Chemoprevention assessment, genotoxicity and cytotoxicity of flavonoids from Inga laurina leaves (FABACEAE).

This study aimed to identify and evaluate the cytotoxicity, genotoxicity, antigenotoxicity and chemoprevention assessment of flavonoids myricetin-3-O-(2″-O-galloyl)-α-rhamnopyranoside and myricetin-3-rhamnoside from Inga laurina leaves extract. The Quinone reductase induction as a biomarker for cancer chemoprevention was evaluated in murine hepatocellular carcinoma, the cytotoxicity was evaluated by sulforhonamide B assay and genotoxicity was evaluated by comet assay using HepG2 cell line. The results demonstrated that the flavonoids didn´t show cytotoxicity in HepG2 cells. In the chemoprevention evaluations were not able to promote the induction of Quinone Reductase and also no genotoxic effect was observed by evaluation of the comet assay in none of the concentrations tested. In the antigenotoxicity test, all compounds had a protective effect against damage induced by hydrogen peroxide and were repaired against damage. Although none of the flavonoids were capable of inducing the enzyme Quinone Reductase at the concentrations tested, the antigenotoxicity results showed a powerful chemoprotective action.

Genotoxicity, cytotoxicity and chemical profile from Inga laurina (Fabaceae).

This study aimed to evaluate the cytotoxicity and genotoxicity from Inga laurina leaves extracts and fractions and obtain their chemical profile. The chemical profile of the crude extract from I. laurina leaves and its fractions was investigated through 1H NMR, RP-HPLC-PDA by co-injection with authentic standards and HPLC-MS. The quinone reductase induction as a biomarker for cancer chemoprevention was evaluated in murine hepatocellular carcinoma line, whereas the cytotoxicity was evaluated by sulforhodamine B assay (SRB) using HepG2 cell line and genotoxicity was evaluated by comet assay. The phytochemical analysis of the leaves crude extract and its fractions showed the presence of 2-hydroxyethyl-dodecanoate and the phenolic compounds: gallic acid, methyl gallate, p-coumaric acid, cinnamic acid, myricetin-3-O-(2″-O-galoyl)-α-rhamnopyranoside, proanthocyanidin A-2 and myricetrin. All the fractions tested were not considered cytotoxic against the selected human cancer cell lines, they did not cause genotoxic in some concentrations damage and induced the enzyme quinone reductase.

Favourable response to therapy with the anti-CD20 monoclonal antibody rituximab in primary chronic cold agglutinin disease.

The 'primary' form of chronic cold agglutinin disease is a clonal B-cell lymphoproliferative disorder that is notoriously difficult to treat with drugs, including corticosteroids, alkylating agents, alpha-interferon and purine analogues. We performed a small, open, uncontrolled, prospective study to evaluate the effect of therapy with the monoclonal anti-CD20 antibody rituximab. Six patients with clonal CD20(+)kappa(+) B-cell proliferation received seven courses of rituximab 375 mg/m(2), d 1, 8, 15, and 22. One patient achieved a complete response. Four partial responses were observed, including a response to re-treatment in one patient. Two patients were categorized as non-responders. Haemoglobin levels increased by a median of 4.1 g/dl in the total group and 4.7 g/dl in the responders, who also experienced a substantial improvement of clinical symptoms. The treatment was well tolerated. We discuss the effect of rituximab therapy compared with other treatment options, and try to explain why two individual patients did not respond. Despite the small numbers, the results are very encouraging. Further studies of rituximab therapy for chronic cold agglutinin disease are warranted.

Recurrent venous thromboembolism after deep vein thrombosis: incidence and risk factors.

