Strategies for atopy prevention.

Allergic diseases, particularly in childhood, have become one of the epidemics of the 21st century. Whereas previous strategies for allergy prevention focused on the avoidance of risk factors, more recent approaches are addressing attempts to provide protective factors to infants and young children to achieve immune modulation and tolerance to harmless nutritional or environmental allergens. This change of paradigm for allergy prevention might lead to more effective interventions, which hopefully contribute to reversing the epidemiologic trend of the last decades. In many industrialized countries, the increased prevalence of atopy and asthma has become a serious public health issue. If preventive intervention could be effective at all, it would have to be applied early in life, most probably in early infancy. Unfortunately, our understanding of the natural history of the process of atopic sensitization, atopic dermatitis, and allergic airway disease is still very limited. On the other hand, the evaluation of risk factors and determinants is a necessary prerequisite for any effective intervention.

Effect of arachidonic and eicosapentaenoic acids on acute lung injury induced by hypochlorous acid.

BACKGROUND: Hypochlorous acid (HOCl) is the main oxidant of activated polymorphonuclear neutrophil granulocytes (PMN) and generated by myeloperoxidase during respiratory burst. This study investigates the effects of HOCl on pulmonary artery pressure (PAP) and vascular permeability and characterises the influence of arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the observed effects. METHODS: HOCl (500, 1,000, 2,000 nmol/min) was continuously infused into the perfusate (Krebs-Henseleit buffer solution, KHB). AA or EPA in subthreshold doses (both 2 nmol/min) or buffer were simultaneously infused using a separate port. PAP, pulmonary venous pressure (PVP), ventilation pressure, and lung weight gain were continuously recorded. The capillary filtration coefficient (Kf,c) was calculated before and 30, 60, and 90 minutes after starting the HOCl infusion. RESULTS: HOCl application resulted in a dose dependent increase in PAP and Kf,c. The onset of these changes was inversely related to the HOCl dose used. The combined infusion of AA with HOCl resulted in a significant additional rise in pressure and oedema formation which forced premature termination of all experiments. The combination of EPA with HOCl did not result in an enhancement of the HOCl induced rise in pressure and oedema formation. CONCLUSIONS: Changes in the pulmonary microvasculature caused by HOCl are differently influenced by omega-6 and omega-3 polyunsaturated free fatty acids, suggesting a link between neutrophil derived oxidative stress and pulmonary eicosanoid metabolism.

Influence of cyclooxygenase and lipoxygenase inhibitors on oxidative stress-induced lung injury.

OBJECTIVE: Hypochlorous acid (HOCl) is the main oxidant of activated neutrophil granulocytes. It is generated by their myeloperoxidase during respiratory burst. This study investigates the effects of HOCl on vascular permeability and pulmonary artery pressure (PAP) and characterizes the influence of the cyclooxygenase inhibitor acetylsalicylic acid (ASA) and the 5-lipoxygenase inhibitor caffeic acid (CaA) on the observed alterations. DESIGN: Prospective experimental study using isolated perfused rabbit lungs. SETTING: Experimental laboratory in a university teaching hospital. INTERVENTIONS: HOCl was infused into the perfusate containing either no inhibitors, ASA (500 micromol/L), or CaA (1 micromol/L). MEASUREMENTS AND MAIN RESULTS: PAP, pulmonary venous pressure, and ventilation pressure as well as lung weight gain were continuously recorded. Capillary filtration coefficient [Kf,c (10(-4) cm3 x sec(-1) x cm H2O(-1) x g(-1)]) was calculated before and 30, 60, and 90 mins after start of HOCl application. Continuous HOCl application (500, 1000, and 2000 nmol/min) resulted in a time- and dose-dependent increase in Kf,c and PAP with a threshold dose at 500 nmol/min. The onset of these changes was inversely related to the HOCl dose used. Both inhibitors, CaA and ASA, exhibited protective effects on the HOCl-induced alterations in pulmonary microcirculation. ASA predominantly reduced the HOCl-induced pressure response and had a minor but also significant inhibitory effect on edema formation as measured by Kf,c and fluid retention. CaA reduced significantly the rise in Kf,c and subsequent edema formation without effects on pulmonary pressure response. CONCLUSIONS: Cyclooxygenase and 5-lipoxygenase are involved in oxidative stress induced acute lung injury, suggesting a link between neutrophil-derived oxidative stress and endothelial eicosanoid metabolism.

Acute changes of myocardial creatine kinase gene expression under beta-adrenergic stimulation.

