Synthetic surfactant with a recombinant surfactant protein C analogue improves lung function and attenuates inflammation in a model of acute respiratory distress syndrome in adult rabbits.

AIM: In acute respiratory distress syndrome (ARDS) damaged alveolar epithelium, leakage of plasma proteins into the alveolar space and inactivation of pulmonary surfactant lead to respiratory dysfunction. Lung function could potentially be restored with exogenous surfactant therapy, but clinical trials have so far been disappointing. These negative results may be explained by inactivation and/or too low doses of the administered surfactant. Surfactant based on a recombinant surfactant protein C analogue (rSP-C33Leu) is easy to produce and in this study we compared its effects on lung function and inflammation with a commercial surfactant preparation in an adult rabbit model of ARDS. METHODS: ARDS was induced in adult New Zealand rabbits by mild lung-lavages followed by injurious ventilation (VT 20 m/kg body weight) until P/F ratio < 26.7 kPa. The animals were treated with two intratracheal boluses of 2.5 mL/kg of 2% rSP-C33Leu in DPPC/egg PC/POPG, 50:40:10 or poractant alfa (Curosurf®), both surfactants containing 80 mg phospholipids/mL, or air as control. The animals were subsequently ventilated (VT 8-9 m/kg body weight) for an additional 3 h and lung function parameters were recorded. Histological appearance of the lungs, degree of lung oedema and levels of the cytokines TNFα IL-6 and IL-8 in lung homogenates were evaluated. RESULTS: Both surfactant preparations improved lung function vs. the control group and also reduced inflammation scores, production of pro-inflammatory cytokines, and formation of lung oedema to similar degrees. Poractant alfa improved compliance at 1 h, P/F ratio and PaO2 at 1.5 h compared to rSP-C33Leu surfactant. CONCLUSION: This study indicates that treatment of experimental ARDS with synthetic lung surfactant based on rSP-C33Leu improves lung function and attenuates inflammation.

Open lung approach ventilation abolishes the negative effects of respiratory rate in experimental lung injury.

BACKGROUND: We recently reported that a high respiratory rate was associated with less inflammation than a low respiratory rate, but caused more pulmonary edema in a model of ARDS when an ARDSNet ventilatory strategy was used. We hypothesized that an open lung approach (OLA) strategy would neutralize the independent effects of respiratory rate on lung inflammation and edema. This hypothesis was tested in an ARDS model using two clinically relevant respiratory rates during OLA strategy. METHODS: Twelve piglets were subjected to an experimental model of ARDS and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated for 6 h according to an OLA strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, wet/dry ratio, regional histology, and cytokines were evaluated. RESULTS: After the ARDS model was established, Cdyn,rs decreased from 21 ± 3.3 to 9.0 ± 1.8 ml/cmH2 O (P < 0.0001). After the lung recruitment maneuver, Cdyn,rs increased to the pre-injury value. During OLA ventilation, no differences in respiratory mechanics, hemodynamics, or EVLW were observed between groups. Wet/dry ratio and histological scores were not different between groups. Cytokine quantification was similar and showed a homogeneous distribution throughout the lung in both groups. CONCLUSION: Contrary to previous findings with the ARDSNet strategy, respiratory rate did not influence lung inflammatory response or pulmonary edema during OLA ventilation in experimental ARDS. This indicates that changing the respiratory rate when OLA ventilation is used will not exacerbate lung injury.

High respiratory rate is associated with early reduction of lung edema clearance in an experimental model of ARDS.

