The sensitivity and specificity of the complement fixation (CF) test for the diagnosis of Babesia infections in equines was assessed, using the indirect fluorescent antibody (IFA) test as a reference. Antibodies were first detected between 11 and 20 days post infection (dpi) in the CF test and between 7 and 14 dpi in the IFA test in ponies infected experimentally with B. equi (USDA strain). The CF test became negative in four of five ponies 63-174 dpi although B. equi was demonstrated microscopically in two of these four ponies up to 364 and 455 dpi. The IFA test remained positive up to 476 dpi (end of the examination period). Ponies infected experimentally with B. caballi (USDA strain) showed positive reactions in the CF test at first between 13 and 15 dpi and in the IFA test 10 or 11 dpi. The CF test became negative in two of three ponies 80 and 140 dpi, whereas the IFA test remained positive up to 190 dpi (end of the examination period). Cross-reactions of sera with heterologous antigens occurred at dilutions of 1/5 in the CF test and up to 1/20 in the IFA test. A total of 3944 CF tests was performed on 3765 horses from various European countries during 1980-1984. Sera that gave positive or trace CF reactions were retested in the IFA test. All 123 CF-positive sera were also IFA-positive and 26 of 31 sera (B. equi) and 11 of 32 sera (B. caballi) showing CF trace reactions were positive in the IFA test. Sera of two CF-negative horses were positive in the IFA test (B. equi); one of these horses was also positive upon microscopic examination. In seven of 21 horses repeatedly examined over longer periods the IFA titers (B. equi) persisted for up to 454 days longer than the CF titers. Sera of horses from highly endemic areas gave the following reactions: Sudan, 62 of 91 sera CF- and 86 of 91 IFA-positive; Zaire, 58 of 75 sera CF- and 72 of 75 IFA-positive; Columbia, 51 of 56 sera CF- and 56 of 56 IFA-positive; Brazil, 17 of 25 sera CF- and 21 of 25 IFA-positive. Only B. equi infections were demonstrated in Zaire. The combined use of the CF and IFA tests is recommended for safe identification of equine Babesia infections.

Chronic pulmonary infections contribute significantly to the morbidity and mortality of patients with CF. The primary pathogens are Pseudomonas aeruginosa (PA) and Staphylococcus aureus. Hemophilus influenzae has been isolated from a significant number of patients also. A number of the beta-lactam and aminoglycoside antibiotics reportedly have altered pharmacokinetic variables in CF. Therapy of acute pulmonary deterioration consists of intravenous antibiotics for two weeks. Antibiotic selection is based on culture and sensitivity results. Currently, the combination of a broad-spectrum penicillin and an aminoglycoside seems to provide the best results. Prophylactic antibiotics are effective if the primary isolates are sensitive to the agents used. Chronic PA infections are problematic because effective oral agents are not available. Aerosolized antibiotics do not improve results over adequate systemic therapy for acute exacerbations. Questions regarding optimal dosages, frequency, and duration of therapy remain.

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosa strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r>>/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r>>/= 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0. 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- secretion by airway epithelial cells. In CF, cAMP does not activate Cl- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[beta-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl- currents and restore cAMP-activated Cl- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[gamma-thio]triphosphate and AlF4- reduce Cl- currents and inhibit cAMP from activating Cl- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[beta-thio]diphosphate and in normal cells, cAMP activates a Cl- conductance that has properties similar to CF transmembrane-conductance regulator Cl- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl- secretion in CF.

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K+ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K+ channel blockers. Barium (5.10(-3) mol/l), tetraethylammoniumchloride (5.10(-3) mol/l), quinidine (10(-3) mol/l) and lidocaine (5.10(-3) mol/l), when added to the cytosolic side, inhibited K+ channels reversibly. An increase in the calcium concentration from 10(-7) mol/l to 10(-6) mol/l led to a marked increase in the open channel probability (Po). Further increases in Ca2+ concentration increased Po only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10(-4) mol/l to 10(-3) mol/l. Non hydrolysable adenosine 5'-[beta, gamma-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K+ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na+ + K+)-pump and the basolateral K+ conductance.

