Autoimmunity in CD73/Ecto-5′-Nucleotidase Deficient Mice Induces Renal Injury

Extracellular adenosine formed by 5′-ectonucleotidase (CD73) is involved in tubulo-glomerular feedback in the kidney but is also known to be an important immune modulator. Since CD73−/−mutant mice exhibit a vascular proinflammatory phenotype, we asked whether long term lack of CD73 causes inflammation related kidney pathologies. CD73−/−mice (13 weeks old) showed significantly increased low molecule proteinuria compared to C57BL6 wild type controls (4.8≥0.52 vs. 2.9±0.54 mg/24 h, p<0.03). Total proteinuria increased to 5.97±0.78 vs. 2.55±0.35 mg/24 h at 30 weeks (p<0.01) whereas creatinine clearance decreased (0.161±0.02 vs. 0.224±0.02 ml/min). We observed autoimmune inflammation in CD73−/−mice with glomerulitis and peritubular capillaritis, showing glomerular deposition of IgG and C3 and enhanced presence of CD11b, CD8, CD25 as well as GR-1-positive cells in the interstitium. Vascular inflammation was associated with enhanced serum levels of the cytokines IL-18 and TNF-α as well as VEGF and the chemokine MIP-2 (CXCL-2) in CD73−/−mice, whereas chemokines and cytokines in the kidney tissue were unaltered or reduced. In CD73−/−mice glomeruli, we found a reduced number of podocytes and endothelial fenestrations, increased capillaries per glomeruli, endotheliosis and enhanced tubular fibrosis. Our results show that adult CD73−/−mice exhibit spontaneous proteinuria and renal functional deterioration even without exogenous stress factors. We have identified an autoimmune inflammatory phenotype comprising the glomerular endothelium, leading to glomeruli inflammation and injury and to a cellular infiltrate of the renal interstitium. Thus, long term lack of CD73 reduced renal function and is associated with autoimmune inflammation

Evaluating platelet function disorders in children with bleeding tendency – A single center study

Platelet function disorders (PFDs) are a common cause of mild bleeding tendency. However, they cannot be recognized by standard screening studies. The gold standard test for PFD is platelet aggregation, performed by light transmission aggregometry (LTA). A newer and less validated method is the closure time (CT), performed by the platelet function Analyzer 100 (PFA-100). Data regarding the validity of these tests in children are limited. The aim of this study was to evaluate the usefulness of LTA and PFA-100 for the diagnosis of pediatric patients with bleeding tendency. This retrospective study included patients one month–18 year old that had LTA tests performed at the coagulation laboratory of Rabin Medical Center between the years 2006–2015. Bleeding severity was assessed using a pediatric bleeding score. Patients were excluded from analysis if they had thrombocytopenia, thrombocytosis or coagulation factors deficiencies. One hundred and thirty-seven (137) patients were included in the analysis. The median age was 7.5 years (range one month–18 years). Most patients (93%) had a bleeding score of 2 or more. Abnormal LTA was found in 40% and prolonged CT in 23% of the patients. Abnormal LTA was significantly more common in patients with a bleeding score of 2 or more compared to patients with a lower bleeding scores (P = 0.04). No significant correlation was found between the bleeding severity and the number of agonists which induced abnormal responses (p = 0.52) or the CT (p = 0.35). Furthermore, no correlation was found between abnormal LTA and prolonged CT. To conclude, we were able to diagnose 40% of children who presented with bleeding tendency with platelet aggregation defects by LTA. Abnormal LTA was significantly more prevalent in patients with a bleeding score of 2 and above. In contrast, CT was not found to be sensitive as a screening tool for PFD. Therefore, our data extend the validity of the use of LTA for the evaluation of pediatric patients with bleeding tendency

Improved Methods for Thermal Rearrangement of Alicyclic α-Hydroxyimines to α-Aminoketones: Synthesis of Ketamine Analogues as Antisepsis Candidates

Ketamine is an analgesic/anesthetic drug, which, in combination with other drugs, has been used as anesthetic for over 40 years. Ketamine induces its analgesic activities by blocking the &lt;em&gt;N&lt;/em&gt;-methyl-D-aspartate (NMDA) receptor in the central nervous system (CNS). We have reported that low doses of ketamine administrated to patients before incision significantly reduced post-operative inflammation as reflected by reduced interleukin-6 (IL-6) sera-levels. Our data demonstrated in a rat model of Gram-negative bacterial-sepsis that if we inject a low dose of ketamine following bacterial inoculation we reduce mortality from approximately 75% to 25%. Similar to what we have observed in operated patients, the levels of TNF-α and IL-6 in ketamine-treated rats were significantly lower than in septic animals not treated with ketamine. On the base of these results, we have designed and synthesized series of new analogues of ketamine applying a thermal rearrangement of alicyclic α-hydroxyimines to a-aminoketones in parallel arrays. One of the analogues (compound &lt;strong&gt;6e&lt;/strong&gt;) displayed high activity in down-regulating the levels of IL-6 and TNF-α&lt;em&gt; in vivo&lt;/em&gt; as compared to ketamine

Effect of adenosine receptor subtype autoregulation on the inflammatory process.

<p>(A) Early expression of A₁R after bacterial inoculation decreases cAMP levels, enhances production of local pro-inflammatory cytokines and promotes leukocyte migration. (B) In a later phase of peritonitis A_2AR expression increase by A₁R which leads to increase in cAMP levels. High cAMP markedly decreases local pro-inflammatory cytokines and leukocyte recruitment, hence restraining inflammatory flames.</p

A₁R and A_2AR expression in peritoneal leukocytes during inflammation <i>in vivo.</i>

<p>Peritonitis was induced in mice by <i>E. coli</i> inoculation at a sub-lethal dose. To examine the dynamic expression of the two high-affinity adenosine receptors, A₁R and A_2AR, peritoneal lavage was performed at indicated time points. A₁R and A_2AR mRNA levels in peritoneal leukocytes were analyzed by real time PCR and normalized to β-actin levels. Data represent three experiments and are expressed as mean±SEM. * <i>p</i><0.05, between expression levels of each receptor to expression at time 0, <i>n</i> = 5 for each experiment.</p

The effect of A₁R agonist, in A_2AR^−/− and in the presence of A_2AR antagonist.

<p>Mice were administrated with A₁R agonist (CHA, 0.1 mg/kg) or vehicle 24 prior to bacterial inoculation. 30 min before inoculation the A_2AR antagonist (ZM241385, 1 mg/kg) or the A_2AR agonist (CGS21680, 1 mg/kg) were administered to the same animals or to untreated animals. (A) sera IL-6 and TNFα (12 hours) and (B) lavage fluids IL-6 and TNFα (12 hours). (C) A_2AR⁻^/− mice or their WT littermates were treated with the A₁R agonist (CHA, 0.1 mg/kg) i.p. or vehicle 24 hours prior to bacterial inoculation. 12 hours following inoculation sera were collected and analyzed for IL-6 and TNFα levels. Data are representative of three individual experiments and are expressed as mean±SEM. * <i>p</i><0.05, ** <i>p</i><0.01 between vehicle and CHA or CGS21680 and between CHA with or without ZM241385, <i>n</i> = 5 for each experiment.</p