Longitudinal study of a mouse model of familial porphyria cutanea tarda.

Most rodent models of porphyria cutanea tarda (PCT) share in common the administration of iron and agents that induce transcription of cytochrome P450s. Dissection of changes related to porphyrin accumulation required generation of a genetic model free from exogenous precipitants. Mice heterozygous for a null Urod mutation and homozygous for null Hfe alleles spontaneously develop major increases in hepatic and urinary porphyrins several months after weaning but the high % uroporphyrin signature of PCT is established earlier, before total hepatic and urinary porphyrins rise. Total porphyrin levels eventually plateau at higher levels in females than in males. Porphyrinogens were the dominant tetrapyrroles accumulating in hepatocytes. Hepatic Urod activity is markedly reduced but total hepatic heme content does not diminish. Microsomal heme, however, is reduced and in vitro metabolism of prototype substrates showed that some but not all cytochrome P450 activities are reduced. High hepatic levels of uroporphyrinogen are also associated with increased glutathione S-transferase activity and elevated mRNA of 2 transporters, Abcc1 and Abcc4. This murine model of familial PCT affords the opportunity to study changes in porphyrinogen and porphyrin accumulation and transport in the absence of exogenous factors that alter P450 activity and transmembrane transporters.

von Willebrand factor: evidence for variable clearance in vivo according to Y/C1584 phenotype and ABO blood group.

BACKGROUND: The plasma von Willebrand factor (VWF) level (VWF:Ag) is known to correlate with the VWF Y/C1584 variation and with ABO blood group. The ratio of the VWF propeptide (VWFpp) to VWF:Ag and the ratio of coagulation factor VIII (FVIII:C) to VWF:Ag have previously been used as indicators of VWF clearance and/or secretion. OBJECTIVES AND METHODS: To investigate the mechanism underlying the relationship between VWF phenotype and VWF:Ag, the VWFpp/VWF:Ag ratio and FVIII:C/VWF:Ag ratio were determined for plasmas of phenotype Y/C1584, Y/Y1584, blood group O and blood group A (n = 50 for each set). The blood group O plasmas comprised two sets of 25 with low and high mean VWF levels (Low-O and High-O), respectively; similarly for group A (Low-A and High-A). RESULTS AND CONCLUSIONS: The VWFpp/VWF:Ag ratio was greater than 1 (unity) for Y/C1584 plasmas and significantly higher than for Y/Y1584 plasmas; however, the FVIII:C/VWF:Ag ratio was near unity for both and was not significantly different. These results are consistent with increased clearance for Y/C1584 VWF. Similarly, the VWFpp/VWF:Ag ratio and FVIII:C/VWF:Ag ratio in combination were consistent with increased VWF clearance in blood group O compared with blood group A, and in Low-O and Low-A, respectively, compared with High-O and High-A. The data indicate that in vivo C1584 and blood group O are associated with increased VWF clearance, and that clearance contributes to differing VWF level within a given blood group.

Measurement of global haemostasis in severe haemophilia A following factor VIII infusion.

Patients with haemophilia requires different amounts of FVIII to prevent and treat bleeds. We hypothesise that this is because FVIII has variable effects on individual patients' global haemostasis. Twelve patients with severe haemophilia A were infused with 50 IU/kg FVIII and thrombin generation in platelet rich (PRP) and platelet poor plasma (PPP) and velocity of changing clot elasticity were measured preinfusion and at nine subsequent time points over 72 h. Despite a close correlation between median FVIII and median initial rate of thrombin generation (R(2) 0.94), endogenous thrombin potential (ETP; R(2) 0.94) and peak thrombin (R(2) 0.91) in PPP, there was wide inter-patient variability at each time point. There was, however, a highly predictable intra-patient relationship between FVIII level and thrombin generation. Inter-patient variability was due to both differences in FVIII levels and the variable effect FVIII had on an individuals' thrombin generation. The utility of PRP was limited because, at low-FVIII levels, only rate of thrombin generation was measurable. At low-FVIII concentrations, the rate of thrombin generation in PPP was the most useful test whilst at higher levels ETP and peak thrombin could also be used.

Oral L-arginine in patients with coronary artery disease on medical management.