BACKGROUND: The independent impact of respiratory rate on ventilator-induced lung injury has not been fully elucidated. The aim of this study was to investigate the effects of two clinically relevant respiratory rates on early ventilator-induced lung injury evolution and lung edema during the protective ARDSNet strategy. We hypothesized that the use of a higher respiratory rate during a protective ARDSNet ventilation strategy increases lung inflammation and, in addition, lung edema associated to strain-induced activation of transforming growth factor beta (TGF-ß) in the lung epithelium. METHODS: Twelve healthy piglets were submitted to a two-hit lung injury model and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated during 6 h according to the ARDSNet strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, the lungs were excised and wet/dry ratio, TGF-ß pathway markers, regional histology, and cytokines were evaluated. RESULTS: No differences in oxygenation, PaCO2 levels, systemic and pulmonary arterial pressures were observed during the study. Respiratory system compliance and mean airway pressure were lower in LRR group. A decrease in EVLW over time occurred only in the LRR group (P < 0.05). Wet/dry ratio was higher in the HRR group (P < 0.05), as well as TGF-ß pathway activation. Histological findings suggestive of inflammation and inflammatory tissue cytokines were higher in LRR. CONCLUSION: HRR was associated with more pulmonary edema and higher activation of the TGF-ß pathway. In contrast with our hypothesis, HRR was associated with less lung inflammation.

Blood catalsase polymorphism: some immunological aspects.

The immunological properties of the erythrocyte catalase of mice-normal (wild type) strain, one lacking catalase (acatalasemic), and four with only slight catalase activity (hypocatalasemic strains)-have been investigated. Agardiffusion tests and antigen titration of red-cell lysates against rabbit antiserum to catalase from normal mouse blood showed that immunologically identical catalase protein was present in large amounts in the acatalasemic as well as in the hypocatalasemic mutant strains. Despite lack of catalatic activity, the erythrocytes lacking catalase as well as those with only a little catalase contain catalase protein that has been modified at the site of enzyme activity, although the antigenic determinants are identical with those of normal catalase protein. This mutation is purely structural, being characterized by modification of the enzyme active site but not of the antigenic site.

Carcinogenic and antitumor effects of aminotriazole on acatalasemic and normal catalase mice.

Dietary 3-amino-1H-1,2,4-triazole (AT), although carcinogenic when administered alone, was an antitumor agent when combined with certain other carconogenic stimuli. The carcinogenic effect was prominent in the livers of C3H mice; thyroid tumors were less common because they required a longer period of development, and the life-span of the animal was shortened by the AT diet. The antitumor effects of AT included: delay in appearance of mammary tumors, striking reduction in gamma-radiation-induced lymphomas, and sharp reduction in neutron radiation-induced harderian gland and ovarian tumors. On an AT diet, the inbred C3H acatalasemic mouse substrain developed more liver tumors, starting earlier, than did the C3H normal catalase substrain. We suggest that our findings pointed to a possible relevance of catalase and H2O2 in carcinogenesis. The most probable mechanism for the increased incidence of liver tumors in AT-treated acatalasemic mice was the diminished rate of degradation of endogenous H2O2.

Purification and immunochemical characterization of aldehyde dehydrogenase from 2-acetylaminofluorene-induced rat hepatomas.

1. A series of aldehyde dehydrogenase isozymes (aldehyde:NAD (P)+ oxidoreductase, EC 1.2.1.5), has been purified from hepatomas induced in Sprague-Dawley rats by 2-acetylaminofluorene. 2. The functional hepatoma-specific aldehyde dehydrogenase isozymes exist as 105 000-dalton dimers composed to two subunits of 53 000 daltons. Isoelectric points of the purified isozymes are 6.9-7.2. 3. Antiserum to these purified hepatoma-specific aldehyde dehydrogenases has been produced and the immunological relationships of these isozymes to their normal liver counterpart have been studied. Results of Ouchterlony double diffusions, agar-gel immunoelectrophoresis and polyacrylamide gel and agar immunoelectrophoresis indicate that anti-hepatoma aldehyde dehydrogenase antiserum cross-reacts with normal liver aldehyde dehydrogenase.

Characterization of a chicken cDNA encoding the retinoblastoma gene product.

We have isolated a chicken cDNA that encodes the retinoblastoma susceptibility gene product (RB). The predicted amino acid sequence of the chicken RB protein is highly similar to that of the mouse, human and Xenopus RB proteins in regions of known functions; however, chicken RB has distinct species-specific differences, including a shorter N-terminal region as compared to the mouse and human RB proteins. In vitro-translated chicken RB co-migrates on SDS-polyacrylamide gels with endogenous RB synthesized in transformed chicken spleen cells. Finally, chicken RB is located in the nucleus of chicken embryo fibroblasts when overexpressed from a retroviral vector.

