Changes in peripheral immune populations during pregnancy and modulation by probiotics and ω-3 fatty acids.

Allergic diseases have become a major health problem, partly due to reduced microbial stimulation and a decreased dietary ω-3/ω-6 long-chain polyunsaturated fatty acid ratio. Prenatal exposures have been reported to influence allergy development, possibly induced via changes in maternal immune regulation. In a randomized double-blind placebo-controlled multicenter allergy prevention trial (PROOM-3), pregnant women were recruited at gestational week 20, and randomized to four study groups, one receiving both L. reuteri oil drops and ω-3 PUFA capsules (n = 22), the second receiving ω-3 PUFA supplementation and placebo regarding L. reuteri (n = 21), the third receiving L. reuteri and placebo regarding ω-3 PUFA (n = 22) and the fourth group receiving placebo capsules and placebo oil drops (n = 23). In this substudy, supplemental and pregnancy-related effects on maternal peripheral immune cell populations during pregnancy were assessed by flow cytometry immune phenotyping at gestational week 20, 32 and 4 days after delivery. The numbers of activated and regulatory T (Treg) cells (CD45RA- Foxp3++/CD45RA+Foxp3+) were reduced after delivery, with the lowest count in the L. reuteri supplemented group compared with the placebo group 4 days after delivery, while the ω-3 PUFA group did not differ from the placebo group. Several treatment-independent changes were observed during and after pregnancy in lymphocytes (CD4+/8+/19+/56+/45RA+/-), CD14+16+/- monocytes, and in subpopulations of T helper cells (Th) CD4+CD45RA-Tbet+ (Th1) and CD4+CD45RA-RORC+ (Th17) cells. In conclusion, probiotic supplementation to the mother during the second half of pregnancy resulted in immunomodulatory effects among activated and resting Treg cells. Furthermore, several systemic immune modifying effects of pregnancy were observed.

Oral microbiota maturation during the first 7 years of life in relation to allergy development.

BACKGROUND: Allergic diseases have become a major public health problem in affluent societies. Microbial colonization early in life seems to be critical for instructing regulation on immune system maturation and allergy development in children. Even though the oral cavity is the first site of encounter between a majority of foreign antigens and the immune system, the influence of oral bacteria on allergy development has not yet been reported. OBJECTIVE: We sought to determine the bacterial composition in longitudinally collected saliva samples during childhood in relation to allergy development. METHODS: Illumina sequencing of the 16S rDNA gene was used to characterize the oral bacterial composition in saliva samples collected at 3, 6, 12, 24 months, and 7 years of age from children developing allergic symptoms and sensitization (n = 47) and children staying healthy (n = 33) up to 7 years of age. RESULTS: Children developing allergic disease, particularly asthma, had lower diversity of salivary bacteria together with highly divergent bacterial composition at 7 years of age, showing a clearly altered oral microbiota in these individuals, likely as a consequence of an impaired immune system during infancy. Moreover, the relative amounts of several bacterial species, including increased abundance of Gemella haemolysans in children developing allergies and Lactobacillus gasseri and L. crispatus in healthy children, were distinctive during early infancy, likely influencing early immune maturation. CONCLUSION: Early changes in oral microbial composition seem to influence immune maturation and allergy development. Future experiments should test the probiotic potential of L. gasseri and L. crispatus isolates.

How do probiotics and prebiotics function at distant sites?

The realisation that microbes regarded as beneficial to the host can impart effects at sites distant from their habitat, has raised many possibilities for treatment of diseases. The objective of a workshop hosted in Turku, Finland, by the International Scientific Association for Probiotics and Prebiotics, was to assess the evidence for these effects and the extent to which early life microbiome programming influences how the gut microbiota communicates with distant sites. In addition, we examined how probiotics and prebiotics might affect the skin, airways, heart, brain and metabolism. The growing levels of scientific and clinical evidence showing how microbes influence the physiology of many body sites, leads us to call for more funding to advance a potentially exciting avenue for novel therapies for many chronic diseases.