Antibodies against a lysosomal membrane antigen (A-Ly-M) have recently been obtained and characterized (Reggio, H., D. Bainton, E. Harms, E. Coudrier, and D. Louvard, 1984, J. Cell Biol., 99:1511-1526). They recognize a 100,000-mol-wt antigen immunologically related to a purified [H+,K+]ATPase from pig gastric mucosa. In the present study, we have localized this antigen during adsorptive endocytosis in rat prolactin cells in culture using cationized ferritin (CF) as a tracer. CF was rapidly internalized (after 5 min) in coated pits and vesicles that were labeled by antibodies against clathrin. The tracer was then delivered (after 15 min) to vacuoles and multivesicular bodies. These structures were labeled with A-Ly-M. These organelles were devoid of acid phosphatase activity. At later stages (after 30 min) CF was observed within larger structures that were strongly stained by A-Ly-M and displayed a strong acid phosphatase activity. These findings clearly indicate that A-Ly-M react with prelysosomal and lysosomal compartments involved in the endocytic pathway in cultured prolactin cells. The membrane of these structures therefore contains antigenic determinant(s) related to the 100,000-mol-wt polypeptide. Our results suggest that the prelysosomal structure stained by A-Ly-M may represent in GH3 cells the acidic prelysosomal compartment recently described in the early steps of endocytosis in other cell types (Tycko, B., and F. R. Maxfield, 1982, Cell, 28:643-651).

We investigated the utility of PCR to detect Burkholderia cepacia directly in sputum samples at two cystic fibrosis (CF) centers serving children and adults. Following liquefaction of the sputa by using N-acetyl-L-cysteine, DNA was isolated and analyzed by PCRs with three different primer pairs directed toward bacterial rRNA loci. Two primer pairs were putatively specific for B. cepacia. The other pair, which universally amplifies a band from all bacteria, served as a control. Sputum samples were obtained from 219 patients and analyzed independently by culture and by PCR to detect B. cepacia. The analyses were performed blinded with respect to each other. The results of the PCR with sputa demonstrated that the primers directed to the 16S loci demonstrated approximately 95% concordance with culture results and were more specific than those amplifying the 16S to 23S spacer region. In addition, the 16S primer pair putatively identified B. cepacia in seven patients whose sputa were culture negative at this time. Of these culture-negative patients, five had sputum samples that were culture positive for B. cepacia either prior or subsequent to this study. The results of this study indicate the utility of PCR as a diagnostic method for the rapid identification of B. cepacia in sputum samples of CF patients. We anticipate that improvements in our taxonomic understanding may allow the design of more specific primers for detection of each species of the B. cepacia complex in sputum samples.

There is a significant phenotypic variance among cystic fibrosis (CF) patients. Due to the role of TGF-betaCF pathogenesis. TGF-beta 1 codons 10 and 25 were genotyped in 118 Czech CF patients and 268 controls by PCR-ARMS. Difference between CF and controls was found at codon 10, lower frequency of T/T homozygotes, and codon 25, higher frequency of G/C heterozygotes. We did not prove the association of TGF-betaCF, however, the TT (codon 10)/GG (codon 25) genotype was preferentially associated with CF-related liver disease and diabetes. Independent of the TGF-betaCF, both extremes, highest or lowest TGF-beta 1 production, were associated with impaired lung function.