Creatine kinase (CK) plays a crucial role in myocardial energy metabolism. Alterations in CK gene expression are found in hypertrophied and failing heart, but the mechanisms behind these changes are unclear. This study tests the hypothesis that increased adrenergic stimulation, which is observed in heart failure, induces changes of myocardial CK-activity, -isoenzyme distribution and -gene expression that are characteristic of the failing and hypertrophied heart. Isolated rat hearts were perfused (constant pressure of 80 mmHg) with red cell suspensions. Following a 20-min warm-up period, perfusion for 3 h with 10(-8) M (iso 3 h) or without (control 3 h) isoproterenol was started or experiments were immediately terminated (control 0 h). Left ventricular tissue was analyzed for total CK-activity, CK-isoenzyme distribution and, by use of quantitative RT-PCR, for B-CK, M-CK, mito-CK and GAPDH- (as internal standard) mRNA. After beta-adrenergic stimulation (iso 3 h) but not after control perfusion (control 3 h) a roughly threefold increase in B-CK mRNA levels and a decrease in M-CK mRNA levels by 18% was found. There were no significant differences among the three groups in total CK-activity and in distribution of CK-MM, CK-BB, CK-MB and mito-CK. Thus, beta-adrenergic stimulation induces a switch in CK gene expression from M-CK to B-CK, which is characteristic for the hypertrophied and failing heart. This may be interpreted as an adaptive mechanism making energy transduction via CK more efficient at times of increased metabolic demand.

[Acute liver failure after therapeutic hyperthermia]. / Akutes Leberversagen nach Hyperthermiebehandlung.

HISTORY AND ADMISSION FINDINGS: A 49-year-old women with malignant neoplasia had undergone whole body hyperthermia under sedation to a rectal temperature of 42.5 to 42.7 degrees C for about one hour. She failed to awaken afterwards and was admitted in a coma and transferred to the intensive care unit (ICU). There was slight improvement in consciousness in the following 30 hours. INVESTIGATIONS: Clinical and laboratory findings gave a constellation indicating acute liver and renal failure (transaminases ca. 1400 U/l, bilirubin 7.27 mg/dl, serum creatinine 3.15 mg/dl, Quick thromboplastin time under 3.5%, antithrombin III 57%, partial thromboplastin time 70.7 s, platelets 23,000/microliter). Other causes of acute liver failure, especially drug effects, and septicaemia were excluded. TREATMENT AND COURSE: Treatment consisted of infusion of fresh plasma, neomycin and lactulose by mouth, medication to prevent stress ulcer, and total parenteral nutrition which included branched-chain amino acids. The patient regained full consciousness on the regimen and on the 6th day after admission was transferred to an ordinary ward. She was discharged after a further 3 weeks, by which time results of laboratory tests were practically normal. CONCLUSION: The course of the illness as well as the exclusion of any other cause of the acute liver failure in a patients who, 7 days before whole-body hyperthermia had been induced, had shown no signs of liver disease, makes a causal relationship between the hyperthermia and the described abnormalities highly probable. These serious, not previously reported, side effects of whole-body hyperthermia treatment underline the importance of undertaking this form of treatment only if strictest specific criteria are met.

Inhibition of PMN- and HOC1-induced vascular injury in isolated rabbit lungs by acetylsalicylic acid: a possible link between neutrophil-derived oxidative stress and eicosanoid metabolism?

Neutrophils are involved in the pathogenesis of acute lung injury. The neutrophil-derived enzyme myeloperoxidase (MPO) catalyzes the formation of the oxidant hypochlorous acid (HOCl). This study characterizes the effects of (A) continuous HOCl infusion, and (B) stimulated neutrophils on pulmonary circulation in an isolated rabbit lung model. Furthermore, the effect of cyclooxygenase inhibition by acetylsalicylic acid (ASA, 0.5 mM) on these effects was investigated. (A) Infusion of HOCl (in nmol min-1, groups: 0, 0+ASA, 1000, 1000+ASA, 2000, and 2000+ASA) into the isolated organ was started after a 45-min steady-state period (t=0). (B) Neutrophils (PMN group: 1480+/-323 and ASA group 1294+/-320 microliter-1) were added into the perfusate between (t=-45 min) and stimulated with FMLP (1 microM) after two 45-min steady-state periods (t=0). Perfusate MPO activity was measured at t=-90, -45, 0, 1, 2, 3, 5, 10, 15, 30, 60, and 90 min. For both groups, pulmonary artery pressure (PAP) and lung weight were continuously recorded and the capillary filtration coefficient (Kf,c in 10(-4) cm(3) s(-1) cm H2O(-1) g(-1) was calculated from the slope of weight gain after a hydrostatic challenge at t=-45, -15, 30, 60 and 90 min. (A) Continuous HOCl infusion (1000/2000 nmol min-1) evoked a significant increase in DeltaPAP and an up to 10-fold increase in Kf,c reaching the maximum extent of the observed effects significant earlier in the 2000 nmol min-1 group. ASA reduced DeltaPAPmax significantly to about 50% in corresponding groups and the increase in PAP and Kf,c occurred later in the ASA groups. (B) Neutrophil stimulation (PMN group/ASA group) evoked a rapid increase in DeltaPAP and MPO activity, while the changes in vascular permeability were rather moderate, but still significant. The release of MPO activity was similar in both groups. ASA significantly reduced the increase in DeltaPAP without affecting the release of MPO activity. Compared to baseline values, the preventive effects on vascular permeability increase reached level of significance as well. In summary, the described changes in pulmonary circulation caused by HOCl infusion or by neutrophil stimulation are significantly reduced by ASA. An involvement of cyclooxygenase products in the mediation of neutrophil-derived oxidative stress could be concluded.