Correlates of Nine-Month Retention following Interim Buprenorphine-Naloxone Treatment in Opioid Dependence: A Pilot Study.

Interim medication-only treatment has been suggested for the initiation of opioid maintenance treatment (OMT) in opioid-dependent subjects, but this rarely has been studied using buprenorphine instead of methadone. Following a pilot trial assessing interim buprenorphine-naloxone treatment in order to facilitate transfer into OMT, we here aimed to study retention, and potential correlates of retention, in full-scale treatment. Thirty-six patients successfully referred from a waiting list through an interim treatment phase were followed for nine months in OMT. Baseline characteristics, as well as urine analyses during the interim phase and during full-scale OMT, were studied as potential correlates of retention. The nine-month retention in OMT was 83 percent (n = 30). While interim-phase urine samples positive for benzodiazepines did not significantly predict dropout from full-scale OMT (p = 0.09), urine samples positive for benzodiazepines within full-scale OMT were significantly associated with dropout (p < 0.01), in contrast to other substances and baseline characteristics. Retention remained high through nine months in this pilot study sample of patients referred through buprenorphine-naloxone interim treatment, but use of benzodiazepines is problematic, and the present data suggest that it may be associated with treatment dropout.

Low gut microbiota diversity in early infancy precedes asthma at school age.

BACKGROUND: Low total diversity of the gut microbiota during the first year of life is associated with allergic diseases in infancy, but little is known how early microbial diversity is related to allergic disease later in school age. OBJECTIVE: To assess microbial diversity and characterize the dominant bacteria in stool during the first year of life in relation to the prevalence of different allergic diseases in school age, such as asthma, allergic rhinoconjunctivitis (ARC) and eczema. METHODS: The microbial diversity and composition was analysed with barcoded 16S rDNA 454 pyrosequencing in stool samples at 1 week, 1 month and 12 months of age in 47 infants which were subsequently assessed for allergic disease and skin prick test reactivity at 7 years of age (ClinicalTrials.gov ID NCT01285830). RESULTS: Children developing asthma (n = 8) had a lower diversity of the total microbiota than non-asthmatic children at 1 week (P = 0.04) and 1 month (P = 0.003) of age, whereas allergic rhinoconjunctivitis (n = 13), eczema (n = 12) and positive skin prick reactivity (n = 14) at 7 years of age did not associate with the gut microbiota diversity. Neither was asthma associated with the microbiota composition later in infancy (at 12 months). Children having IgE-associated eczema in infancy and subsequently developing asthma had lower microbial diversity than those that did not. There were no significant differences, however, in relative abundance of bacterial phyla and genera between children with or without allergic disease. CONCLUSION AND CLINICAL RELEVANCE: Low total diversity of the gut microbiota during the first month of life was associated with asthma but not ARC in children at 7 years of age. Measures affecting microbial colonization of the infant during the first month of life may impact asthma development in childhood.

Oral administration of Lactobacillus reuteri during the first year of life reduces caries prevalence in the primary dentition at 9 years of age.

The aim of this study was to evaluate the effect on oral health, at age 9 years, of daily oral supplementation with the probiotic Lactobacillus reuteri, strain ATCC 55730, to mothers during the last month of gestation and to children through the first year of life. The study was a single-blind, placebo-controlled, multicenter trial involving 113 children: 60 in the probiotic and 53 in the placebo group. The subjects underwent clinical and radiographic examination of the primary dentition and carious lesions, plaque and gingivitis were recorded. Saliva and plaque were sampled for determination of mutans streptococci (MS) and lactobacilli (LB) in saliva and plaque as well as salivary secretory IgA (SIgA). Forty-nine (82%) children in the probiotic group and 31 (58%) in the placebo group were caries-free (p < 0.01). The prevalence of approximal caries lesions was lower in the probiotic group (0.67 ± 1.61 vs. 1.53 ± 2.64; p < 0.05) and there were fewer sites with gingivitis compared to the placebo group (p < 0.05). There were no significant differences between the groups with respect to frequency of toothbrushing, plaque and dietary habits, but to intake of fluoride supplements (p < 0.05). There were no intergroup differences with respect to L. reuteri, MS, LB or SIgA in saliva. Within the limitation of this study it seems that daily supplementation with L. reuteri from birth and during the first year of life is associated with reduced caries prevalence and gingivitis score in the primary dentition at 9 years of age.

