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<p>Autoantibody production and autoantibody-mediated inflammation are hallmarks of a number of autoimmune diseases. The K/BxN serum-transfer arthritis is one of the most widely used models of the effector phase of autoantibody-induced pathology. Several hematopoietic lineages including neutrophils, platelets, and mast cells have been proposed to contribute to inflammation and tissue damage in this model. We have previously shown that the Syk tyrosine kinase is critically involved in the development in K/BxN serum-transfer arthritis and bone marrow chimeric experiments indicated that Syk is likely involved in one or more hematopoietic lineages during the disease course. The aim of the present study was to further define the lineage(s) in which Syk expression is required for autoantibody-induced arthritis. To this end, K/BxN serum-transfer arthritis was tested in conditional mutant mice in which Syk was deleted in a lineage-specific manner from neutrophils, platelets, or mast cells. Combination of the MRP8-Cre, PF4-Cre, or Mcpt5-Cre transgene with floxed Syk alleles allowed efficient and selective deletion of Syk from neutrophils, platelets, or mast cells, respectively. This has also been confirmed by defective Syk-dependent in vitro functional responses of the respective cell types. In vivo studies revealed nearly complete defect of the development of K/BxN serum-transfer arthritis upon neutrophil-specific deletion of Syk. By contrast, Syk deletion from platelets or mast cells did not affect the development of K/BxN serum-transfer arthritis. Our results indicate that autoantibody-induced arthritis requires Syk expression in neutrophils, whereas, contrary to prior assumptions, Syk expression in platelets or mast cells is dispensable for disease development in this model.</p

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<p>Autoantibody production and autoantibody-mediated inflammation are hallmarks of a number of autoimmune diseases. The K/BxN serum-transfer arthritis is one of the most widely used models of the effector phase of autoantibody-induced pathology. Several hematopoietic lineages including neutrophils, platelets, and mast cells have been proposed to contribute to inflammation and tissue damage in this model. We have previously shown that the Syk tyrosine kinase is critically involved in the development in K/BxN serum-transfer arthritis and bone marrow chimeric experiments indicated that Syk is likely involved in one or more hematopoietic lineages during the disease course. The aim of the present study was to further define the lineage(s) in which Syk expression is required for autoantibody-induced arthritis. To this end, K/BxN serum-transfer arthritis was tested in conditional mutant mice in which Syk was deleted in a lineage-specific manner from neutrophils, platelets, or mast cells. Combination of the MRP8-Cre, PF4-Cre, or Mcpt5-Cre transgene with floxed Syk alleles allowed efficient and selective deletion of Syk from neutrophils, platelets, or mast cells, respectively. This has also been confirmed by defective Syk-dependent in vitro functional responses of the respective cell types. In vivo studies revealed nearly complete defect of the development of K/BxN serum-transfer arthritis upon neutrophil-specific deletion of Syk. By contrast, Syk deletion from platelets or mast cells did not affect the development of K/BxN serum-transfer arthritis. Our results indicate that autoantibody-induced arthritis requires Syk expression in neutrophils, whereas, contrary to prior assumptions, Syk expression in platelets or mast cells is dispensable for disease development in this model.</p

Inflammation after surgery induced NGAL production, but NGAL was not derived from neutrophils.

<p>Systemic inflammation markers (A) IL-6 and the (B) p40 subunit of IL-12/-23 peaked at 3 and 6 hours after both 30-min renal I/R injury and control. (C) In the Mcl-1^ΔMyelo bone marrow chimeric mice neutrophil deficiency was verified by FACS. There was no difference in (D) BUN, (E) pNGAL and (F, G) urinary NGAL levels normalized to urinary creatinine and to plasma NGAL in Mcl-1^ΔMyelo compared to wild type (WT) bone marrow chimeras. *, **, ***: p<0.05, 0.01, 0.001 vs. the groups indicated. (IL-6 and p40 kinetics n = 5/group/time-point; WT-control-op n = 7; WT-IR20 n = 17; Mcl-1^ΔMyelo-control-op n = 6; Mcl-1^ΔMyelo-IR20 n = 8).</p

Urinary NGAL was sensitive enough to detect 10-min ischemia.

<p>(A) Urinary NGAL normalized to urinary creatinine correlated significantly with 24-hour urinary NGAL (r = 0.9082, p<0.0001). (B) Plasma NGAL levels and (C) NGAL renal mRNA expression increased both after 20-min ischemia and control operation (# p<0.01 vs. non-op, 20- and 30-min ischemia; *# p<0.01 vs. non-op and ctrl-op; † p<0.0001 vs. non-op, 20- and 30-min ischemia, ‡ p<0.0001 vs. non-op, ctrl-op and 10-min ischemia). (D) Urinary NGAL increased significantly after 10-min ischemia, but also after control operation (* p<0.0001 vs. non-op, p<0.05 vs. 10-min ischemia, p<0.0001 vs. 20-min ischemia; *& p<0.0001 vs. non-op and p<0.05 vs. ctrl-op; & p<0.0001 vs. non-op and ctrl-op). (E) Urinary NGAL further normalized to (divided by) plasma NGAL increased significantly after ischemia-reperfusion injury, but not after control operation (¶ p<0.001 vs. non-op and ctrl-op). (F) The ratio between urinary NGAL and calculated filtrered NGAL was significantly higher only after 10-min ischemia and not after ctrl-op vs. non-op ($ p<0.001 vs. ctrl-op and non-op). (non-op: urine and plasma n = 23, kidney RNA n = 10; ctrl-op: n = 6; 10 min: n = 6; 20 min: n = 5; 30 min: plasma and kidney RNA n = 17).</p

The ability of plasma and urinary NGAL and BUN levels to discriminate between 10-min renal ischemia and control operation compared to non-operated (non-op.) group.