BACKGROUND: Vascular nitric oxide (NO) bioavailability is reduced in patients with coronary artery disease (CAD). We investigated whether oral L-arginine, the substrate for NO synthesis, improves homeostatic functions of the vascular endothelium in patients maintained on appropriate medical therapy and thus might be useful as adjunctive therapy. METHODS AND RESULTS: Thirty CAD patients (29 men; age, 67+/-8 years) on appropriate medical management were randomly assigned to L-arginine (9 g) or placebo daily for 1 month, with crossover to the alternate therapy after 1 month off therapy, in a double-blind study. Nitrogen oxides in serum (as an index of endothelial NO release), flow-mediated brachial artery dilation (as an index of vascular NO bioactivity), and serum cell adhesion molecules (as an index of NO-regulated markers of inflammation) were measured at the end of each treatment period. L-Arginine significantly increased arginine levels in plasma (130+/-53 versus 70+/-17 micromol/L, P<0.001) compared with placebo. However, there was no effect of L-arginine on nitrogen oxides (19.3+/-7.9 versus 18. 6+/-6.7 micromol/L, P=0.546), on flow-mediated dilation of the brachial artery (11.9+/-6.3% versus 11.4+/-7.9%, P=0.742), or on the cell adhesion molecules E-selectin (47.8+/-15.2 versus 47.2+/-14.4 ng/mL, P=0.601), intercellular adhesion molecule-1 (250+/-57 versus 249+/-57 ng/mL, P=0.862), and vascular cell adhesion molecule-1 (567+/-124 versus 574+/-135 ng/mL, P=0.473). CONCLUSIONS: Oral L-arginine therapy does not improve NO bioavailability in CAD patients on appropriate medical management and thus may not benefit this group of patients.

The role of M cells in mucosal immunity.

Mucosa-associated lymphoid tissue in the respiratory and digestive tracts are covered by a specialized epithelium, the follicle-associated epithelium, which includes M cells, which are specialized for the uptake and transcytosis of macromolecules and microorganisms. Following transcytosis, antigens are released to cells of the immune system in lymphoid aggregates beneath the epithelium where antigen processing and presentation and stimulation of specific B and T lymphocytes are achieved. Circulation of the lymphoid cells enables their homing to their original, and other, mucosal sites where they exert the effector function. Such a response may be dominated by secretory immunoglobulin A release and may include cytotoxic T lymphocyte action. Binding of particles to the apical M cell membrane may be nonspecific or due to specific interaction between molecules such as integrins and lectins. Exploiting the specific binding to M cells is an aim for mucosal vaccination, for example to increase the efficiency of uptake of an oral vaccine by its conjugation to an M-cell-specific molecule. Alternatively, an M-cell-specific live vector, such as attenuated Salmonella bacteria, may be used to deliver epitopes of other organisms. Mucosal vaccination efficiency may also be enhanced by a temporary increase in the number of M cells. Therefore, investigation of the properties and ontogeny of M cells must be pursued to allow the development of better mucosal vaccines for the future.

Effects of oral L-arginine on endothelium-dependent vasodilation and markers of inflammation in healthy postmenopausal women.

OBJECTIVES: We examined whether oral administration of L-arginine, the substrate for nitric oxide (NO) synthesis, increases NO bioactivity in healthy postmenopausal women. BACKGROUND: Nitric oxide may protect arteries against atherosclerosis, as suggested by experimental studies in animals. Estrogen therapy, which has been shown to increase NO bioactivity in the vasculature of healthy postmenopausal women, is not acceptable for long-term use by many women. METHODS: In a randomized, double-blind, crossover study, 10 postmenopausal women without additional risk factors for atherosclerosis received L-arginine 9 g or placebo daily for one month, with treatment periods separated by one month. Nitric oxide levels in serum (as an index of endothelial NO release), brachial artery endothelium-dependent dilator responses to hyperemia by ultrasonography (as an index of vascular NO bioactivity) and markers of inflammation in blood that are inhibited by NO in cell culture experiments were measured at the end of each treatment period. RESULTS: L-arginine levels in plasma were increased in all women during L-arginine treatment compared with placebo (136.8 +/- 63.1 vs. 75.2 +/- 16.2 micromol/liter, p = 0.009). However, there was no change in serum nitrogen oxide levels (42.1 +/- 24.5 vs. 39.1 +/- 16.6 micromol/liter, p = 0.61), nor was there an effect of L-arginine on flow-mediated dilation during hyperemia (3.8 +/- 3.0% vs. 4.9 +/- 4.8%, p = 0.53) compared with placebo. Our study had sufficient power (beta = 0.80) to detect a true absolute treatment difference in flow-mediated brachial artery dilation of 1.7% or larger as statistically significant at alpha = 0.05. There was no effect of L-arginine on serum levels of soluble cell adhesion molecules compared with placebo: E-selectin (50.6 +/- 14.8 vs. 52.1 +/- 17.0 ng/ml, p = 0.45), intercellular adhesion molecule-1 (230 +/- 51 vs. 230 +/- 52 ng/ml, p = 0.97) and vascular cell adhesion molecule-1 (456 +/- 62 vs. 469 +/- 91 ng/ml, p = 0.53). CONCLUSIONS: Oral administration of L-arginine may not augment endothelial NO synthesis and release in postmenopausal women and is thus unlikely to be of general benefit to healthy postmenopausal women in protection from the development of atherosclerosis.