The effect of the oxidant hypochlorous acid on the L-type calcium current in isolated ventricular cardiomyocytes.

Disturbances of cellular calcium homeostasis due to oxidative stress are involved in reperfusion associated phenomena like myocardial stunning and reperfusion induced arrhythmias. This study investigates the effect of the major neutrophil-derived oxidant hypochlorous acid (HOCl) on the l-type calcium current (ICa,L) of hamster ventricular cardiomyocytes. Using the whole-cell recording configuration of the patch-clamp technique, I Ca,L was recorded over 12.5 min (0.1 Hz). Application of HOCl or buffer (for control) via a second micropipette in close proximity to the cell was started at t=1 min. To study the influence of increased intracellular calcium buffer concentration and of ATP on HOCl-induced effects, internal solutions were composed as follows (EGTA/ATP in mmol/l): group I (standard) 0.5/0.0, group II 5.0/0.0, group III 0.5/1.0, and group IV 5.0/1.0. Application of 10, 20 and 40 micromol/l HOCl (under group I-conditions) caused a dose-dependent decrease in peak ICa,L to 82+/-3.2, 66+/-4.2 and 36+/-4.3% of baseline value (v 94+/-4.8% in controls, mean+/-s.e.m., P<0.05), and integrated ICa,L without affecting apparent reversal potential, activation and inactivation kinetics. HOCl-induced (40 micromol/l) decrease in ICa,L was partially inhibited in group II and III. Peak currents of these groups averaged 51+/-4.7 and 52+/-4.2% of baseline after 11.5 min administration of HOCl. Peak current in group IV cells decreased to 65+/-3.8% of baseline value (P<0.05 between group I-IV and v controls). Oxidative stress-induced decrease in ICa,L may be explained by energy depletion or calcium overload rather than by direct oxidative inactivation of channel proteins. A decrease in ICa, L may contribute to the shortening of action potential during reperfusion.

Comparable effects of HOCl and of FMLP-stimulated PMN on the circulation in an isolated lung model.

Polymorphonuclear leukocytes (PMN) are involved in acute lung injury during adult respiratory distress syndrome (ARDS) via several mechanisms. This study focuses on neutrophil-derived oxidative stress. The influence of (A), continuous hypochlorous acid (HOCl) infusion over 105 min and (B) stimulation of PMN having been delayed in the pulmonary microvasculature were studied. Therefore pulmonary artery pressure (PAP), capillary filtration coefficient (Kf,c), and fluid retention (delta W) were monitored using isolated rabbit lungs. These models (A/B) were compared with each other to assess the reproducibility of neutrophil-derived oxidative stress by HOCl. A: Infusion of 250/500/1,000/2,000 nmol/min HOCl (n = 6/group) evoked a delta PAPmax of 0.4 +/- 0.07/2.4 +/- 0.21/4.9 +/- 0.29/4.6 +/- 0.25 mm Hg at 105/105/56.4 +/- 5.6/21.5 +/- 0.8 min and a tenfold increase in Kf,c/delta W at 60 min. B: Stimulation of PMN (1,480 +/- 323/microliter, n = 8), which were added into the perfusate and sequestrated in the microvasculature, with 1 microM FMLP resulted in a delta PAPmax = 8.4 +/- 1.1 torr (t = 3.7 +/- 0.19 min) and a twofold increase in Kf,c/delta W (t = 60 min) that were accompanied by a myeloperoxidase (MPO)-release (MPOmax = 56.1 +/- 7.3 mU/l, after 1 to 3 min). There was a strong correlation between delta PAPmax and MPOmax (r = 0.97, p < 0.01). Both models of neutrophil-derived oxidative stress evoked changes in pulmonary circulation providing evidence for an involvement of PMN via their major oxidant HOCl in pulmonary hypertension and edema during ARDS.