Targeted disruption of the mouse phospholipase C beta3 gene results in early embryonic lethality.

In order to investigate the biological function of phosphatidylinositol-specific phospholipase C (PLC) we generated mutant mice by gene targeting. Homozygous inactivation of PLCbeta3 is lethal at embryonic day 2.5. These mutants show poor embryonic organization as well as reduced numbers of cells. Identical phenotypes were recorded in homozygous mutants generated from two independently targeted embryonic stem cell clones. Heterozygous mutant mice, however, are viable and fertile for at least two generations. We also showed that mouse PLCbeta3 is expressed in unfertilized eggs, 3-cell and egg cylinder stages of embryos. In conclusion, these results indicate that PLCbeta3 expression is essential for early mouse embryonic development.

Structural requirements for neuropeptide Y18-36-evoked hypotension: a systematic study.

It has been shown that NPY and select C-terminal fragments of NPY that evoke a hypotensive response upon intraarterial administration in the rat also cause mast cell degranulation and histamine release in vitro. Additionally, elevation of plasma histamine levels has been observed concomitant with the hypotensive effect of NPY and various C-terminal fragments. In order to investigate whether the hypotensive response to NPY18-36 is correlated to this observed elevation of histamine in vivo, we sought to characterize the structural requirements for each activity. We conducted a systematic replacement of each amino acid in NPY18-36 by its D-isomer. Additionally, various modifications were made to the N- or C-terminii of NPY18-36. The following rank order of potency was obtained for the hypotensive action of these analogues of NPY18-36 relative to NPY18-36. Only one analogue ([D-Tyr21]NPY18-36) exhibited significantly enhanced potency. Eleven analogues of NPY18-36, ([D-Thr32]-, [D-Arg35]-, [D-Ile31]-, [D-Leu30]-, [D-Tyr27]-, [D-Ser22]-, [D-Tyr36]-, [D-Gln34]-, [D-Asn29]-, [D-Ala23]-, and [D-Arg33]NPY18-36) were equipotent with NPY18-36. Four analogues ([D-His26]-, [D-Ile28]-, and [D-Ala18]NPY18-36 and -NPY18-27) had reduced potency (10-80%) while eight analogues ([D-Arg19]-, [D-Tyr20], [D-Leu24]-, [D-Arg25]-, [Ac-Ala18]-, [Me-Ala18]-, [desamino-Ala18]NPY18-36 and NPY18-36 free acid) failed to produce a significant hypotensive response (less than 10%) at the doses tested. The sensitivity of NPY18-36 to chiral inversion of single residues or other modifications at the N-terminus suggested the presence of a conformationally well defined N-terminal pharmacophore. Additionally, five NPY18-36 analogues were tested for elevation of plasma histamine levels. The rank order of potency ([D-Thr32]NPY18-36 = [D-Tyr21]NPY18-36 much greater than NPY18-36 greater than [D-Ala18]NPY18-36 greater than [Ac-Ala18]NPY18-36) was correlated with each analogue's potency at evoking a hypotensive response. In contrast, NPY1-36 failed to evoke an elevation in plasma histamine levels despite its hypotensive effects. Hence, we conclude that the magnitude of the hypotensive response evoked by an NPY18-36 analogue is primarily a function of its ability to elevate plasma histamine levels. However, the mechanism underlying NPY1-36-evoked hypotension appears to be different.

Defining structural requirements for neuropeptide Y receptors using truncated and conformationally restricted analogues.