Pre- and post-natal Lactobacillus reuteri supplementation decreases allergen responsiveness in infancy.

BACKGROUND: We have previously shown that Lactobacillus reuteri supplementation from pregnancy week 36 and to the infant through the first year of life decreased the prevalence of IgE-associated eczema at 2 years. The underlying immunological mechanisms are unknown, however. OBJECTIVE: To investigate the immunomodulatory effect of probiotic supplementation on allergen- and mitogen-induced immune responses in children until 2 years of age. METHODS: Blood mononuclear cells were collected at birth, 6, 12 and 24 months from 61 children (29 probiotic and 32 placebo treated) and cultured with ovalbumin, birch and cat extract and Phytohaemagglutinin (PHA). Cytokine and chemokine secretion was determined using an in-house multiplexed Luminex assay and ELISA. Real-time PCR was performed to investigate the Ebi3, Foxp3, GATA-3 and T-bet mRNA expression. RESULTS: Probiotic treatment was associated with low cat-induced Th2-like responses at 6 months (IL-5, P = 0.01, and IL-13, P = 0.009), with a similar trend for IL-5 at 12 months (P = 0.09). Cat-induced IFN-γ responses were also lower after probiotic than after placebo treatment at 24 months (P = 0.007), with similar findings for the anti-inflammatory IL-10 at birth (P = 0.001) and at 12 months (P = 0.009). At 24 months, Th2-associated CCL22 levels were lower in the probiotic than in the placebo group after birch stimulation (P = 0.02), with a similar trend after ovalbumin stimulation (P = 0.07). Lower CCL22 levels were recorded at 12 and 24 months (P = 0.03 and P = 0.01) after PHA stimulation. CONCLUSION AND CLINICAL RELEVANCE: Lactobacillus reuteri supplementation decreases allergen responsiveness and may enhance immunoregulatory capacity during infancy. L. reuteri supplementation from week 36 and during the first year of life significantly decreases IgE-associated eczema and lowers allergen and mitogen responsiveness.

A Th1/Th2-associated chemokine imbalance during infancy in children developing eczema, wheeze and sensitization.

BACKGROUND: Analyses of circulating chemokines offer novel tools to investigate the T helper (Th)1/Th2 imbalance in allergic disease in vivo. OBJECTIVE: To relate circulating Th1- and Th2-associated chemokines in infancy to allergic disease, sensitization and probiotic supplementation. METHODS: Circulating levels of Th1-associated CXC-chemokine ligand (CXCL)9, CXCL10 and CXCL11 and Th2-associated CC-chemokine ligand (CCL)17 and CCL22 were assessed with Luminex and CCL18 with enzyme-linked immunosorbent assay at birth (n=109), 6 (n=104), 12 (n=116) and 24 months (n=123) in 161 infants completing a double-blind placebo-controlled allergy prevention trial with Lactobacillus reuteri during the last month of gestation and through the first year of life. The infants were followed regarding the development of allergic disease and sensitization until 2 years of age. RESULTS: The Th2-associated chemokines CCL17 and CCL22 were the highest at birth and then decreased, whereas CCL18 and the Th1-associated chemokines increased with age. High Th2-associated chemokine levels were observed in children developing allergic disease. Sensitization was preceded by elevated levels of the Th2-associated CCL22 and reduced levels of the Th1-associated CXCL11 already at birth. The Th2-associated CCL17 was also elevated at birth in infants developing recurrent wheeze. A high Th2/Th1 ratio (CCL22/CXCL10) at birth associated with both sensitization and eczema development. The presence of L. reuteri in stool in the first week of life was associated with low CCL17 and CCL22 and high CXCL11 levels at 6 months of age. High Th1-associated chemokine levels were associated with day-care. CONCLUSION AND CLINICAL RELEVANCE: Allergic disease and sensitization in infancy was associated with low circulating Th1- and high Th2-associated chemokine levels already from birth. Circulating chemokines are useful for investigating the Th1/Th2 imbalance in allergic disease in vivo. Elucidation of the role of chemokines in allergic diseases may lead to future treatments (ClinicalTrials.gov NCT01285830).