Synthesis of 4- and 5-series leukotrienes in the lung microvasculature challenged with Escherichia coli hemolysin: critical dependence on exogenous free fatty acid supply.

Escherichia coli hemolysin (HlyA) has been identified as a potent inductor of phosphoinositide hydrolysis and related metabolic responses in neutrophils (Grimminger and colleagues, 1991, J. Clin. Invest. 88:1531-1539). In isolated perfused rabbit lungs, which harbor a large number of entrapped microvascular leukocytes, we investigated the effect of a low dose of HlyA on lipoxygenase product formation in the presence of exogenous free arachidonic acid (AA), eicosapentaenoic acid (EPA), or both precursor fatty acids. Leukotrienes (LT) and hydroxyeicosatetra(penta)enoic acids (HET[P]E) in the recirculating perfusate were quantified using high-performance liquid chromatography techniques. In the absence of exogenous precursor fatty acid supply, 0.02 hemolytic units/ml HlyA elicited only minor amounts of LTs and 5-HETE. AA, 10 microM, provoked the generation of limited quantities of LTB4, LTE4, and 5-HETE. Combined application of HlyA and AA caused a manifold amplification of 4-series LT and 5-HETE generation, with predominance of cysteinyl-LTs. EPA, 10 microM, elicited the synthesis of 5-series LTs accompanied by marked quantities of 5-HEPE. Dual stimulation with HlyA and EPA provoked exclusive generation of excessive quantities of all 5-series 5-lipoxygenase products. When HlyA was administered in the presence of both AA (10 microM) and EPA (10 microM), the n-3 fatty acid clearly turned out to be the preferred substrate, with ratios of the various 5-series to 4-series products ranging between 1.8 and 14.5. Moreover, the absolute quantities of AA-derived metabolites and the total sum of all 5-lipoxygenase products was markedly reduced under these conditions. We conclude that the HlyA-evoked 5-lipoxygenase product formation in the pulmonary vasculature of the rabbit is critically dependent on the presence of free precursor fatty acids. The profile of LTs suggests neutrophil (PMN)-related transcellular eicosanoid synthesis as a major underlying metabolic pathway. EPA represents the preferred substrate as compared with AA, resulting in a marked suppression of AA metabolite formation. Therapeutic attempts to provide n-3 fatty acids via the intravenous route may have a major impact on lipid mediator profiles in PMN-related inflammatory events.

Impact of arachidonic versus eicosapentaenoic acid on exotonin-induced lung vascular leakage: relation to 4-series versus 5-series leukotriene generation.

Escherichia coli hemolysin (HlyA) is a proteinaceous pore-forming exotoxin that is implicated as a significant pathogenicity factor in extraintestinal E. coli infections including sepsis. In perfused rabbit lungs, subcytolytic concentrations of the toxin evoke thromboxane-mediated vasoconstriction and prostanoid-independent protracted vascular permeability increase (11). In the present study, the influence of submicromolar concentrations of free arachidonic acid (AA) and eicosapentaenoic acid (EPA) on the HlyA-induced leakage response was investigated. HlyA at concentration from 0.02 to 0.06 hemolytic units/ml provoked a dose-dependent, severalfold increase in the capillary filtration coefficient (Kfc), accompanied by the release of leukotriene(LT)B4, LTC4, and LTE4 into the recirculating buffer fluid. Simultaneous application of 100 nmol/L AA markedly augmented the HlyA-elicited leakage response, concomitant with an amplification of LTB4 release and a change in the kinetics of cysteinyl-LT generation. In contrast, 50 to 200 nmol/L EPA suppressed in a dose-dependent manner the HlyA-induced increase in Kfc values. This was accompanied by a blockage of 4-series LT generation and a dose-dependent appearance of LTB5, LTC5, and LTE5. In addition, EPA fully antagonized the AA-induced amplification of the HlyA-provoked Kfc increase, again accompanied by a shift from 4-series to 5-series LT generation. We conclude that the vascular leakage provoked by HlyA in rabbit lungs is differentially influenced by free AA versus free EPA, related to the generation of 4- versus 5-series leukotrienes. The composition of lipid emulsions used for parenteral nutrition may thus influence inflammatory capillary leakage.

