Protective effect of EDTA preadministration on renal ischemia.

BACKGROUND: Chelation therapy with sodium edetate (EDTA) improved renal function and slowed the progression of renal insufficiency in patients subjected to lead intoxication. This study was performed to identify the underlying mechanism of the ability of EDTA treatment to protect kidneys from damage. METHODS: The effects of EDTA administration were studied in a rat model of acute renal failure induced by 60 minutes ischemia followed or not by 60 minutes reperfusion. Renal ischemic damage was evaluated by histological studies and by functional studies, namely serum creatinine and blood urea nitrogen levels. Treatment with EDTA was performed 30 minutes before the induction of ischemia. Polymorphonuclear cell (PMN) adhesion capability, plasmatic nitric oxide (NO) levels and endothelial NO synthase (eNOS) renal expression were studied as well as the EDTA protection from the TNFalpha-induced vascular leakage in the kidneys. Data was compared by two-way analysis of variance followed by a post hoc test. RESULTS: EDTA administration resulted in the preservation of both functional and histological parameters of rat kidneys. PMN obtained from peripheral blood of EDTA-treated ischemized rats, displayed a significant reduction in the expression of the adhesion molecule Mac-1 with respect to controls. NO was significantly increased by EDTA administration and eNOS expression was higher and more diffuse in kidneys of rats treated with EDTA than in the controls. Finally, EDTA administration was able to prevent in vivo the TNFalpha-induced vascular leakage in the kidneys. CONCLUSION: This data provides evidence that EDTA treatment is able to protect rat kidneys from ischemic damage possibly through the stimulation of NO production.

Baicalin is a major component of PC-SPES which inhibits the proliferation of human cancer cells via apoptosis and cell cycle arrest.

BACKGROUND: PC-SPES is an eight-herb mixture that was shown to have activity against prostate cancer. Recently, we isolated a major component (6% of the total ethanolic extract) known as baicalin from PC-SPES by high performance liquid chromatography (HPLC). METHODS: Baicalin was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of various cancer types (PC-3, DU145, LNCaP prostate cancer cell lines, MCF-7 breast cancer cell line, HL-60 myeloblastic leukemia cell line, and NB4 promyelocytic leukemia cell line). The ability of baicalin to induce apoptosis of cancer cells was examined by both staining with Annexin V and detection of cleavage of Poly (ADP-ribose) polymerase (PARP)(3). Western blot analysis examined the effect of baicalin on levels of p21(waf1) and p27(kip1) in those cells. Futhermore, induction of differentiation in HL-60 cells was measured by expression of CD11b. RESULTS: Baicalin inhibited the clonal proliferation of LNCaP and PC3 prostate cancer cell lines, and the HL-60 and NB4 myeloblastic/promyelocytic leukemia cell lines with a 50% inhibition (ED(50)) that ranged between 6.4 x 10(-6) to 12 x 10(-6) mol/L. Cell cycle analysis showed that baicalin (2 x 10(-5) mol/L, 4 days) caused a G(0)/G(1) and G(2)/M accumulation of LNCaP and HL-60 cells, respectively. Concomitantly, differentiation and apoptosis were induced in HL-60 cells, as measured by expression of CD11b antigen, staining with annexin V, and detection of cleavage of PARP. Moreover, baicalin enhanced the expression of the cyclin-dependent kinase inhibitor, p27(kip1) in LNCaP and HL-60 cells. CONCLUSIONS: Baicalin inhibited the proliferation of cancer cells via apoptosis and cell cycle arrest, in which p27(kip1) may play a role. Baicalin may be a novel, adjunctive therapy for selected malignancies including prostate cancer.

Combined defect in membrane expression and activation of platelet GPIIb--IIIa complex without primary sequence abnormalities in myeloproliferative disease.

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.

Nitric oxide-mediated immunosuppression following murine Echinococcus multilocularis infection.

