Evidence for a role of regulatory T cells in mediating the atheroprotective effect of apolipoprotein B peptide vaccine.

OBJECTIVES: Autoimmune responses against oxidized low-density lipoprotein are considered to play an important pro-inflammatory role in atherosclerosis and to promote disease progression. T-regulatory cells (Tregs) are immunosuppressive cells that have an important part in maintaining self-tolerance and protection against autoimmunity. We investigated whether aBp210, a prototype atherosclerosis vaccine based on a peptide sequence derived from apolipoprotein B, inhibits atherosclerosis through the activation of Tregs. DESIGN: Six-week-old Apoe(-/-) mice were immunized with aBp210 and received booster immunizations 3 and 5 weeks later, as well as 1 week before being killed at 25 weeks of age. RESULTS: At 12 weeks, immunized mice had increased expression of the Treg marker CD25 on circulating CD4 cells, and concanavalin A (Con A)-induced interferon-γ, interleukin (IL)-4, and IL-10 release from splenocytes was markedly depressed. At 25 weeks, there was a fivefold expansion of splenic CD4+ CD25+ Foxp3 Tregs, a 65% decrease in Con A-induced splenic T-cell proliferation and a 37% reduction in the development of atherosclerosis in immunized mice. Administration of blocking antibodies against CD25 neutralized aBp210-induced Treg activation as well as the reduction of atherosclerosis. CONCLUSIONS: The present findings demonstrate that immunization of Apoe(-/-) mice with the apolipoprotein B peptide vaccine aBp210 is associated with activation of Tregs. Administration of antibodies against CD25 results in depletion of Tregs and blocking of the atheroprotective effect of the vaccine. Modulation in atherosclerosis-related autoimmunity by antigen-specific activation of Tregs represents a novel approach for treatment of atherosclerosis.

Treatment with apo B peptide vaccines inhibits atherosclerosis in human apo B-100 transgenic mice without inducing an increase in peptide-specific antibodies.

OBJECTIVES: Autoantibodies to apolipoprotein (apo) B-100 peptides are present in human plasma and have been shown to be associated with decreased cardiovascular risk. The present study aimed to determine if apo B-100 peptide vaccines are atheroprotective in mice expressing human apo B-100 and if the effectiveness of the vaccines is influenced by the level of pre-existing peptide-specific autoantibodies. DESIGN: LDL receptor(-/-)/human apo B-100 transgenic mice were immunized with native human apo B-100 peptides p45 or p210 at 6, 9 and 11 weeks and the extent of atherosclerosis determined by en face Oil Red O staining of the aorta at 25 weeks. Autoantibody levels were determined by enzyme-linked immunosorbent assay, and RNA expression in the spleen was assessed by real time PCR. RESULTS: Control mice had high levels of autoantibodies against p210 but only low levels against p45. Immunization with native p45 and p210 reduced atherosclerosis by 66% (P < 0.02) and 59% (P = 0.06), respectively. The atheroprotective effect of apo B peptide immunization occurred in the absence of an increase in peptide-specific IgG, but was associated with an increase in IgM recognizing native and copper-oxidized LDL. CONCLUSIONS: Immunization with apo B peptide-based vaccines inhibits atherosclerosis in mice expressing human apo B-100 suggesting that they can interact with their target as expressed in humans. The protective effect is independent of the pre-existing level of apo B peptide autoantibodies and can occur without activating an increase in peptide-specific antibodies suggesting that atheroprotection can be mediated by cellular immune responses.

Sustained elevated plasma aprotinin concentration in mice following intraperitoneal injections of w/o emulsions incorporating aprotinin.

This study was initiated to test the feasibility of w/o emulsions as a sustained release system for aprotinin following intraperitoneal injection in mice. The emulsion was well tolerated in mice and sustained release was observed over a period of 96 h. The time for maximum plasma concentration of aprotinin was 10 min and 12 h after injection of a control solution and the emulsion dosage form, respectively. Furthermore, the hemolytic activity of the emulsion constituents was low indicating a low acute toxicological potential of the emulsion. The present study also showed that the lipolytic activity in peritoneal exudate from mice is important for the clearance of oily vehicles from the peritoneal cavity with lipolytic rate constants ranging from 50 to 130 nmol free fatty acid released/min/mg exudate protein at 37 degrees C, pH 8.5. It was concluded that the w/o emulsion was well suited to provide sustained elevated plasma aprotinin concentrations in mice.

The effect of controlled osmotic stress on release and swelling properties of a water-in-oil emulsion.

The purpose of this study was to investigate the effect of osmotic gradients in a water-in-oil (w/o) emulsion on release properties in order to control the release of hydrophilic drugs. The magnitude and direction of the osmotic gradient was shown to have a pronounced effect on the apparent permeability of the hydrophilic marker, [3H]glucose. The apparent permeability coefficient of glucose could be varied between 1.0x10(-5) and 5.0x10(-8) cm s-1 using osmotic gradients. The release rate of glucose was related to the swelling properties. The larger the degree of swelling, the lower the release rate. Furthermore the present w/o emulsion has a low viscosity and a long-term physical stability. This makes the emulsion a promising parenteral drug delivery system in which the release of hydrophilic drugs such as peptides, can be controlled.

Formulation and evaluation of release and swelling mechanism of a water-in-oil emulsion using factorial design.