Genetic markers on chromosome 19p and prenatal diagnosis of HLA class II-deficient combined immunodeficiency.

HLA class II-deficient combined immunodeficiency (CID) is an inherited disease characterized by a total lack of HLA class II gene expression, due to a regulatory defect affecting these genes. In the family investigated the disease phenotype occurs parallel to an abnormal structural feature of the CD23 antigen. We sequenced parts of the FCER2 gene coding for CD23 and found a restriction fragment length polymorphism (RFLP) that cosegregates with the disease. Analysis of recombinant haplotypes by microsatellites mapping to the chromosomal region 19p13.3 suggests that the disease locus maps between FCER2 and the microsatellite marker D19S424, probably close to D19S216 and D19S177. These data may offer the possibility of a rapid and early prenatal diagnosis of a subgroup of patients with HLA class II-deficient CID.

Differential influence of arachidonic vs. eicosapentaenoic acid on experimental pulmonary hypertension.

The impact of the 2- and 3-series prostanoid precursors arachidonic acid (AA) and eicosapentaenoic acid (EPA) on experimental pulmonary hypertension was investigated. The model of buffer-perfused rabbit lungs was stimulated by infusion of Escherichia coli hemolysin (HlyA), which is known to provoke sustained thromboxane (Tx)-mediated pulmonary hypertension. Release of di- and trienoic Tx into the recirculating perfusate was quantified by a post-high-performance liquid chromatography enzyme-linked immunosorbent assay technique. HlyA at 0.08 hemolytic unit/ml caused a sustained rise in pulmonary arterial pressure (PAP; maximum increase 14 +/- 2 mmHg) accompanied by progressive TxB2 liberation (maximum perfusate concn 33 +/- 4 pg/ml, baseline < 2 pg/ml). Between 5 and 30 nM, AA provoked a transient monophasic rise in PAP (maximum pressor response 1.5-15 mmHg) and concomitant TxB2 release (peak concn 2-30 pg/ml). Simultaneous administration of HlyA and AA exhibited additive effects with regard to mediator release and pressor responses. EPA at 200-2,000 nM caused a transient rise in PAP similar to that provoked by 5-30 nM AA (maximum pressor response 3-18 mmHg). This was accompanied by liberation of TxB2 (peak concn 16 +/- 5 and 28 +/- 4 pg/ml after 1,000 and 2,000 nM EPA) and TxB3 (peak concn 9 +/- 4 and 30 +/- 3 pg/ml). Combined application of HlyA and EPA resulted in approximate addition of the TxB2 release reaction to each single compound, and TxB3 liberation more than doubled (maximum concn 59 +/- 12 pg/ml). The pressor responses to HlyA-EPA (200-2,000 nM) did not, however, surpass those to HlyA-AA (5-30 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of transcription initiation in vivo on Xenopus laevis ribosomal DNA.

We report the results of a novel method for locating sites of transcription initiation using a complex of capping enzymes from vaccinia virions that catalyze the reaction pppG + S-adenosylmethionine + (p)ppXpYpZp..... -->(7m)GpppXpYpZp..... [Ensinger, M. J., Martin, S. A., Paoletti, E. and Moss, B. (1975) Proc. Natl. Acad. Sci. USA 72, 2525-2529]. This enzyme complex will cap di- or triphosphate termini but will not cap monophosphate or hydroxyl termini. Xenopus laevis 40S precursor rRNA from oocytes is capped by these enzymes, and we conclude that it has 5'-polyphosphate termini. Therefore, 40S RNA must represent the primary transcript of amplified X. laevis ribosomal DNA. The majority of 40S molecules with polyphosphate termini begin with the sequence (p)ppAAG. There is evidence, however, that the 5' terminus may be heterogeneous. The majority of all detectable initiation events were localized close to the region coding for the 5' end of the 40S RNA. No initiation sites were detected in the nontranscribed spacer, but an apparent initiation site in the middle of the transcribed region was also observed.

Adenosine 3',5'-monophosphate, morphogenetic movements and embryonic neural differentiation in Pleurodeles waltlii.

Evidence is presented for the following hypothesis dealing with dibutyryl-c-AMP induction of neural differentiation in Pleurodeles waltlii embryos: (1) Dibutyryl-c-AMP induces the determination for neural differentiation. (2) Additional dibutyryl-c-AMP is not needed for cytodifferentiation. (3) Cytodifferentiation of induced cells can only take place after morphogenetic movements (in vivo) or extensive cell migrations (in vitro). It is suggested that cell migrations, both in vivo and in vitro, lead to the separation of the induced cells from one another or from unidentified inhibitory cells.