Vascular effects of estrogen and vitamin E therapies in postmenopausal women.

BACKGROUND: Estrogen and vitamin E therapies have been suggested to reduce cardiovascular risk, but comparison of the vascular effects of these therapies to determine mechanisms of potential benefit has not been performed in postmenopausal women. METHODS AND RESULTS: In a double-blind, 3-period crossover study, we randomly assigned 28 healthy postmenopausal women to conjugated equine estrogens (CE) 0. 625 mg/d, vitamin E 800 IU/d, and their combination, with measurements made before and after each 6-week treatment period. The ratio of LDL to HDL cholesterol and lipoprotein(a) decreased on therapies including CE but increased on vitamin E alone (P<0.001 and P=0.002, respectively, by ANOVA). Brachial artery flow-mediated dilation improved on all therapies (all P<0.001 versus pretreatment values) and to a similar degree (P=0.267 by ANOVA). No therapy improved the dilator response to nitroglycerin. CE lowered serum levels of cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 (all P<0.05 versus pretreatment values). Vitamin E had no significant effect on levels of these markers of inflammation (P<0. 001 by ANOVA for E-selectin). CE alone or combined with vitamin E but not vitamin E alone lowered or showed a trend for lowering plasma levels of plasminogen activator inhibitor type-1 (P=0.069 by ANOVA). CONCLUSIONS: Estrogen and vitamin E therapies similarly improved arterial endothelium-dependent vasodilator responsiveness consistent with increased nitric oxide in healthy postmenopausal women, despite divergent effects on atherogenic lipoproteins. However, only estrogen reduced markers of vascular disease.

Mechanism by which quinapril improves vascular function in coronary artery disease.

Angiotensin-converting enzyme (ACE) inhibition has been shown to improve endothelium-dependent vasodilator responsiveness, but the contribution and mechanism of enhanced nitric oxide (NO) bioactivity to this effect in patients with coronary artery disease are unknown. We investigated the effect of ACE inhibition on brachial artery dilator responsiveness to increased shear stress after forearm ischemia by ultrasonography as a bioassay for endothelial NO available to vascular smooth muscle in 9 men with coronary artery disease. Serum nitrogen oxides were measured after 3 days of nitrate-restricted diet as an index of endothelial NO release. Patients received quinapril 20 to 40 mg/day for 8 weeks. Relative to pretreatment measurements, quinapril increased flow-mediated dilation (from 2.4+/-0.4 to 10.8+/-2.2, p <0.001), with significant improvement persisting 1 week after discontinuation of therapy (6.7+/-2.5%, p <0.01). However, quinapril decreased serum nitrogen oxide levels by 19+/-17% compared with pretreatment values (from 58.2+/-19.0 to 46.0+/-13.3 micromol/L, p <0.01). Thus, ACE inhibitor therapy with quinapril selectively improves endothelium-dependent vasodilator responsiveness by increased NO bioactivity in relation to vascular smooth muscle in patients with coronary artery disease, an effect achieved at a lower rate of NO release from the endothelium. These findings suggest that ACE inhibitors may reduce angiotensin II-induced oxidant stress within the vessel wall and protect NO from oxidative inactivation. This effect may reduce endothelial NO synthesis required for vasomotor regulation.

Vascular effects of estrogen and cholesterol-lowering therapies in hypercholesterolemic postmenopausal women.