In some parasitic infections immunosuppression is a prominent characteristic of the host-parasite interplay. We have used a murine alveolar echinococcosis (AE) model in susceptible C57BL/6 mice to document a suppressed splenocyte proliferative response to concanavalin A (Con A) at the early (1-month) stage and to Echinococcus multilocularis-crude antigen (Emc-antigen) at the late (4-6-month) stage of chronic infection. Despite proliferative suppression, splenic cytokine production [interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma)] in response to Con A or Emc-antigen stimulation was not suppressed at 1 month postinfection (p.i.). Infection resulted in a strong Mac-1+ cell infiltration of the peritoneal cavity and spleen. Peritoneal cells (PEC) from mice infected at the 1-month stage were rich in macrophages and expressed significantly higher levels of transcripts for the inflammatory cytokine IL-1beta and for tumour necrosis factor-alpha and inducible nitric oxide synthase (iNOS), when compared with PEC from non-infected control mice. Conversely, the IL-10 transcript level remained low and did not change during infection. Spleen cells supplemented with PEC from infected mice induced a marked increase in the levels of nitrite in response to Con A and Emc-antigen stimulation, and also a complete suppression of splenic proliferation. The spleen cells from late-stage infected mice expressed only background levels of IL-10 but greatly increased levels of iNOS, when compared with normal spleen cells. This observation correlated with the immunosuppression demonstrated at the late stage of murine AE. Furthermore, the suppressed splenic proliferative responses observed at the early and late stage were reversed to a large extent by the addition of NG-monomethyl-l-arginine and partially by anti-IFN-gamma. Thus, our results demonstrated that the immunosuppression observed in chronic AE was not primarily dependent on IL-10 but rather on nitric oxide production by macrophages from infected animals.

Delayed administration of low-dose NPC18915 ameliorates lung ischemia-reperfusion injury.

BACKGROUND: NPC18915, a member of new antiinflammatory agent called nactins (neutrophil activation inhibitors), has been shown to reduce reperfusion injury in rat lung transplantation at high dosage. In vitro studies have demonstrated effectiveness of this compound even at low dosage. We hypothesized that this compound ameliorates lung ischemia reperfusion injury even at low dosage levels if administration is optimally timed. The aim of this study was to determine the efficacy and the best timing for administration of low-dose NPC18915. METHODS: Forty syngeneic rat left lung transplantations were performed. All isografts were flushed with low-potassium dextran-1% glucose solution 20 ml and preserved for 18 hours at 4 degrees C. Animals were divided into four groups. Group I animals (n = 10) served as control subjects. In groups II (n = 10), III (n = 10), and IV (n = 10), NPC18915 (0.04 mg) was added to the flush solution and was administered intravenously (0.4 mg/kg) immediately before reperfusion (group II) and 60 minutes (group III) and 120 minutes (group IV) after reperfusion. Pulmonary function was assessed 24 hours after reperfusion. RESULTS: In group III, oxygenation improved in comparison to group I (247.2 +/- 59.8 versus 76.6 +/- 16.0 mm Hg, p < 0.002). Wet-to-dry weight ratio and graft myeloperoxidase activity were significantly improved (group III versus group I, 6.02 +/- 0.21 versus 7.19 +/- 0.41, p = 0.013) (group III versus group I, 0.093 +/- 0.019 versus 0.207 +/- 0.023 delta optical density/min/mg, p < 0.002). There were no significant differences in CD11b expression. CONCLUSION: These data suggest that delayed administration of NPC18915, 60 minutes after reperfusion, dramatically improves pulmonary graft function.

Enhancement of peritoneal leukocyte function by granulocyte colony-stimulating factor in rats with abdominal sepsis.

OBJECTIVE: To investigate the therapeutic effects of granulocyte colony-stimulating factor (G-CSF) on the functional activities of circulating and peritoneal neutrophils during intra-abdominal sepsis. DESIGN: Placebo, controlled study, using a rat model of intra-abdominal sepsis. SETTING: Animal research facility. SUBJECTS: Male specific pathogen-free Sprague-Dawley rats. INTERVENTIONS: Abdominal sepsis was produced in rats by cecal ligation and puncture. The control animals received a sham operation. G-CSF (subcutaneous injection at 50 microg/kg) or vehicle (100 microL of 5% dextrose) treatment was initiated at 1 hr after cecal ligation and puncture or sham operation and repeated at 12-hr intervals thereafter. MEASUREMENTS AND MAIN RESULTS: Six hours after cecal ligation and puncture, CD11b/c and CD18 expression on circulating neutrophils was significantly up-regulated when compared with those in the sham operated control animals. Peritoneal neutrophils exhibited a further up-regulation of these adhesion molecules than did the circulating neutrophils. A sustained up-regulation of CD11b/c and CD18 was found in peritoneal neutrophils even at 24 hrs after cecal ligation and puncture. G-CSF treatment increased CD11b/c expression on circulating neutrophils in 6-hr sham-operated rats, but did not further up-regulate CD11b/c or CD18 expression on circulating or peritoneal neutrophils in cecal ligation and puncture rats. Phagocytic activities of circulating neutrophils assessed by uptake of fluorescent latex microspheres were lower in 24-hr cecal ligation and puncture rats when compared with the sham-operated controls. G-CSF treatment prevented this inhibition. Furthermore, G-CSF enhanced the phagocytic activities of peritoneal neutrophils in both 6- and 24-hr cecal ligation and puncture rats when compared with those of the vehicle-treated animals. Spontaneous hydrogen peroxide generation by circulating neutrophils was increased in 6-hr cecal ligation and puncture rats, but not in 24-hr cecal ligation and puncture rats. Peritoneal neutrophils exhibited an inhibition of phorbol myristate acetate-stimulated hydrogen peroxide generation. G-CSF treatment did not up-regulate neutrophil hydrogen peroxide generation. CONCLUSIONS: Circulating and peritoneal neutrophils exhibit marked polymorphism in their functional activities during the host response to abdominal sepsis. G-CSF treatment significantly enhanced the phagocytic function of both circulating and peritoneal neutrophils which may be one mechanism underlying its protective effect in abdominal sepsis.