Local proCPU (TAFI) activation during thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecule thrombin inhibitor (melagatran).

To test the hypothesis that the direct thrombin inhibitor, melagatran is able to inhibit local pro-carboxypeptidase U (proCPU) activation that occurs during thrombolytic treatment, t-PA alone, or in combination with melagatran, was given to dogs with a coronary artery thrombosis. Blood samples from the great cardiac vein and aorta were collected at baseline, during thrombus formation, throughout the t-PA+/-melagatran infusion and during the patency period, for analysis of CPU activity using a novel assay. A higher CPU activity in venous compared to arterial blood (V-A difference) indicates CPU activation in coronary vessels. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Dogs (n = 26) were randomized to receive either 1) t-PA, 1 mg/kg as an intravenous 20-min infusion; 2) t-PA as in group 1, +melagatran bolus, 0.3 mg/kg, followed by a 3-h infusion (0.15 mg/kg per h); 3) sham-operated but no coronary thrombus, and administered t-PA as for Group 1. All groups had similar baseline characteristics. Significant increases in CPU activity were observed in Groups 1 and 2 during thrombus formation, with V-A differences of 5.5 and 4.5 U/L, respectively. No significant V-A difference was observed in the sham-operated group. CPU activity increased in Group 1 during the t-PA infusion (V-A difference 15.9 U/L), whereas the V-A difference in Group 2 decreased to 2.6 U/L following melagatran treatment. These results demonstrate that melagatran attenuates generation of CPU in the coronary circulation. The mechanism is probably indirect, via inhibition of thrombin-mediated activation of proCPU.

Candesartan Causes Long-lasting Antagonism of the Angiotensin II Receptor-mediated Contractile Effects in Isolated Vascular Preparations: a Comparison with Irbesartan, Losartan and its Active Metabolite (EXP-3174).

Candesartan is a new angiotensin II type 1 (AT 1 ) receptor blocker. It displays insurmountable antagonism of angiotensin II responses, binding tightly to and dissociating slowly from the AT 1 -receptor. The purpose of this study was to compare the duration of angiotensin II antagonism by the AT 1 -receptor blockers candesartan, irbesartan, losartan and its active metabolite EXP-3174 in an isolated tissue preparation. The contractile response to angiotensin II was studied in the isolated portal vein of the rat, during incubation with AT1-receptor blockers and after an extensive washout period. The portal vein preparation was pre-stretched to a passive force of 5 mN in an organ bath filled with oxygenated Krebs' buffer at 37°C. The contractile tension developed by the vascular smooth muscles was monitored using a force-displacement transducer. The contractile response to repeated administration of angiotensin II was recorded before, during and following exposure (for 30-180 min) to candesartan, 0.1-1 nmol/l, irbesartan, 1-50 nmol/l, losartan, 30-100 nmol/l, and EXP-3174, 1-10 nmol/l. Drug exposure was followed by washing for up to 2 h. Candesartan produced a long-lasting blockade of the vascular contractile response to angiotensin II, as shown by maintenance of inhibition during the washout period. This effect of candesartan was independent of drug concentration and exposure time prior to washing. Irbesartan, losartan and EXP-3174 also blocked the angiotensin II-mediated contraction. However, in contrast to candesartan, the responses to angiotensin II rapidly returned towards baseline values during the washout period. The relatively short-lasting blockade by irbesartan, losartan and EXP-3174 was also independent of drug concentration and exposure time prior to washout. It is concluded that the AT 1 -receptor blockers differ in their ability to inhibit angiotensin II-mediated vascular contraction, with candesartan producing longer-lasting blockade than irbesartan, losartan and EXP-3174. The mechanism of the persistent inhibitory effect of candesartan is at present unclear. Possible explanations include tight binding and slow dissociation from the AT 1 -receptor, tissue accumulation resulting in 'local( dissociation and reassociation to the AT 1 -receptor, and stimulation of internalization of the AT 1 -receptor.