BACKGROUND: Lipoproteins affect endothelium-dependent vasomotor responsiveness. Because lipoprotein effects of estrogen and cholesterol-lowering therapies differ, we studied the vascular responses to these therapies in hypercholesterolemic postmenopausal women. METHODS AND RESULTS: We randomly assigned 28 women to conjugated equine estrogen (CE) 0.625 mg, simvastatin 10 mg, and their combination daily for 6 weeks. Compared with respective baseline values, simvastatin alone and combined with CE reduced LDL cholesterol to a greater extent than CE alone (both P<0.05). CE alone and combined with simvastatin raised HDL cholesterol and lowered lipoprotein(a) to a greater extent than simvastatin alone (all P<0.05). Flow-mediated dilation of the brachial artery (by ultrasonography) improved (all P<0.001 versus baseline values) on CE (4.0+/-2.6% to 10.2+/-3.9%), simvastatin (4.3+/-2.4% to 10.0+/-3.9%), and CE combined with simvastatin (4.6+/-2.0% to 9.8+/-2.6%), but similarly among therapies (P=0.507 by ANOVA). None of the therapies improved the dilator response to nitroglycerin (all P>/=0.184). Only therapies including CE lowered levels of plasminogen activator inhibitor type 1 and the cell adhesion molecule E-selectin (all P<0. 05 versus simvastatin). CONCLUSIONS: Although estrogen and statin therapies have differing effects on lipoprotein levels, specific improvement in endothelium-dependent vasodilator responsiveness is similar. However, only therapies including estrogen improved markers of fibrinolysis and vascular inflammation. Thus, estrogen therapy appears to have unique properties that may benefit the vasculature of hypercholesterolemic postmenopausal women, even if they are already on cholesterol-lowering therapy.

Protection against measles virus-induced encephalitis by antibodies from mice immunized intranasally with a synthetic peptide immunogen.

Balb/c mice were immunized intranasally (i.n.) with a chimeric synthetic peptide containing two copies of a T- and one copy of a B-cell epitope (TTB) from measles virus (MV) fusion protein, plus cholera toxin B (CTB) adjuvant. The antibodies induced cross-reacted with, and neutralized MV and on passive transfer, protected mice against encephalitis induced by neuroadapated MV. Immunization with TTB alone induced antibodies which increased survival but not significantly compared to controls. Furthermore, i.n. immunization with TTB plus CTB induced TTB-specific IgA antibodies in saliva and nasal washes. Co-administration of CTB increased the affinity of antibodies to the B-cell epitope of TTB and caused a relative increase in the level of anti-peptide antibodies of the IgG2a subclass and the overall titre of IgG antibodies. These results indicate the potential of the i.n. route for immunization with synthetic peptide immunogens for induction of both local and systemic anti-peptide antibody responses.

Induction of systemic immune responses to measles virus synthetic peptides administered intranasally.

A systemic antibody response was induced when a chimeric peptide containing two copies of a promiscuous T-cell epitope and one copy of a B-cell epitope (TTB) from the fusion protein of measles virus (MV) was administered to mice intranasally without adjuvant. A higher antibody titre was produced when the peptide was administered intranasally with cholera toxin B subunit (CTB) as an adjuvant and these antibodies crossreacted with the MV. Furthermore, splenocytes from intranasally immunized mice proliferated in vitro in the presence of the TTB peptide. The immune response following intranasal immunization with the peptide was influenced by the MHC haplotype of the strain of mice used. Thus CBA and BALB/c mice were high responders whereas C57BL/6 mice were low responders. Although peptide administered intranasally with CTB to CBA mice induced an immune response, no significant protection was observed against intra-cranial challenge with canine distemper virus which is antigenically related to MV.

Progestin stimulation of thymidine kinase in the human breast cancer cell line T47D.

Our laboratory has previously reported that progestins stimulate growth of the human breast cancer cell line T47D. In an attempt to probe further into this stimulation, we are investigating progestin effects on thymidine kinase (EC 2.7.1.21), an enzyme known to be involved in growth regulation. This report relates our finding that progestins stimulate thymidine kinase activity, at physiological progestin concentrations, in a dose-responsive manner. Estradiol-17 beta also stimulates, but testosterone, hydrocortisone and aldosterone do not. The antiprogestin RU486 inhibits progestin stimulation, but also stimulates on its own. Maximal by 24 h, the progestin stimulation then falls off with time. Experiments with actinomycin D and cycloheximide suggest that the thymidine kinase stimulation depends on new RNA and protein synthesis. These data shed further light on progestin stimulation of the growth of human breast cancer. To our knowledge, this is the first report of progestin stimulation of thymidine kinase in human breast cancer cells.