Effects of growth hormone and insulin-like growth factor I on opsonin receptor expression on local and systemic phagocytes in a lethal peritonitis model.

OBJECTIVE: To investigate the effects of pretreatment with growth hormone (GH) and insulin-like growth factor I (IGF-I) on phagocyte exudation and bacterial clearance, focusing on CD11b and CD32/CD16 expression on local and systemic phagocytes, in a lethal peritonitis model. DESIGN: Prospective randomized experimental study. SETTING: Research laboratory in a university hospital. SUBJECTS: Balb/c mice (n = 21). INTERVENTIONS: Mice were challenged intraperitoneally with 1 x 10(8) Escherichia coli, after 6 days of pretreatment with saline (control), GH (4.8 mg/kg/day), or IGF-I (24 mg/kg/day). Samples were harvested at 4 hrs after the challenge. MEASUREMENTS AND MAIN RESULTS: Viable bacterial counts in peritoneal lavage fluid (PLF) and blood were determined. Peritoneal exudative cells and peripheral blood leukocytes were counted and analyzed for receptor expressions by flow cytometry. GH reduced viable bacterial counts in PLF, as compared with the saline control. GH (three-fold) and IGF-I (two-fold) increased the number of peritoneal exudative neutrophils (PENs), as compared with the saline control. The number of PENs showed an inverse correlation with PLF viable bacterial counts. By contrast, there were no differences in peripheral blood neutrophil (PN) counts among the three groups, nor was there a correlation between PN and PEN counts. CD11b expression was greater on PENs than on PNs in all three groups. CD11b expression on PNs did not differ among the three groups. However, GH increased CD11b expression on PENs, as compared with saline and IGF-I, and this expression showed a positive correlation with PEN numbers and an inverse correlation with PLF viable bacterial counts. CD11b expression on peritoneal macrophages and peripheral blood monocytes did not differ among the three groups. There were no differences in phagocyte CD32/CD16 expression among the three groups. CONCLUSIONS: GH pretreatment enhanced CD11b expression on PENs, but not PNs, possibly in association with enhanced neutrophil recruitment, phagocytosis, and bacterial elimination by PENs, without activation of PNs. GH prophylaxis may be useful for reducing the frequency rate and severity of septic complications, via modulation of CD11b expression on local and systemic neutrophils.

Induction of macrophagic and granulocytic differentiation of murine bone marrow progenitor cells by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was recently shown to promote maturation of 5-fluorouracil (5FU)-treated bone marrow cells by up-regulating macrophage-colony stimulating factor (M-CSF) receptors in the presence of interleukin 1 alpha (IL-1 alpha). In order to reveal how 1,25(OH)2D3 interacts with colony-stimulating factors and regulates the differentiation of bone marrow progenitor cell populations, in the present study, natural bone marrow cells were isolated from untreated mice and used in alpha-minimum essential medium supplemented with 20% heat-inactivated horse serum without added appropriate cytokines. Under the conditions, cells spontaneously differentiated gradually with days of culture, as assessed by expression of macrophage differentiation antigens such as Mac-1 (CD11b) and F4/80. Both M-CSF and granulocyte macrophage-colony stimulating factor (GM-CSF) induced only Mac-1 antigen expression. Simultaneous treatment with M-CSF and 1,25(OH)2D3 enhanced the M-CSF's effect on expression of both antigens, although 1,25(OH)2D3 per se has no effect on the expression for up to 11 days. In addition, successive treatment with 1,25(OH)2D3 and M-CSF or GM-CSF dramatically enhanced expression of both antigens or Mac-1 antigen, respectively. Similarly, both simultaneous and successive treatment with 1,25(OH)2D3 and M-CSF significantly enhanced phagocytic activity and H2O2 production, whereas successive treatment with 1,25(OH)2D3 and GM-CSF significantly enhanced only phagocytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)