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Background and Aims Hemodialysis (HD) patients suffer from devastatingly high rates of morbidity and mortality due to infections. Neutrophils isolated from HD patients were shown in the past to exhibit impaired phagocytosis in a mechanism yet to be completely elucidated. In 2004, Brinkmann et al. were the first to describe a new form of cell death which they termed Neutrophil Extracellular Trap Formation, or NETosis, in which neutrophils expulse batches of DNA and proteins in response to bacterial or chemical stimuli in order to trap and remove the stimulus. NETosis is further divided into two pathways, NADPH oxidase (NOX)-dependent and NOX-independent, induced in vitro by phorbol 12-myristate 13-acetate (PMA) and Calcium Ionophore (CI), respectively. In this research, we aim to assess the capacities of HD neutrophils to engage in NETosis, hypothesizing they might be diminished similarly to their phagocytic capacities, and to elucidate the underlying mechanism behind this impairment. Method Neutrophils were isolated from whole venous blood of normal controls and from the arterial line of HD patients before the onset of a dialysis session using EasySepTM direct human neutrophils isolation kit. Then, NETosis was induced with either PMA, Calcium Ionophore A23187 or Phosphate-buffered saline (PBS) as negative control. cfDNA released from the cells was quantified by measuring SYTOXTM-green nucleic acid stain fluorescence levels in the supernatant after stimulation using Elisa plate reader and morphological analysis was done under fluorescence microscope. Reactive oxygen species levels were quantified using flow cytometry and superoxide dismutase (SOD) activity was measured using the SOD Assay Kit (Sigma-Aldrich). Protein arginine deiminase 4 (PAD4) expression was assessed by western blotting. Hydrogen peroxide (H2O2) was added exogenously in order to restore NETosis. Results HD isolated neutrophils exhibit decreased NETosis compared with normal controls in response to both PMA in the NOX-dependent pathway and CI in the NOX-independent pathway, as measured by immunofluorescence and cfDNA quantification. In the NOX-dependent pathway SOD activity was found to be 14% decreased in HD patients, resulting in the accumulation of superoxide radicals and decreased production of H2O2. In the NOX-independent pathway, PAD4 expression was found to be significantly decreased as well. NET formation was restored in vitro in HD neutrophils by the addition of exogenous H2O2. Conclusion To date, impaired NETosis was described only in congenital conditions such as chronic granulomatous disease and myeloperoxidase deficiency. To our knowledge, our research is the first to describe an acquired defect in NETosis in end stage renal disease patients undergoing chronic hemodialysis. An intervention aimed to improve neutrophil function in these patients may reduce the morbidity and mortality due to infection-related disease.

Background and Aims Pro-inflammatory cytokines are one of several factors which contribute to the progression of diabetic kidney disease (DKD), a condition characterized by chronic kidney inflammation which results in the tubulointerstitial fibrosis which contributes to the progression of DKD. Interleukin 1 (IL-1) two main agonists IL-1α and IL-1β activate a pro-inflammatory cascade in response to different inflammatory stimuli, including hyperglycemia. It was previously shown that a deficiency of NLRP3 which is required for the conversion of IL-1 to its active state, protects mice from the development and progression of DKD. We hypothesize that the chronic hyperglycemia in diabetic patients triggers the activation and release of IL1α and/or IL-1β from renal tubular cells and that this activation leads to the tissue fibrosis. We aim to assess Il-1 and fibronectin expression in an immortalized proximal tubule epithelial cell line from normal adult human kidney (HK-2). In addition, we evaluate the influence of Anakinra™, a pharmaceutical inhibitor of the Il-1 receptor, currently indicated mainly for rheumatoid diseases, on the levels of fibronectin expression in this model. Methods HK-2 cells were cultured and treated with either physiological glucose concentration (5.5mM), high glucose (30mM) or 30mM mannitol as osmotic control for 24 hours to evaluate their effects on Il-1 expression and fibronectin expression. mRNA levels of IL-1α, IL-1β and fibronectin were assessed in q-PCR, and protein expression levels were quantified by western blotting. Immunofluorescence was used to visually demonstrate the presence of IL-1α and IL-1β upon stimulation. Finally, Anakinra™ was added to the tissue cultures in a range of physiologic prescribed concentrations and its effect on cell fibrosis was assessed by the measurement of fibronectin expression 24 hours later by western blotting. Results mRNA and protein expression of IL-1α but mostly IL-1β was elevated in HK-2 cells under hyperglycemic conditions but not in physiological glucose environment or under high osmotic conditions. Fibronectin levels were elevated in the high glucose treated cells compared with control. Finally, Anakinra™ was found to attenuate fibronectin expression under high glucose conditions, compared with the untreated cells. Conclusion Proinflammatory IL-1α and IL-1β cytokines are expressed by HK-2 cells upon stimulation with glucose and result in the fibrosis on the cells measured by the production of fibronectin. The addition of Anakinra™, an IL-1 receptor blocker, to the cell culture attenuate the expression of fibronectin by the tubular cells. Our research is the first to describe a causation between hyperglycemia, IL-1 elevated levels and fibrosis in HK-2 cells, as demonstrated by the beneficial effect of Anakinra™ on lowering fibronectin expression.

A 14-year-old girl, a rear seat passenger on a jet-ski not wearing suitable protective gear, was ‘jumping’ waves at a low velocity when she was ejected backwards off the vehicle, suffering a complex and unusual hydrostatic perineal injury as a result of the high-pressure water stream propelling the jet-ski. She presented to the emergency room with rectal bleeding and perineal and abdominal pain. Initial investigations revealed signs of anorectal injury and both intraperitoneal and extraperitoneal free air and fluid, suggesting a possible rectal perforation. Proctoscopy confirmed the primary diagnosis and exploratory laparotomy revealed an intraperitoneal tear in the rectal wall. The tear was repaired, and protective loop colostomy was performed. Initial results of anal manometry, transrectal ultrasound and anal electromyography were unfavourable. However, 17 months after pelvic floor physiotherapy and biofeedback, the colostomy was reversed, and her continence has returned to her normal (preinjury) state.

The immunotherapy treatments offered for Chronic lymphocytic leukemia (CLL) activate various cellular and biochemical mechanisms, including the complement system. Recently it was shown that the classical complement pathway (CP) in CLL patients is persistently activated at a low level through (Michelis et al. PlosOne, 2019), and that the mechanism of chronic CP activation involves the formation of IgG-hexamers (Michelis et al. PlosOne, 2020). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur only after antigen binding. The study investigated the formation of IgG-hexamers in CLL patients and normal controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and NC were used for separation of IgG-hexamers. The obtained IgG-hexamers were measure and used for assessment of CP activity. For evaluation of the presence of IgG-hexamers on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG-hexamers were higher in patients compared to NC. Also, they activated the complement system to a higher extent in patients than in NC. Alpha 2 macroglobulin (A2M) was recognized as the antigen causing the hexamerization of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the surface of the cells. The exact course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens. Disclosures Tadmor: Neopharm: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Medison: Consultancy, Speakers Bureau; 6. Novartis Israel Ltd., a company wholly owned by Novartis Pharma AG: Consultancy, Speakers Bureau. Aviv:Abbvie: Honoraria.