Mechanistic differences of various AT1-receptor blockers in isolated vessels of different origin.

The functional inhibitory characteristics of the angiotensin II type 1 receptor blockers (ARB) candesartan; irbesartan; and losartan and its active metabolite EXP 3174 (EXP) were studied in rabbit aortic strips and rat portal vein preparations in vitro. Moreover, plasma-protein binding was determined, and the binding was high (>98. 5%) for all ARBs. These values were needed to relate the concentrations of the ARBs used in vitro to the nonprotein bound concentrations in clinical use. In both vascular preparations, candesartan caused a marked decrease in the maximal contractile response of the angiotensin II (Ang II) concentration-response curve. Losartan, EXP, and irbesartan caused a rightward parallel shift without any major effects on the maximal response to Ang II. The inhibitory effect of candesartan developed slowly (maximal effect after >30 minutes) and lasted >2 hours despite repeated washing of the vessels. The effect of losartan, irbesartan, and EXP had a faster onset, and most of the inhibitory effect disappeared after washing. The duration of the inhibitory effects of the ARBs were not related to lipophilicity of the compounds. Cooling of the rat portal vein preparations to 4 degrees C before administration of candesartan prevented the persistent inhibition of Ang II response seen at 37 degrees C. For the other ARBs studied, the magnitude of inhibition and the speed of recovery of the Ang II response were independent of the incubation temperature before washing. In addition, when candesartan was given to conscious rats, the inhibitory effect on Ang II-induced blood pressure responses persisted during the 24-hour period despite nondetectable plasma concentrations of candesartan at 24 hours. It is concluded that functional inhibitory characteristics of candesartan differ from those of the other ARBs tested. At clinically relevant concentrations, candesartan is an insurmountable and long-lasting antagonist of the vascular contractile responses to Ang II.

PAI-1 inhibition enhances the lysis of the platelet-rich part of arterial-like thrombi formed in vitro. A comparative study using thrombi prepared from rat and human blood.

We studied the effects of PRAP-1, a PAI-1-inhibiting polyclonal antibody, on tissue-type plasminogen activator (t-PA)-induced lysis of arterial-like thrombi prepared in vitro from rat and human blood in a Chandler loop device. The t-PA induced lysis of the thrombus head and tail was studied over 5 h. For thrombi prepared from rat blood, the lysis ratio head:tail was (mean +/- SD): 0.71 +/- 0.16 (platelet-poor blood), 0.49 +/- 0.22 (whole blood), and 0.22 +/- 0.12 (platelet-rich blood). PRAP-1 increased the lysis ratio to 0.68 +/- 0.22 for whole-blood thrombi (P < 0.01) and to 0.46 +/- 0.15 for platelet-rich thrombi (P < 0.001). For thrombi formed from human whole blood, PRAP-1 increased the lysis ratio head:tail from 0.61 +/- 0.18 to 0.95 +/- 0.24 (P < 0.05). These effects of PRAP-1 were due to a selective increase in the lysis of the thrombi head. The plasminogen activator inhibitor (PAI-1) activity was 7.5 +/- 2.8 (rat) and 3.4 +/- 1.3 (human) times higher in the thrombus head compared with the tail. In thrombi prepared from rat whole blood, PRAP-1 decreased the PAI-1 activity of the thrombus head only by 38 +/- 7% (P < 0.01), while in thrombi prepared from human whole blood the PAI-1 activity of the thrombus head decreased by 83 +/- 4% (P < 0.001). In conclusion, the lysis resistance of the head of an arterial-like thrombus formed in vitro can be partially (rat) or totally (human) normalized by inhibition of the PAI-1 activity. The results further suggest that in rat arterial-like thrombi a t-PA-inhibiting component, different from PAI-1, is present.

