Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1β were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

Simultaneous Measurement of Changes in Neutrophil Granulocyte Membrane Potential, Intracellular pH, and Cell Size by Multiparametric Flow Cytometry

Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with cellular changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

Complement Factor H Family Proteins Modulate Monocyte and Neutrophil Granulocyte Functions

Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1β and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.

Effects of Propofol and Ketamine as Induction Agent to Isoflurane and as Continuous Rate Infusion on Haematobiochemical Parameters in Dogs Premedicated with Glycopyrrolate, Dexmedetomidine and Butorphanol

Background: The study was conducted to evaluate the effect of propofol and ketamine as induction agent to isoflurane and as continuous rate infusion on haematobiochemical parameters in dogs premedicated with glycopyrrolate, dexmedetomidine and butorphenol. Methods: Twenty four animals were randomly divided into four Groups (A, B, C and D) comprising six animals in each. All the animals were premedicated with glycopyrrolate, dexmedetomidine and Butorphanol. Induction was done with propofol in group A and B and with ketamine in group C and D. Anaesthesia was maintained with isoflurane in group A and C and with propofol and ketamine continuous rate infusion in group B and D, respectively. Blood samples were collected at 0(before premedication), 20, 40 and 60 min (during maintenance) to evaluate different haematobiochmical parameters. Result: The haemoglobin levels decreased in all the groups but significantly in Group C and Group D. The total erythrocyte count decreased in all the groups with significant change in Group D. The TLC, PCV, platelet, lymphocyte and monocyte count decreased non-significantly. The neutrophil, granulocyte count in all the groups increased non-significantly over the different observation period. The serum glucose and LDH increased significantly in all the groups. The GGT, alkaline phosphatise, BUN and serum creatinine increased non-significantly in all the groups.

Histological evaluation of periprosthetic infection using HOES scale and CD15 expression analysis at the stage of the hip revision arthroplasty

Background.The effectiveness improvement and standardization of the methods of histological diagnosing periprosthetic infection (PPI) is an urgent task in the treatment of complications after large joint arthroplasty. Purpose of the study— Histopathological evaluation of the infection involvement of periprosthetic tissues at the stage of revision arthroplasty for deep infection of the hip using HOES scale and immunohistochemical analysis of CD15 expression.Materials and Methods.A single-center prospective study was performed on the clinical intraoperative material obtained at the stage of revision arthroplasty of the hip in 27 patients at the age of 65 (55÷69) years. The group of examination included patients with acute and chronic forms of deep periprosthetic infection. Light-optical microscopic investigation of the samples of periprosthetic connective-tissue membrane and bone tissue from the foci of infectious involvement was made on paraffin sections stained with hematoxylin and eosin; with the immunohistochemical reaction to determine the expression of CD15 neutrophil granulocyte markers. HOES Scale for pathohistological assessment was used in order to objectify osteomyelitis signs in periprosthetic bone tissue.Results. The signs of acute and chronic stages of periprosthetic osteomyelitis were observed in 9/16 patients with PPI chronic course within 1–30 months of postoperative period, from one to 18 months after manifestation of the symptoms. The signs of subsided osteomyelitis were determined in 12/27 patients with PPI of acute and chronic forms. Infected periprosthetic membranes were found in 19/27 clinical cases in the early and longterm time periods after arthroplasty surgery. A direct significant correlation was revealed between histopathological signs of infecting the periprosthetic bone and the connective-tissue periprosthetic membrane, especially strong one in patients with acute and chronic PPI osteomyelitis.Conclusion. The use of HOES Scale and the analysis of CD15 expression ensure the objectivity of PPI histological diagnosing. The results obtained indicate an increased risk of osteomyelitis development in patients with chronic periprosthetic infection after the hip arthroplasty.

Selection for feed efficiency elicits different postprandial plasma metabolite profiles in response to poor hygiene of housing conditions in growing pigs