BACKGROUND: The recurrence rate after deep vein thrombosis (DVT) is high and the risk factors for recurrent thromboembolic events have only been investigated on a small scale. OBJECTIVES: To estimate the cumulative incidence of recurrent venous thromboembolic events after a first or a second DVT and to identify possible risk factors for recurrent venous thromboembolism. METHODS: We prospectively followed up 738 consecutive patients with an objectively verified symptomatic DVT for 3.7 to 8.8 years. Medical records and death certificates for all patients were reviewed during follow-up and recurrent DVT and pulmonary embolism were registered. RESULTS: The 5-year cumulative incidence of recurrent venous thromboembolic events was 21.5% (95% confidence interval [CI], 17.7%-25.4%) after a first DVT and 27.9% (95% CI, 19.7%-36.1%) after a second DVT. The 5-year cumulative incidence of fatal pulmonary embolism was 2.6% (95% CI, 1.1%-4.1%) after a first DVT. Proximal DVT (relative risk [RR], 2.40; 95% CI, 1.48-3.88; P<.001), cancer (RR, 1.97; 95% CI, 1.20-3.23; P<.001), and history of a venous thromboembolism (RR, 1.71; 95% CI, 1.16-2.52; P<.01) predicted an independently increased risk of recurrent events in multivariate survival analysis. Postoperative DVT (RR, 0.27; 95% CI, 0.13-0.55; P<.001) and a long duration of oral anticoagulation therapy (RR, 0.95; 95% CI, 0.92-0.98; P<.01) involved a smaller risk of recurrent events. Sex, age, initial antithrombotic therapy, or immobilization did not affect the risk of a recurrent event. CONCLUSIONS: The recurrence rate after a symptomatic DVT is high. Patients with proximal DVT, diagnosed cancer, short duration of oral anticoagulation therapy, or a history of thromboembolic events had a higher risk of recurrent events, while patients with postoperative DVT had a lower recurrence rate. This knowledge could help identify patients who might benefit most from prolonged prophylactic treatment in various risk situations.

Endotoxin is angiogenic.

The formation of new blood vessels, angiogenesis, is an important event in inflammation, wound healing and tumour growth. Mediators produced by various cells when exposed to endotoxin include cytokines (tumour necrosis factor, interleukins 1 and 6, and basic fibroblast growth factor) which, it has been suggested, stimulate angiogenesis. The angiogenic effect of endotoxin (lipopolysaccharide, LPS) was studied in rats using the quantitative mesenteric window assay. Adult rats were injected intraperitoneally with Escherichia coli LPS (5 pg/ml-20,000 ng/ml) twice daily for 4.5 consecutive days and were sacrificed 14 days after the start of this treatment. An angiogenic response was observed at concentrations of > 2 ng/ml in a dose-dependent manner. No inflammatory cellular exudate was seen in the test tissue at the time of angiogenesis analysis. Suppressed body-weight gain, a marker of the systemic effect of LPS in the rat, was significant only at the highest dose tested. The data suggest that endotoxin-mediated neovascularization could be a component of inflammation and wound healing.

Mast-cell histamine is angiogenic through receptors for histamine1 and histamine2.

The activation of mast-cells in situ induces angiogenesis in normally vascularized, adult mammalian tissue. Since the secreting mast-cell characteristically releases histamine, we studied the possible role of histamine in the outcome of mast-cell mediated angiogenesis using the rat mesenteric window assay. One H1-receptor antagonist, brompheniramine maleate (BPA), and one H2-receptor antagonist, metiamide, were separately administered systemically (s.c.) at non-toxic doses during the period of angiogenesis induction. Angiogenesis was effected by i.p. injections of the mast-cell secretagogue compound 48/80 for 5 consecutive days. The animals were killed 14 days after the start of the i.p. and s.c. treatment, close to the middle of the expanding angiogenic phase of the angiogenic reaction studied. Angiogenesis was quantified in terms of (a) the number of vessel profiles per unit tissue length (No/UL), which reflects mainly the degree of branching and/or tortuosity, (b) the relative vascularized area (VA), which is a measure of spatial extension, and (c) the vascular density (VD), a measure of vessel density per unit area of vascularized tissue. Whereas BPA significantly suppressed No/UL, metiamide significantly reduced No/UL and VD in statistical terms suggesting that endogenous mast-cell histamine is angiogenic through both H1- and H2-receptors. This appears to be the first paper to report that the occupancy of H2-receptors is angiogenic.