To further elucidate the minimum bioactive conformation of neuropeptide Y (NPY), a series of truncated and conformationally constrained analogues has been prepared. The synthesis and purification of these peptides was achieved using routine laboratory strategies and techniques. Parent molecules consisted of the native NPY N-terminal 1-4 and C-terminal 25-36 segments, having the residue 5-24 core replaced by either a single flexible omega-aminoalkanoic acid, or a more rigid Pro-Gly or Pro-DAla sequence which was expected to constrain a putative turn, and allow the N- and C-termini to align. Cross-linking between residues 2 and 27 through lactamization using side-chain length and chirality suggested by computer simulations, resulted in cyclo-(2/27)-des-AA7-24[Glu2,Gly6,DDpr27]NPY that exhibited very high affinity (Ki = 0.3 versus 0.3 nM for NPY) for the Y2 receptor using SK-N-BE2 human neuroblastoma cells, yet very low affinity for the Y1 receptor using SK-N-MC human neuroblastoma cells (Ki = 130 versus 2.0 nM for NPY). The added constraint resulting from bridging in this analogue as well as in others suggested that the combination of the deletion of residues 5-24 and the introduction of an internal ring produced exclusive selectivity for the Y2 receptor with little or no loss of affinity. The tolerance of structural recognition was further demonstrated as a second ring was introduced which was expected to constrain the amphiphilic alpha-helix, resulting in the full Y2 agonist dicyclo (2/27,28/32)-des-AA7-24 [Glu2,32,DAla6,DDpr27,Lys28]NPY. Improvement of Y1 binding activity was achieved only by including more residues (des-AA10-17) in the central PP-fold region, while allowing limited flexibility of the termini. Although the length of the bridge seemed to have little effect on binding potency, changes in the location of and chirality at the bridgehead resulted in analogues with different binding affinities. Combination of optimum structural modifications resulted in cyclo-(7/21)-des-AA10-18[Cys7,21]NPY, an analogue shortened by 25% but retaining comparable binding properties to that of native NPY at Y1 and Y2 receptor types (Ki = 5.1 and 1.3 nM, respectively).

Estimation of the C-cell numbers in rat thyroid glands using the optical fractionator.

We estimated the total number of calcitonin-immunoreactive C-cells in rat thyroid gland using the optical fractionator, the unbiased stereological method for estimation of numbers. It was necessary first to use a fixative composed of formalin, acetic acid, and ethanol to distinctly visualize the C-cells. The 40-microm-thick sections had to adhere to chromalum-gelatin-coated Superfrost Plus glass slides, and the immunostaining technique had to stain the C-cells evenly throughout the whole sections. Because the C-cells were irregularly distributed in the thyroid tissues, their counting required screening of about 500 fields per lobe, but the number of C-cells counted need not be high, about 90 per lobe. We estimated that rats have 185,000 +/- 42,000 C-cells (mean +/- SD; n - 7). The C-cell population did not differ significantly between the two lobes of a given rat, but it varied markedly among rats. The biological differences among the animals contributed 83% to the observed variability, whereas the methodological uncertainty contributed 17%. The serum levels of calcitonin and calcium were not closely correlated to the C-cell numbers. Our results indicate that variability in C-cell experiments can be reduced most effectively by increasing the number of animals used. However, the similar C-cell frequency found in the two thyroid lobes of each rat allows the use of one uniformly sampled lobe for quantification and the other lobe for further analysis.

American Academy of Pediatrics. Committee on Adolescence. Contraception in adolescents.

The risks and negative consequences of adolescent sexual intercourse are of national concern, and promoting sexual abstinence is an important goal of the American Academy of Pediatrics. In previous publications, the American Academy of Pediatrics has addressed important issues of adolescent sexuality, pregnancy, sexually transmitted diseases, and contraception. The development of new contraceptive technologies mandates a revision of this policy statement, which provides the pediatrician with an updated review of adolescent sexuality and use of contraception by adolescents and presents current guidelines for counseling adolescents on sexual activity and contraceptive methods.

Blood sampling procedures influence serum calcitonin concentrations in rats.

The aim of this study was to determine whether serum calcitonin (S-CT) in rats is influenced by the method of taking blood samples. Sampling during halothane anaesthesia, after repeated administration of anaesthesia after a 14-day interval, and sampling without the use of anaesthetics (i.e. after the rats were made unconscious by stunning), resulted in different S-CT values (P < or = 0.001), whereas Ca2+ levels were not affected. In thyroidectomized rats, the S-CT values after stunning were not significantly different whereas those in sham-operated rats were different (P < or = 0.01). The possibility that anaesthesia may suppress stunning-induced changes in S-CT was explored in three other groups of rats subjected to halothane anaesthesia, stunning and stunning under halothane anaesthesia respectively. Although the S-CT level was highest after stunning and lowest in halothane-anaesthetized rats (P < or = 0.001), anaesthesia did not suppress the effect of stunning on S-CT. In conclusion the effect of sampling procedures must be considered in studies on the levels of S-CT in rats.

Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: parameters involved in standardization.

Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody.

Clinical and histologic features of pityriasis lichenoides et varioliformis acuta in children.

Pityriasis lichenoides et varioliformis acuta (PLEVA) is commonly thought of as a disease of young adults, yet we identified five cases, involving patients who were 3, 5, 6, 8, and 11 years of age, among 13,000 consecutive specimens submitted to a general dermatopathology laboratory during a 15-week period. The clinical and histologic features of PLEVA in our cases were similar to those reported for adults, except that no lesions were observed on the scalp or mucous membranes of children. A high index of suspicion and biopsy specimens of suspected lesions are often needed to differentiate PLEVA from other papular and crusted eruptions seen in the pediatric age group. These include reactions to arthropods, Gianotti-Crosti syndrome, varicella, and erythema multiforme. Histologically, papular eczema and pityriasis rosea may be misdiagnosed as PLEVA.

Systemic antibiotic therapy of secondary infected dermatitis.

Systemic cloxacillin therapy of secondarily infected dermatitis cloxacillin therapy become apparent produces a significant increase in healing when compared to a placebo. The effects of systemic after five days of treatment. A single pretreatment culture was not helpful in directing therapy.

Long-term testosterone stimulation induces hyperplasia in the guinea-pig prostate.

The relation between supraphysiologic circulating testosterone levels and prostatic diseases is unclear and difficult to study in men. Animal models may be advantageous. Based on a pilot study, testosterone enantate 50 mg (n=12) or 25 mg (n=12) was administered to guinea-pigs intramuscularly every 3 weeks, for either 7 or 14 months. The histopathology of the prostate was described. Epithelial hyperplasia was found in 14/21 animals receiving testosterone and in 7/12 very old animals, but no such changes were found in the sham or castrated animals. Testosterone stimulation seems to induce epithelial hyperplasia, but not cancer, in the guinea-pig prostate.

Monitoring of cortical excitability during induced hypotension in aneurysm operations.

The electrical excitability of the cortex was monitored during craniotomy in 10 patients with ruptured aneurysms, to test their ability to tolerate hypotensive anesthesia. Excitability was assessed by measuring the direct cortical response, a response evoked and recorded from the surface of the brain. Previous animal experiments had shown that this response can be used as an index of cerebral blood flow. In the 10 patients the response progressively declined as the blood pressure was lowered and increased when the pressure was restored. Observation of the direct cortical response during aneurysm operations is a practical method for evaluating the electrophysiological responsiveness of the cortex during hypotension, and the authors suggest that decreases in the amplitude of the response are related to decreased in local cerebral blood flow.

Applications of random sampling to virtual screening of combinatorial libraries.

We describe statistical techniques for effective evaluation of large virtual combinatorial libraries (> 10(10) potential compounds). The methods described are used for computationally evaluating templates (prioritization of candidate libraries for synthesis and screening) and for the design of individual combinatorial libraries (e.g., for a given diversity site, reagents can be selected based on the estimated frequency with which they appear in products that pass a computational filter). These statistical methods are powerful because they provide a simple way to estimate the properties of the overall library without explicitly enumerating all of the possible products. In addition, they are fast and simple, and the amount of sampling required to achieve a desired precision is calculable. In this article, we discuss the computational methods that allow random product selection from a combinatorial library and the statistics involved in estimating errors from quantities obtained from such samples. We then describe three examples: (1) an estimate of average molecular weight for the several billion possible products in a four-component Ugi reaction, a quantity that can be calculated exactly for comparison; (2) the prioritization of four templates for combinatorial synthesis using a computational filter based on four-point pharmacophores; and (3) selection of reagents for the four-component Ugi reaction based on their frequency of occurrence in products that pass a pharmacophore filter.