<p>The ability of plasma and urinary NGAL and BUN levels to discriminate between 10-min renal ischemia and control operation compared to non-operated (non-op.) group.</p

Severity of AKI after various renal ischemia times.

<p>The most sensitive parameter was (P) the tubular dilation score, which increased already after 10-min ischemia (C, H and M) compared to control-operated (ctrl-op; B, G and L) (* p<0.05 vs. non-operated (non-op) and p<0.001 vs. control-operated (ctrl-op); ** p<0.001 vs. non-op and ctrl-op). (Q) Tubular necrosis and (R) casts were present mostly after 20- (D, I and N) and 30-min (E, J and O) ischemia († p<0.05 vs. non-op, p<0.01 vs. ctrl-op, 10- and 30-min ishemia; ‡ p<0.0001 non-op, ctrl-op and 10-min ischemia, p<0.01 vs. 20-min ischemia; ¶ p<0.05 vs. non-op, p<0.01 vs. ctrl-op and 10-min ischemia, p<0.0001 vs. 30-min ischemia; *¶ p<0.0001 vs. non-op, ctrl-op, 10- and 20-min ischemia). There was no significant histologic change between non-op (A, F and K) and ctrl-op. (S) Renal function measured by blood urea nitrogen retention worsened after 20-min ischemia (# p<0.0001 vs. non-op, ctrl-op (ctrl-op) and 10-min ischemia). (non-op: n = 4; ctrl-op: n = 6; 10-min: n = 7; 20 min: n = 7; 30 min: n = 16).</p

The intensity and extent of the NGAL immunostaining increased progressively with renal ischemia time.

<p>In the non-op kidneys (A, F and K) only the cortex (A) was NGAL positive, where the intracellular punctate staining pattern intensified after ischemia-reperfusion injury (R) compared to non-op (P) and control-op (Q). After control operation (ctrl-op; B, G and L) slight staining of the outer (G) and inner stripe (L) could be also observed. However, NGAL staining score increased further after 10- (C, H and M), 20- (D, I and N) and 30-min (E, J and O) renal ischemia in the outer medulla (U and V). Without the primary antibody no nonspecific staining was visible (S). * p<0.0001 vs. non-op; † p<0.05 vs. non-op and to 20-min, and p<0.001 vs. 30-min ischemia; ‡ p<0.001 vs. non-op, and p<0.05 vs. 30-min ischemia; ¶ p<0.0001 vs. non-op, and p<0.05 vs. ctrl-op; § p<0.0001 vs. non-op, p<0.001 compared ctrl-op, and p<0.05 vs. 10-min ischemia; †* p<0.01 vs. 20-min, and p<0.001 vs. 30-min ischemia; ¶* p<0.0001 vs. non-op, and p<0.01 vs. ctrl-op. (non-op: n = 4; ctrl-op: n = 7; 10 min: n = 9; 20 min: n = 8; 30 min: n = 17).</p

The ability of plasma and urinary NGAL and BUN levels to discriminate between the severity of renal ischemia-reperfusion injury compared to control operation.

<p>The ability of plasma and urinary NGAL and BUN levels to discriminate between the severity of renal ischemia-reperfusion injury compared to control operation.</p

Study of pheonotype of the H9c2 cells derived from rat cardiac myoblasts

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Martina Kutichová Supervisor: Doc. PharmDr. Tomáš Šimůnek, Ph.D. Supervisor - specialist: Doc. MUDr. Michaela Adamcová, Ph.D. Title of diploma thesis: Study of phenotype H9c2 cells derived from rat cardiac myoblasts The cell-based in vitro investigation represents an important and frequently employed platform in current biomedical research. In experimental cardiovascular research, enzymatically dispersed neonatal or adult cardiomyocytes from various animal species are considered as a "gold standard" for such experiments. However, besides isolated primary cultures, permanent cell lines are becoming extremely popular among cardiovascular scientists. H9c2(2-1) is the first permanent cardiomyocyte-derived cell line. Although these cells are known since the mid-seventies, there is not much information about their phenotype. This study followed the results of the shotgun proteomic analysis, which identified about 1500 proteins. The aim of this thesis was to continue with obtained proteomic results with focus on detection of specific proteins by Western blot method. H9c2 cell were studied for the presence of several muscle and/or cardiac specific proteins in both non-proliferating H9c2..