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Changes in adenosine triphosphate (ATP) and peroxynitrite (ONOO - ) concentrations have been correlated in a number of diseases including ischemia-reperfusion injury and drug-induced liver injury. Herein, we report the development of a fluorescent probe ATP-LW, which enables the simultaneous detection of ONOO - and ATP. ONOO - selectively oxidizes the boronate pinacol ester of ATP-LW to afford the fluorescent 4-hydroxy-1,8-naphthalimide product NA-OH (λ ex = 450 nm, λ em = 562 nm or λ ex = 488 nm, λ em = 568 nm). In contrast, the binding of ATP to ATP-LW induces the spirolactam ring opening of rhodamine to afford a highly emissive product (λ ex = 520 nm, λ em = 587 nm). Due to the differences in emission between the ONOO - and ATP products, ATP-LW allows ONOO - levels to be monitored in the green channel (λ ex = 488 nm, λ em = 500-575 nm) and ATP concentrations in the red channel (λ ex = 514 nm, λ em = 575-650 nm). The use of ATP-LW as a combined ONOO - and ATP probe was demonstrated using hepatocytes (HL-7702 cells) in cellular imaging experiments. Treatment of HL-7702 cells with oligomycin A (an inhibitor of ATP synthase) resulted in a reduction of signal intensity in the red channel and an increase in that of the green channel as expected for a reduction in ATP concentrations. Similar fluorescence changes were seen in the presence of SIN-1 (an exogenous ONOO - donor).

Aim: To investigate the expression of matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3) and their tissue inhibitors of metalloproteinases 1 and 3 (TIMP-1 and TIMP-3) in the conjunctiva of eyes with conjunctivochalasis (CCh)., Methods: The conjunctival tissue was obtained from the CCh patients and controls, the MMPs/TIMPs expression concentration was determined by enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence staining. The expression levels of MMPs/TIMPs in the CCh fibro-blasts were determined by analyzing its concentration in the cellular supernatant that was abstracted from the in vitro cultured CCh fibroblasts., Results: MMP-1 and MMP-3 levels determined by ELISA were both significantly higher in the CCh group than that in the control group ( P =0.042, 0.022, respectively), so was the levels of TIMP-1 ( P =0.010). No significant difference in the expression of TIMP-3 in conjunctiva was found between the two groups ( P =0.298). The expression of MMP-1 and MMP-3 were both up-regulated significantly in the CCh group ( P =0.040, 0.001, respectively) on immuno-fluorescence staining. MMP-1 and MMP-3 expression in the fibroblasts were both significantly higher in the CCh group than that in the control group ( P =0.027, 0.001, respectively), while neither the TIMP-1 nor TIMP-3 expression was significantly different between the two groups ( P =0.421, 0.237, respectively)., Conclusion: The overexpression of MMP-1 and MMP-3 in conjunctival tissue and fibroblasts may play an important role in the pathogenesis and development of CCh.

Aim: To investigate the expression of nuclear transcription factor-kappa B (NF-kappaB)/IkappaB in renal tissues of hepatitis B virus-associated nephritis (HBVGN) and to study its role in the pathogenesis of HBVGN., Methods: The expression of NF-kappaB p65 and IkappaB-alpha in renal tissues was examined in biopsy specimens from 42 HBVGN patients, 20 patients with primary membranous nephropathy and 5 normal subjects by immunohistochemical staining., Results: The NF-kappaB p65 expression in glomerular and tubular of HBVGN tissues was notably higher than that in normal control tissues (P<0.05) and in primary membranous nephropathy tissues (P<0.01). The IkappaB-alpha expression in glomerular and tubular of HBVGN tissues was lower than that in primary membranous nephropathy tissues (P<0.05)., Conclusion: There might be protein degradation of IkappaB-alpha and nuclear translocation of NF-kappaB in renal tissues of HBVGN, which suggests that NF-kappaB/IkappaB signal pathway may play an important role in the pathogenesis of HBVGN.

Aim: To explore the role of continuous high-volume renal replacement therapy(HV-CRRT) in serum TNF-alpha removal., Methods: 13 panceatitis patients complicated with acute renal failure were treated by continuous venovenous hemofiltration (CVVH), and they were divided into two groups according to the substitution rate during CVVH. The substitution rate of group I (8 cases) was 2 L/h, the that of group II(5 cases) was 4 L/h. 1ml blood samples were taken from the post-filter at 0,1,2,3,6 and 24 h after CRRT, respectively. The TNF-alpha level produced from endotoxin-induced periphral blood mononuclear cells (PBMCs) was detected by ELISA. Ultrafiltrate samples were co-incubated with donor's whole blood containing ET so as to detect suppression activity of ultrafiltrate., Results: Production of TNF-alpha by ET-induced PBMCs was significantly suppressed in group I and II. During CVVH treatment, suppressed ET-induced TNF-alpha production increased variously in the first 3 h, and then decreased gradually. In contrast, the increased levels of ET-induced TNF-alpha production in group II were higher than that in group I, and maintained a higher level until 24 h after CVVH therapy. The ultrafiltrate from group I had no suppressing activity, but the ultrafiltrate from group II suppressed ET-induced TNF-alpha production, the suppression rate was (15+/-6)%., Conclusion: HV-CRRT is more effective than standard CRRT in removal of inflammatory mediators. The mediators are removed mainly by filter membrane adsorption, and maybe partly by convection. Therefore, HV-CRRT with large-pore filter membranes is an effective way in removal of inflammatory mediators.

Aim: To further explore significance of fluorescent patterns of antineutrophil cytoplasmic antibodies in renal diseases., Methods: ANCA fluorescent patterns and its target antigens in sera of 251 patients with renal diseases were detected by indirect immunofluorescence (IIF) and ELISA., Results: The positive percentage of ANCA is 20.3% (51/251), in which 3.98%(10/251) is cANCA, 11.95% (30/251) pANCA and 4.38%(11/251) aANCA., Conclusion: The simultaneous screening for ANCA and its target antigens by IIF and ELISA can increase the positive percentage of ANCA in renal diseases. This novel screening strategy is able to distinguish atypical ANCA(aANCA) from pANCA with ANA and therefore useful for differential diagnosis between vasculitis and autoimmune diseases.