Isolated CF-1 mouse bone marrow cells were exposed for 1 hr to 5-fluorouracil (FUra) at concentrations from 1.8 to 50 microM and then washed and suspended in a soft agar growth medium to assess the effect on toxicity (measured as reduction in colony growth compared to control). These data were used to determine specific toxic concentrations ranging from 25 to 90% lethal doses. Subsequent studies examined in parallel the effect of these toxic concentrations of FUra on the possible sites of toxicity including: (a) inhibition of thymidylate synthetase activity using a modified 3H release assay; (b) incorporation of FUra into RNA (FUra-RNA); and (c) incorporation of FUra into DNA (FUra-DNA). Thymidylate synthetase activity was slightly decreased (75% of control) after 1-hr exposure to a 50% lethal dose and was not significantly further reduced as the FUra concentration was increased to an 85% lethal dose. Furthermore, subsequent exposure of FUra-treated cells to a nontoxic thymidine dose (5 microM) failed to reverse toxicity. FUra-RNA increased during 1-hr exposure to increasing concentrations of FUra (25 to 90% lethal doses). Although initially suggesting a relationship between the level of FUra-RNA and toxicity, subsequent studies in cells exposed to FUra in the presence of uridine demonstrated a significantly decreased toxicity while, at the same time, a minimal decrease of FUra in RNA. In contrast, FUra-DNA was significantly decreased in the presence of uridine and correlated with decreased toxicity. In additional subsequent studies, an apparent decrease in subsequent DNA synthesis was observed (measured by 32P or [3H]thymidine incorporation into DNA) as the level of FUra-DNA increased. In conclusion, FUra is demonstrated to be incorporated into DNA of isolated CF-1 mouse bone marrow cells, and the level of FUra-DNA appears to be closely associated with toxicity and inhibition of further DNA synthesis. The parallel studies of thymidylate synthetase activity and FUra-RNA suggest that FUra-DNA may be an unrecognized mechanism of FUra toxicity in these cells.

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection>> or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection>> or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by>> or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB> or =80% inhibition; PD98059,>> or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.

OBJECTIVE

To evaluate prospectively the prevalence and pathophysiology of anorectal dysfunction following radiation therapy (RTH) for localized carcinoma of the prostate.

METHODS

The following parameters of anorectal function were evaluated in each of 35 patients (aged 55-82 years) with localized prostatic carcinoma treated with RTH either to a dose of 55 Gy/20 fractions/4 weeks (18 patients) or 64 Gy/32 fractions/6.5 weeks (17 patients), before RTH and 4-6 weeks and at a mean (+/- SD) of 1.4 (+/- 0.2) years after its completion: (1) anorectal symptoms (questionnaire), (2) anorectal pressures at rest and in response to voluntary squeeze and increases in intra-abdominal pressure (multiport anorectal manometry), (3) rectal sensation (balloon distension) and (4) anal sphincteric morphology (endoanal ultrasound).

RESULTS

All but 1 patient completed three series of measurements. RTH had no effect on anal sphincteric morphology. The increase in frequency of defecation and fecal urgency and incontinence scores previously reported in the patients 4-6 weeks after RTH were sustained 1 year later (p < 0.001, p < 0.001, and p < 0.05, cf. baseline, respectively). At this time, 56% (19 of 34), 50% (17 of 34) and 26% (9 of 34) of the patients had increased frequency of defecation, fecal urgency, and incontinence, respectively. Decreases in anal sphincteric pressures at rest and in response to voluntary squeeze recorded in the patients 4-6 weeks after RTH were not sustained 1 year later but the volumes of rectal distension associated with perception of the stimulus and desire to defecate were lower compared with baseline volumes (p < 0.01 and p < 0.05, respectively), reflecting heightened rectal sensitivity in the patients. There was no difference in measurements between the two radiation dose regimens. Univariate logistical regression analysis was performed on patients who had experienced increased symptom scores or decreases in recorded motor and sensory manometric parameters at 1 year, cf. baseline. The predictor variables used included individual patient tumor and treatment characteristics as well as individual patient symptom scores and parameters of anorectal motor and sensory function at baseline and 4-6 weeks after RTH. The results of the univariate logistical regression analysis showed that (1) frequency of defecation at 4-6 weeks and (2) rectal volumes at baseline both for (a) perception (p < 0.001) and (b) desire to defecate (p < 0.001), predicted significantly for the patients who had symptoms and signs of anorectal dysfunction at 1 year. Individual patient tumor and treatment-related variables tested, in contrast, had no predictive significance.

CONCLUSIONS

Anorectal symptoms following RTH for prostatic carcinoma are common and persist at least until 1 year after its completion and are associated with objective evidence of heightened rectal sensitivity.