The Fab-fragment of a PAI-1 inhibiting antibody reduces thrombus size and restores blood flow in a rat model of arterial thrombosis.

The effect of PRAP-1, a Fab-fragment of a PAI-1-inhibiting polyclonal antibody, on thrombus size and arterial blood flow was studied in a rat model of arterial thrombosis. It was shown that exposure of the carotid artery to FeCl3 led to the rapid formation of an occlusive thrombus with a morphology similar to that of arterial thrombi found in humans. Tranexamic acid (50 mg/kg), an inhibitor of fibrinolysis, increased thrombus size (p = 0.014) when given intravenously (i.v.) prior to the FeCl3-exposure. Heparin (1000 U), when given i.v. after FeCl3, did not affect the thrombus size per se, but caused a reduction in the interindividual variation of the size of the thrombus (p < 0.05). Thus, heparin was included in all the subsequent experiments. An i.v. infusion of t-PA (1 mg/kg/h), starting before thrombus formation, induced a 3.3 fold increase in the perfusion rate (p = 0.006) and a 67% reduction in the thrombus size (P < 0.001). PRAP-1, an inhibitor of rat PAI-1 activity, was given i.v. as a bolus followed by an infusion. Two doses of PRAP-1 were studied (7.5 and 15 mg/kg/h), and the administration of the PAI-1 inhibitor was started 10 min before FeCl3. The lower PRAP-1 dose caused a 3.8 fold increase in perfusion rate (p = 0.036), a 1.44 fold increase in the time to occlusion (p = 0.034), and the thrombus size was decreased by 18% (p = 0.104). The corresponding effects of the high PRAP-1 dose were a 6.5 fold increase in perfusion rate (p < 0.001), a 1.6 fold increase in time to occlusion (p = 0.038) and a 32% reduction in thrombus size (p = 0.016). It is concluded that an inhibitor of PAI-1 activity, PRAP-1, caused a moderate decrease in thrombus size and partly restores blood flow in a rat model of arterial thrombus. This finding suggests a potential role for an inhibitor of PAI-1 in the treatment of arterial thrombosis.

Anti-thrombotic effect of a PAI-1 inhibitor in rats given endotoxin.

The aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i.v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus+infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.

Vascular versus myocardial selectivity of dihydropyridine calcium antagonists as studied in vivo and in vitro.

The use of in vitro models for the study of cardiovascular effects of drugs may not be representative for the in vivo therapeutic effects. However, drug effects in vivo are often difficult to assess because of counteracting reflexes and auto-regulatory rearrangements. To solve this dilemma, the present study presents a two-step method using both in vivo and in vitro techniques to investigate vascular versus myocardial selectivity of three dihydropyridine calcium antagonists: amlodipine, felodipine and nifedipine. The ratio between intravenous drug doses causing 25% reduction in mean arterial blood pressure (vascular potency) and in heart rate (cardiac chronotropic potency) was determined in anaesthetised spontaneously hypertensive rats during autonomic cardiac blockade. In isolated hearts from spontaneously hypertensive rats, the inotropic versus chronotropic potency ratio was determined between the two drug concentrations producing a 25% reduction in cardiac contractility (dP/dt max) and in heart rate, respectively. The vascular versus chronotropic selectivity in vivo was higher for felodipine (121) than for nifedipine (47) and amlodipine (15). The inotropic versus chronotropic potency ratios obtained from the in vitro studies were: felodipine (1), amlodipine (2) and nifedipine (20). The in vitro results were used to extrapolate the vascular versus cardiac chronotropic selectivity obtained in vivo to a vascular versus myocardial selectivity drug ratio, being 20 and 60 times higher for felodipine than for amlodipine and nifedipine, respectively.