Recurrent venous thrombosis during warfarin treatment related to acquired protein S deficiency.

Failure of warfarin to prevent new thrombotic processes was observed in three patients with very low free protein S concentrations and high C4b-binding protein (C4bBP) concentrations, and in one patient with hereditary protein S deficiency. We suggest that an increase in C4bBP reduces the free Protein S level, and warfarin treatment causes an additional decrease of free protein S. The four patients presented indicate that such reductions are of clinical importance. Heparin seems preferable as an anticoagulant in this situation, as warfarin given alone is ineffective, or may even be harmful. In a group of pancreatic cancer patients with advanced disease, subnormal mean free protein S was found, whereas mean total protein S concentration, and mean C4bBP concentrations were significantly higher (p less than 0.01) than in healthy controls. These findings indicate that an increase in C4bBP may induce free protein S deficiency contributing to the increased thrombotic tendency in this group of patients. The correlation between free protein S and C4bBP was 0.11, (n.s.), between total protein S and C4bBP 0.73 (p less than 0.0001).

Heparin enhances angiogenesis by a systemic mode of action.

A systemically-administered standard sodium heparin, but not an oligosaccharide fraction derived from the heparin, significantly potentiated angiogenesis induced by saline in normal rats, as assessed by the quantitative mesenteric window angiogenesis assay. This is the first unambiguous evidence that any single specific mast-cell product can potentiate angiogenesis in normally vascularized mammalian tissue. Whether systemic treatment with a heparin-like substance may be useful for stimulating neoangiogenic formation of collaterals in situations of relative microvascular insufficiency, such as coronary collaterals in patients suffering from ischaemic heart disease, is briefly discussed.

Increased angiogenesis in diabetes.

Rats with streptozotocin-induced diabetes mellitus showed a 3.4-4.5 times increased angiogenic response following mast-cell activation in situ as compared with age-matched normal controls. The test tissue used was the mesenteric window, which we have previously exploited as a quantitative angiogenesis assay. In the present study two independent techniques for quantifying the angiogenic response showed essentially the same result. The finding of a pathologically increased angiogenic reaction in the diabetic animals is noteworthy since some of the most harmful complications of diabetes in man relate to proliferative vascular lesions.

Quantitative angiogenesis in spreads of intact rat mesenteric windows.

By introducing a new mode of the recently described mesenteric-window angiogenesis assay (K. Norrby, A. Jakobsson, and J. Sörbo, 1986, Virchows Arch. B. Cell Pathol. 52, 195-206) we measured the entire vascular tree in terms of the vascularized area and the vascular density in spreads of intact mesenteric windows in rats receiving a terminal iv infusion of an ink-gelatin solution to visualize the vessels. Adult and prepubescent rats given ip injections of the mast-cell secretagogue Compound 48/80 or the saline vehicle, as well as untreated litter mates, were used. Following mast-cell activation, both prepubescent and adult rats showed a significant angiogenesis taken as the vascularized area and the vascular density in spreads compared with those of the vehicle-treated animals. In fact the 48/80-treatment increased the vasculature 100-fold compared with the untreated controls. The finding of experimentally induced angiogenesis occurring in rats of all ages should make the assay useful in a wide range of angiogenesis experiments.

Protamine and mast-cell-mediated angiogenesis in the rat.

Different doses of protamine sulphate (PS) given s.c. (at 12-h intervals) were tested for signs of non-specific toxicity measured as effect on body weight and small-gut proliferation as well as on mast-cell secretion and mast-cell-mediated mitogenesis in the mesenteric windows following i.p. injection of Compound 48/80, a potent mast cell secretagogue, in normal rats. In a non-toxic dose range, the effect of PS on mast-cell-mediated angiogenesis, effected by 48/80, was quantified as the number of vessels per mm of mesenteric window in histological sections at x 400. No intelligible dose-effect relationship was discernible between the dose of PS given and the effect on angiogenesis. Only in a tight interval, at 40 mg PS/kg but not at 20 or 60 mg PS/kg, was the angiogenesis statistically significantly suppressed. Hence, it was concluded that PS can be angiostatic but does not exert a more general angiostatic effect in the autogenous systems used.

