addition, we measured serum concentration of human myxovirus resistance protein 1, an IFN-a–induced protein, and anti-HLA IgE. Results We observed a significantly higher IgE deposition in tubules and glomeruli in antibody-mediated rejection (1766679 pixels) and SLE (1495643 pixels) compared with interstitial fibrosis/tubular atrophy (5826122 pixels) and control (253650 pixels). Patients with antibody-mediated rejection, but not control patients and patients with interstitial fibrosis/tubular atrophy, presented circulating anti-HLA IgE antibodies, although with a low mean fluorescence intensity. In addition, immunofluorescence revealed the presence of both mast cells and activated basophils in antibody-mediated rejection but not in control and interstitial fibrosis/tubular atrophy. The concentration of circulating basophils was significantly higher in antibody-mediated rejection compared with control and interstitial fibrosis/tubular atrophy. MxA serum levels were significantly higher in antibodymediated rejection compared with control and correlated with the extent of IgE deposition. Concl

Chronic antibody-mediated rejection (CAMR) is the major cause of kidney transplant failure. The molecular mechanisms underlying this event are still poorly defined and this lack of knowledge deeply influences the potential therapeutic strategies. The aim of our study was to analyze the phosphoproteome of peripheral blood mononuclear cells (PBMCs), to identify cellular signaling networks differentially activated in CAMR. Phosphoproteins isolated from PBMCs of biopsy proven CAMR, kidney transplant recipients with normal graft function and histology and healthy immunocompetent individuals, have been investigated by proteomic analysis. Phosphoproteomic results were confirmed by Western blot and PBMCs’ confocal microscopy analyses. Overall, 38 PBMCs samples were analyzed. A differential analysis of PBMCs’ phosphoproteomes revealed an increase of lactotransferrin, actin-related protein 2 (ARPC2) and calgranulin-B in antibody-mediated rejection patients, compared to controls. Increased expression of phosphorylated ARPC2 and its correlation to F-actin filaments were confirmed in CAMR patients. Our results are the first evidence of altered cytoskeleton organization in circulating immune cells of CAMR patients. The increased expression of phosphorylated ARPC2 found in the PBMCs of our patients, and its association with derangement of F-actin filaments, might suggest that proteins regulating actin dynamics in immune cells could be involved in the mechanism of CAMR of kidney grafts.

LPS-induced sepsis is a leading cause of acute kidney injury (AKI) in critically ill patients. LPS may induce CD80 expression in podocytes with subsequent onset of proteinuria, a risk factor for progressive chronic kidney disease (CKD) frequently observed after AKI. This study aimed to investigate the therapeutic efficacy of LPS removal in decreasing albuminuria through the reduction of podocyte CD80 expression. Between January 2015 and December 2017, 70 consecutive patients with Gram-negative sepsis-induced AKI were randomized to either have coupled plasma filtration and adsorption (CPFA) added to the standard care (n = 35) or not (n = 35). To elucidate the possible relationship between LPS-induced renal damage, proteinuria, and CD80 expression in Gram sepsis, a swine model of LPS-induced AKI was set up. Three hours after LPS infusion, animals were treated or not with CPFA for 6 h. Treatment with CPFA significantly reduced serum cytokines, C-reactive protein, procalcitonin, and endotoxin levels in patients with Gram-negative sepsis-induced AKI. CPFA significantly lowered also proteinuria and CD80 urinary excretion. In the swine model of LPS-induced AKI, CD80 glomerular expression, which was undetectable in control pigs, was markedly increased at the podocyte level in LPS-exposed animals. CPFA significantly reduced LPS-induced proteinuria and podocyte CD80 expression in septic pigs. Our data indicate that LPS induces albuminuria via podocyte expression of CD80 and suggest a possible role of timely LPS removal in preventing the maladaptive repair of the podocytes and the consequent increased risk of CKD in sepsis-induced AKI.

Primary membranous nephropathy (PMN) is a renal-specific autoimmune disease caused by circulating autoantibodies that target glomerular podocyte antigens (PLA2R/THSD7A). However, very little is known on the molecular mechanisms controlling B cell response in this nephropathy. The present study was aimed at correlating the serum levels of B cell activators BAFF/BLyS and APRIL with the presence of anti-PLA2R antibodies in PMN patients and with long-term clinical outcome. To this aim, 51 patients with anti-PLA2R-positive biopsy-proven PMN and nephrotic range proteinuria (>3.5 g/24 hours) were enrolled between January 2009 and December 2015 and treated with conventional 6-month immunosuppressive therapy. After 6 months, 29 patients (56.9%) cleared circulating anti-PLA2R, while in remaining 22 (43.1%), they persisted. Intriguingly, in the first group, baseline serum levels of BAFF/BLyS and APRIL were significantly lower than those in the second one. Moreover, after 6 months of immunosuppressive therapy, an overall reduction in both cytokine serum levels was observed. However, in PMN patients with anti-PLA2R clearance, this reduction was more prominent, as compared with those with anti-PLA2R persistence. When related to clinical outcome, lower baseline BAFF/BLyS (<6.05 ng/mL) and APRIL (<4.20 ng/mL) serum levels were associated with significantly higher probability to achieve complete or partial remission after 24-month follow-up. After dividing the entire study cohort into three groups depending on both cytokine baseline serum levels, patients with both BAFF/BLyS and APRIL below the cut-off showed a significantly higher rate of complete or partial remission as compared with patients with only one cytokine above the cut-off, while the composite endpoint was achieved in a very low rate of patients with both cytokines above the cut-off. Taken together, these results provide new insights into the role of BAFF/BLyS and APRIL in both the pathogenesis of anti-PLA2R-pos

An altered metabolism is involved in the development of clear cell - renal cell carcinoma (ccRCC), and in this tumor many altered genes play a fundamental role in controlling cell metabolic activities. We delineated a large-scale metabolomic profile of human ccRCC, and integrated it with transcriptomic data to connect the variations in cancer metabolism with gene expression changes. Moreover, to better analyze the specific contribution of metabolic gene alterations potentially associated with tumorigenesis and tumor progression, we evaluated the transcription profile of primary renal tumor cells. Untargeted metabolomic analysis revealed a signature of an increased glucose uptake and utilization in ccRCC. In addition, metabolites related to pentose phosphate pathway were also altered in the tumor samples in association with changes in Krebs cycle intermediates and related metabolites. We identified NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) as the most highly expressed gene in renal cancer cells and evaluated its role in sustaining angiogenesis, chemoresistance, and mitochondrial dysfunction. Finally, we showed that silencing of NDUFA4L2 affects cell viability, increases mitochondrial mass, and induces ROS generation in hypoxia.