Central nervous and systemic kinetics of ramipril and ramiprilat in the conscious dog.

The central nervous and systemic kinetics of ramipril, an angiotensin-converting enzyme inhibitor prodrug, and its active metabolite ramiprilat were studied in conscious beagle dogs after sampling of cerebrospinal fluid (CSF) and blood during chronic drug administration. The cardiovascular effect of angiotensin-converting enzyme inhibitors have been suggested to be mediated partly by central action. Ramiprilat and ramipril were determined in CSF and plasma by a gas chromatographic-mass spectrometric assay method. After 10 mg/kg of ramipril, given orally to four dogs once daily for 7 days, significant concentrations of ramiprilat were measured in CSF over the 24-h period after both the 1st and 7th day of treatment. The CSF/plasma (unbound) ratios of ramiprilat on day 7 were (mean +/- S.D.): 0.01 +/- 0.003 (2 h after dose), 0.18 +/- 0.05 (12 h after dose) and 0.32 +/- 0.11 (24 h after dose). Measurable concentrations of ramipril were recorded in plasma after oral dosing (bioavailability approximately 45%), whereas in CSF the prodrug concentration was below the minimal determinable levels in most cases. In a second set of experiments, ramiprilat (3 mg/kg) or ramipril (3 mg/kg) were given i.v. to three dogs once daily for 7 days. Ramipril was rapidly cleared from the plasma, clearance being approximately 140 ml/min/kg and half-life about 0.5 h on day 7. The corresponding values for ramiprilat were 8 ml/min/kg and 0.75 h. The CSF/plasma ratios for ramiprilat were essentially the same after i.v. administration of ramiprilat and ramipril and, furthermore, the ratios did not differ significantly from the ratios observed after oral administration of the prodrug.(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of regional flow distribution following reperfusion of the isolated ischaemic rat heart.

1. The regional distribution of flow was studied at different times after the onset of reperfusion in isolated rat heart preparations. The hearts were submitted to 30 min of global ischaemia followed by 60 min of reperfusion. Microspheres labelled with various nuclides were added to the perfusate before ischaemia and 1, 5, 20, and 60 min after the onset of reperfusion. 2. One minute after the start of reperfusion, the flow to the left ventricular inner layer was restricted to 0.5 +/- 0.2 mL/min per g (2-3% of the pre-ischaemic flow). In this segment, the perfusion remained at the same low level during the entire reperfusion period studied. At the onset of reperfusion the flow to the outer layer of the left ventricle was 4.8 +/- 1.7 mL/min per g (37% of the pre-ischaemic flow), and 3.0 +/- 1.3 mL/min per g (27% of the pre-ischaemic value) to the free wall of the right ventricle. The flow was progressively reduced in the outer layer of the left ventricle in the course of reperfusion. After 60 min of reperfusion the flow to the left ventricular outer layer was 2.5 +/- 0.9 mL/min per g (19% of pre-ischaemic flow when compared with the onset of reperfusion [P less than 0.05]). 3. It is concluded that a 'no-reflow' condition develops very early during reperfusion and becomes more marked during this period.

Vascular bound recombinant extracellular superoxide dismutase type C protects against the detrimental effects of superoxide radicals on endothelium-dependent arterial relaxation.