On mast-cell-mediated angiogenesis in the rat mesenteric-window assay.

Testosterone propionate injected s.c. significantly reduced the mast-cell mediated angiogenic response occurring in prepubescent rats, thereby indicating that this angiogenic reaction can be influenced by hormonal stimuli. Since the newly-formed vessels are very small, one could expect that microscopic examination would be needed to quantify the vasculature. When studying the discernment of vessels at optical magnifications of between x 100 and x 1000 in sections of methacrylate-embedded mesenteric window, we found that the number of discernible vessels increased dramatically as the magnification increased. This not only underlines the genuine microvascular character of the reaction but also demonstrates the need for high-power magnification when truly quantifying an angiogenic reaction by optical means.

Mast-cell secretion and angiogenesis, a quantitative study in rats and mice.

The activation of the autogenous mast cells (MCs) in situ in intact mesenterial windows was elicited by the intraperitoneal injection of the MC secretagogue Compound 48/80 over a period of 1, 3 and 5 days in Sprague-Dawley rats and in C57 BL/6 and CBA/Ca mice. As a probe of MC secretion, the release of histamine was quantified fluorometrically at predetermined intervals during the treatment. Fourteen days after the start of the treatment, the angiogenic response was quantified histologically as the number of vessel profiles per unit length of mesenteric window. Both the MC-activating and the angiogenic effect of the 48/80-treatment was greater in the rats than in the mice. The occurrence of MC-mediated angiogenesis in the mouse is demonstrated here for the first time. In the rat, 48/80-induced MC mediated angiogenesis increased in a distinctly dose-dependent manner. Two daily doses of 48/80 was the most efficient angiogenic protocol tested; a single day's treatment increased the number of vessels almost fivefold. The remarkable potency of the angiogenic reaction following MC secretion supports our previous notion that MC-mediated angiogenesis may have therapeutic implications in poorly vascularized tissues.

Age-dependent mast-cell-mediated angiogenesis.

In maturing male rats of approximately 5.5,10.5, and 15 weeks of age (groups I, II, and III), the mast-cell-mediated angiogenesis in the mesenterial windows was quantitatively assessed on days 14,21,28, and 35 after the start of intraperitoneal treatment using the mast-cell secretagogue 48/80, and using the saline vehicle. The number of blood vessels per unit length of the central part of the mesenterial window was virtually unaffected by age as well as by the saline treatment. The number of vessels at the mesenterial-window circumference was, however, increased in the older, untreated animals. The postpubescent animals in groups II and III showed a marked mast-cell-mediated angiogenic response lasting until day 28. The relative angiogenic response over the period of 14-28 days clearly increased with advancing age. The highest mean value in group II was about 8 times (p less than 0.001), whereas the highest mean value observed in group III was about 24 times (p less than 0.001) greater than in corresponding saline-treated controls. In contrast, no statistically significant mast-cell-mediated angiogenesis appeared in the prepubescent rats of group I.

Mast-cell-mediated angiogenesis: a novel experimental model using the rat mesentery.

The angiogenic effect of autogenous secreting mast cells (MCs) was studied using a novel experimental approach. The virtually avascular membranous rat mesentery was used as test tissue. The activation of MCs was elicited by repeated intraperitoneal injections of the MC-secretagogue compound 48/80, which per se appears inert from the proliferogenic and angiogenic point of view. Angiogenesis was quantitated histologically and expressed the number of vessels/unit length of mesentery. The smallest vessels recognized had a luminal area of approximately 7-8 microns 2 (corresponding to a circular diameter of 3.0-3.2 microns). Seven to ten days after MC-activation ended, the number of blood vessels had increased 7- to 6-fold. A retrogressive reaction occurred between days 21 and 38 after treatment, when the number of vessels had essentially normalized, as compared to vehicle-treated controls. The present study, introducing the membranous mesentery as a model for quantitative angiogenetic studies, provides evidence that MCs can induce angiogenesis, which is new. The possible therapeutic implication of this finding is noteworthy.