Water-in-oil emulsions have a potential as a parenteral prolonged release system for hydrophilic drugs. A consistent challenge when developing an emulsion drug delivery system is to obtain a proper release characteristic of the entrapped drug. The aim of the present study was to study the release mechanism from water-in-oil emulsions. Secondly, to study the effects of droplet size, phase ratio and osmotic pressure on the release rate of glucose from water-in-oil emulsions in a factorial experimental design. The release mechanism of glucose was deduced from the release kinetics of two coentrapped marker molecules, glucose and inulin, with a molecule weight of 180 and 5000 g/mol, respectively. The results indicate that release of glucose was dominated by diffusion through the oily barrier as opposed to membrane rupture. Using statistical methodology, the release rate of glucose could be varied 8 fold in a controlled manner with osmotic pressure as the most important parameter. The osmotic behaviour of the emulsions was further studied in a dynamic swelling study. These results show that the release of entrapped hydrophilic drug can be controlled within certain limits using pharmaceutical formulation principles.

Parenteral water/oil emulsions containing hydrophilic compounds with enhanced in vivo retention: formulation, rheological characterisation and study of in vivo fate using whole body gamma-scintigraphy.

The preparation and characterization of parenteral water-in-oil (w/o) emulsions with a potential for sustained release of hydrophilic drugs was described with emphasis on rheological behaviour and spreading phenomenon after intramuscular (i.m.) injection in rabbit thigh muscle. Both steady state and dynamic rheological parameters were investigated showing Newtonian behaviour at low fraction of disperse phase ratio as opposed to viscoelastic and pseudoplastic behaviour at high fraction of disperse phase. Disappearance and spreading behaviour of hydrophilic radioactive markers, aprotinin (6512 g/mol) and pertechnetate (193 g/mol) entrapped in w/o emulsions from an i.m. injection site was studied by whole body gamma-scintigraphy. The retention of entrapped aprotinin 24 h postinjection was 83 +/- 5% for a low spreading emulsion and 76 +/- 6% for a high spreading emulsion. The corresponding values for pertechnetate were 50 +/- 11 and 23 +/- 2%, respectively. The relatively long retention times were suggested to be related to the good physical stability properties of the present emulsions. It was concluded that the presented w/o emulsions are promising vehicles for sustained release of hydrophilic drugs from an i.m. injection site.

Eye strain resulting from VDT work at the Swedish telecommunications administration.

A short experiment was carried out in the sales office and directory enquiries department of the Swedish Telecommunications Administration. VDT workload was set at two levels, a 'normal' level and an 'intensified' level. Visual strain associated with the work on VDTs was assessed by questionnaires and measurements of the near points of accommodation and convergence. When the VDT work routines were intensified an increase in subjective symptoms of visual strain was recorded. Increases in the near points of accommodation and convergence were recorded with intensified VDT workload. The experiment showed that organisational factors such as work spell duration and the rigidity of work routines seemed to play an important role in the frequency with which visual strain occurs in VDT work.

Investigation of visual strain experienced by microscope operators at an electronics plant.

This investigation was concerned with the examination of 75 microscope operators at an electronics plant, with reference to eye function and visual strain. It consisted of three separate parts: (1) standardised interviews dealing with symptoms of visual strain, (2) examinations of the refractive state as well as possible diseases of the eye, and (3) examination of binocular vision. It emerged that 80% of the microscope operators engaged in full time microscope operation experienced various symptoms of visual strain. A statistically verified relationship was found to exist between these manifestations of visual strain on the one hand and uncorrected astigmatism, fusion insufficiency (poorer eye co-ordination) and the length of time spent at the microscope during the working day on the other.

Investigations on the effect of antimicrobial drugs on platelet aggregation in vitro and ex vivo.

The influence of ten betalactam antibiotics and ten other antibiotics on platelet aggregation induced by ADP or adrenaline was investigated in vitro. In concentrations of 900 mg/l most antimicrobial drugs exerted a moderate inhibition that was not seen in concentrations of 90 mg/l that better corresponded to therapeutic levels. The influence on the platelets of a single large intravenous dose was also tested using eight antimicrobial drugs, viz. benzylpenicillin, ampicillin, carbenicillin, piperacillin, cloxacillin, cefuroxime, cefotaxime and erythromycin. Each drug was given to five healthy volunteers but none caused any significant inhibition of platelet aggregation. The second wave of aggregation persisted after administration of the drugs and it was even seen after the administration of such a high dose as 10 g of carbenicillin.

Effect on platelet aggregation of oral administration of 10 non-steroidal analgesics to humans.

Platelet aggregation was investigated in platelet-rich plasma from normal volunteers before and at various times after intake of 10 analgesic drugs. The drugs used were aspirin, piroxicam, naproxen, indomethacin, diclofenac, ibuprofen, diflunisal, paracetamol and oxyphenbutazone. Aggregation of the platelets was induced by adrenaline or ADP and the first and second waves of aggregation were evaluated. It was found that no drug exerted any effect on the first wave of aggregation. The second wave of aggregation was abolished by aspirin that produced a long-lasting effect for 5-8 d. Piroxicam also abolished the second wave of aggregation and this effect persisted on the 2 following d. Naproxen was normalized in half of the volunteers on the 2nd d. The inhibition caused by indomethacin and diclofenac was corrected on the 2nd d. Ibuprofen and diflunisal produced a definite but short-term effect. The effect of salicylic acid was weak. Paracematol and oxyphenbutazone did not affect platelet aggregation.