Extracellular superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme that, in vivo, is bound to heparan sulfate proteoglycans in the glycocalyx of various cell types (e.g., endothelial cells) and in the connective tissue matrix. The aim of this study was to investigate the efficacy of vascular bound EC-SOD C in protecting arterial relaxation mediated by endothelium-derived relaxing factor (EDRF) against the inhibitory effects of superoxide radicals. For comparison, the effect of CuZn SOD was also studied. This SOD isoenzyme lacks affinity toward heparan sulfate and does not bind to cell surfaces. Rings from rabbit aorta were mounted in an organ bath and acetylcholine-induced endothelium-dependent relaxation was then studied in preparations precontracted with phenylephrine. Pyrogallol (10(-4) M), used to generate superoxide radicals, reduced the maximal relaxant effect of acetylcholine from about 65% to 25%. When present in the buffer throughout the experiment, CuZn SOD and EC-SOD C caused a concentration-dependent prevention of the pyrogallol effect on EDRF-mediated relaxation, with a half-maximal effect at about 100 units/ml (KO2 assay). In a second set of experiments, the arterial rings were preincubated with 8,000 units/ml CuZn SOD (50 micrograms/ml) or EC-SOD C (69 micrograms/ml) during 30 minutes, followed by washing, before the effect of pyrogallol on EDRF-mediated relaxation was studied in SOD-free buffer.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide dismutase and catalase do not improve recovery of regional myocardial contractile function when given at the time of reperfusion after reversible regional ischemia in anesthetized dogs.

Earlier studies have demonstrated an improvement in the recovery of the regional myocardial function after reversible myocardial ischemia when dogs were treated with superoxide dismutase (SOD) + catalase (CAT). In all these studies, drug administration was started prior to the ischemic period. The aim of this study was to investigate the effects of SOD and CAT on the recovery of the regional contractile function in anesthetized beagle dogs when the drugs were administered at the time of reperfusion. The animals were subjected to 20 min of left coronary artery occlusion followed by 3 h reperfusion. The regional myocardial contractile function, measured as subendocardial segment shortening (SS, sonomicrometry) decreased to below zero and the regional blood flow in the ischemic subendocardium was reduced to about 5% of pre-ischemic values during the coronary artery occlusion period. The size of the occluded bed was similar in the two groups. Saline (n = 8) or SOD (10 mg/kg) + CAT (3.4 mg/kg) (n = 8) were infused into the left atrium from 2.5 min prior to until 20 min after the start of reperfusion. The peak plasma level of SOD was 102 +/- 15 mg/l at 20 min reperfusion. There were no significant differences in the arterial blood pressure, cardiac contractile function and regional blood flow between the two groups at any time during the experiment. During reperfusion in the dogs given vehicle, SS recovered to 48 +/- 7% (mean +/- SEM) after the first hour of reperfusion, and to 51 +/- 6% of pre-ischemic values after 3 h of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardioprotective effects of recombinant human extracellular-superoxide dismutase type C in rat isolated heart subjected to ischemia and reperfusion.

The cardioprotective effect of recombinant human extracellular-superoxide dismutase type C (rh-EC-SOD C) was studied in isolated perfused rat heart subjected to left coronary artery ligation for 30 or 60 min followed by 30-min reperfusion. A comparison was made with the effects of bovine CuZn-SOD. Reperfusion after 30-min coronary artery ligation was associated with a release of creatine kinase (CK) into the coronary effluent (71 +/- 5.2 IU/30 min), which was markedly reduced (39 +/- 5.5 IU/30 min) in hearts perfused with rh-EC-SOD C (28 mg/L). CuZn-SOD (4 or 20 mg/l) or a lower concentration of rh-EC-SOD C (5.6 mg/l) did not significantly attenuate CK outflow during reperfusion, however. In both vehicle- and SOD-treated hearts, the left ventricular developed pressure (LVDP) and the coronary flow recovered to 80-90% of baseline at the end of the reperfusion period. Increasing the ischemic period from 30 to 60 min caused a much more pronounced cardiac injury measured after 30-min reperfusion. In the hearts that received vehicle, recovery of LVDP (in percentage of baseline values) at the end of reperfusion was 58 +/- 2%, which was increased to 84 +/- 3 and 83 +/- 5% after treatment with rh-EC-SOD C (28 mg/L) and CuZn-SOD (20 mg/L), respectively. The corresponding values for recovery in coronary flow were 54 +/- 3% (vehicle), 69 +/- 4% (rh-EC-SOD C), and 74 +/- 3% (CuZn-SOD).(ABSTRACT TRUNCATED AT 250 WORDS)