This study was conducted to compare postprandial plasma concentrations of insulin, energy-related metabolites, and amino acids measured after a 6-week challenge consisting of exposure to good or poor hygiene of housing conditions of 24 growing pigs divergently selected for low-RFI (LRFI) and high-RFI (HRFI). Blood indicators of immune responses were assessed from samples collected before 0 (W0), and 3 (W3), and 6 weeks (W6) after pigs transfer to their respective hygiene of housing conditions. Plasma haptoglobin concentrations and blood neutrophil granulocyte numbers were greater in poor than in good hygiene of housing conditions at W3. Plasma concentrations of total immunoglobulin G were greater (p = 0.04) in poor than in good hygiene of housing conditions at W6. At W6, pigs were fitted with an intravenous catheter for serial blood samplings. Low-RFI pigs had greater insulin (p < 0.001) and lower triglyceride (p = 0.04) average plasma concentrations than HRFI pigs in both conditions. In poor hygiene of housing conditions, the peaks of insulin and glucose were observed earlier and that of insulin was greater in LRFI than in HRFI pigs. Irrespective of genetic line, average plasma concentrations of histidine, isoleucine, leucine, methionine, threonine, valine, and alanine were greater in poor compared with good hygiene of housing conditions. Only HRFI pigs had greater lysine, asparagine, proline, and tyrosine plasma concentrations in poor than in good hygiene of housing conditions. Conversely, arginine, tryptophan, proline, and tyrosine plasma concentrations were lower only for LRFI pigs housed in poor hygiene conditions. Our results suggest that, contrary to HRFI, LRFI pigs increase or maintain their utilization of tryptophan, arginine, and lysine when housed in poor hygiene conditions. This indicates that this difference may contribute to the better capacity of LRFI to cope with poor hygiene of housing conditions.

Selection for feed efficiency elicits different postprandial plasma metabolite profiles in response to poor hygiene of housing conditions in growing pigs

Selection for residual feed intake (RFI), a measure of feed efficiency, may affect the ability of pigs to adapt their metabolism in response to poor environmental conditions. This study was conducted to compare postprandial plasma concentrations of insulin, energy related metabolites, and amino acids measured after a 6-week challenge consisting of exposure to good or poor hygiene of housing conditions of 24 growing pigs divergently selected for low-RFI (LRFI) and high-RFI (HRFI). Blood indicators of immune responses were assessed from samples collected before (week 0 or W0), and 3 (W3) and 6 weeks (W6) after pigs transfer to their respective housing hygiene conditions. Plasma haptoglobin concentrations and blood neutrophil granulocyte numbers were greater in poor than in good conditions at W3. Plasma concentrations of total immunoglobulin G were greater (p = 0.04) in poor than in good hygiene conditions at W6. At W6, pigs were fitted with an intravenous catheter for serial blood samplings. Low-RFI pigs had greater insulin (p < 0.001) and lower triglyceride (p = 0.04) average plasma concentrations than HRFI pigs in both conditions. In poor hygiene conditions, the peaks of insulin and glucose occured earlier and that of insulin was greater in LRFI than in HRFI pigs. Irrespective of genetic line, average plasma concentrations of histidine, isoleucine, leucine, methionine, threonine, valine, and alanine were greater in poor compared with good conditions. Only HRFI pigs had greater lysine, asparagine, proline, and tyrosine plasma concentrations in poor than in good hygiene conditions. Conversely, arginine, tryptophan, proline, and tyrosine plasma concentrations were lower only for LRFI pigs housed in poor hygiene conditions. The impact of poor hygiene of housing conditions on insulin, triglycerides, and AA profiles differed between RFI lines. More specifically, our results suggest that, contrary to HRFI, LRFI pigs increased or maintained their utilization of Trp, Arg, and Lys when housed in poor hygiene conditions. This difference may contribute to the better capacity of LRFI to cope with the poor hygiene of housing conditions.

Neutrophil Extracellular Traps Promote the Development and Growth of Human Salivary Stones

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. The mechanism behind the formation of sialoliths has been elusive. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland, and often results in sialadenitis with the need for surgical intervention. Here, we show by the use of immunohistochemistry, immunofluorescence, computed tomography (CT) scans and reconstructions, special dye techniques, bacterial genotyping, and enzyme activity analyses that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths in humans. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in the dense aggregation of the latter, and the subsequent mineralization creates alternating layers of dense mineral, which are predominantly calcium salt deposits and DNA. The further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis, in which an immune system-mediated response essentially participates in the physicochemical process of concrement formation and growth.

Development and Growth of Human Salivary Stones by Neutrophil Extracellular Traps

Salivary gland stones, or sialoliths, are the most common cause of the obstruction of salivary glands. Symptomatic sialolithiasis has a prevalence of 0.45% in the general population, is characterized by recurrent painful periprandial swelling of the affected gland and often results in sialadenitis with the need for surgical intervention. The mechanism behind the formation of sialoliths has been elusive. Here we show that neutrophil extracellular traps (NETs) initiate the formation and growth of sialoliths. The deposition of neutrophil granulocyte extracellular DNA around small crystals results in their dense aggregation, and the subsequent mineralization creates alternating layers of dense mineral, predominantly calcium salt deposits and DNA. Further agglomeration and appositional growth of these structures promotes the development of macroscopic sialoliths that finally occlude the efferent ducts of the salivary glands, causing clinical symptoms and salivary gland dysfunction. These findings provide an entirely novel insight into the mechanism of sialolithogenesis in which an immune system mediated response essentially participates in the physico-chemical process of concrement formation and growth.