OBJECTIVE

To evaluate outcomes of intermediate- and high-risk prostate cancer patients on a prospective dose-escalation study of pelvic external-beam radiation therapy (EBRT) combined with high-dose-rate (HDR) brachytherapy boost.

METHODS

From November 1991 to April 2003, 197 patients were treated for intermediate- and high-risk disease features. All patients had prostate-specific antigen>10 ng/ml, Gleason score>or=7, or clinical stage>or=T2b, and all received pelvic EBRT (46 Gy) while receiving either two or three HDR boost treatments. HDR dose fractionation increased progressively and was divided into two dose levels. The mean prostate biologic equivalency dose was 88.2 Gy for the low-dose group and 116.8 Gy for the high-dose group (alpha/beta=1.2). Clinical failure was either local failure or distant metastasis; clinical event-free survival (cEFS) was defined as patients who lived free of clinical failure.

RESULTS

Median follow-up was 4.9 years. The 5-year rates were as follows: biologic failure (BF), 18.6%, clinical failure (CF), 9.8%, cEFS 84.8%, cause-specific survival (CSS), 98.3%, and overall survival (OS), 92.9%. Five-year biochemical failure (68.7% vs. 86%, p<0.001), CF (6.1% vs. 15.6%, p=0.04), cEFS (75.5% vs. 91.7%, p=0.003), CSS (95.4% vs. 100%, p=0.02), and OS (86.2% vs. 97.8%, p=0.002) were significantly better for the high-dose group. Multivariate analysis showed that high-dose group (p=0.01, HR 0.35) and Gleason score (p=0.01, HR 1.84) were significant variables for cEFS. Multivariate analysis showed that high-dose group (p=0.01, HR 0.14) and age (p=0.03, HR 1.09 per year) were significant variables for overall survival.

CONCLUSIONS

There is a strong dose-response relationship for intermediate- to high-risk prostate cancer patients. Improved locoregional control with higher radiation doses alone can significantly decrease biochemical and clinical failures.

Capsid, envelope, and nonvirion-associated soluble components of type 1 and type 2 herpes simplex virus (HSV) were obtained from infected monolayer cell cultures and used as complement fixation (<em>CF</em>) antigens. Capsids were prepared by treatment of cells with the nonionic detergent Nonidet-P40, envelope material by treatment of virions with ether and high pH, and soluble components were obtained from culture fluids of untreated cells. Serological studies with experimental anti-herpesvirus sera indicate that these serotypes share cross-reacting envelope, capsid, and soluble antigens with each other and with herpesvirus <em>B</em> but not with varicella virus. In addition, animals immunized with crude HSV preparations contain high levels of <em>CF</em> antibody (1:32 to 1:64) to soluble antigens, whereas sera from humans who have experienced natural infection contain low levels of antibody (</=1:8) to this antigen. Further testing with reference, capsid, and envelope antigens indicates that antibody levels to reference and capsid antigens are about the same in sera from healthy humans, whereas antibody to the envelope is decidedly lower in these sera. Herpes convalescent-phase sera contain higher levels of antibody to reference and envelope antigens than to capsid antigen.

Patients with combined hyperlipidemia and low high-density lipoprotein (HDL) cholesterol levels may benefit from combination therapy with a statin and niacin; therefore, we assessed the efficacy and safety of rosuvastatin and extended-release (ER) niacin alone and in combination in 270 patients with this atherogenic dyslipidemia. Men and women>> or =18 years with fasting total cholesterol levels>> or =200 mg/dl, triglycerides 200 to 800 mg/dl, apolipoprotein B>> or cf=110 mg/dl, and HDL cholesterol <45 mg/dl were randomized to 1 of 4 treatments in this 24-week, open-label, multicenter trial: rosuvastatin 10 to 40 mg; ER niacin 0.5 to 2 g; rosuvastatin 40 mg/ER niacin 0.5 to 1 g; or rosuvastatin 10 mg/ER niacin 0.5 to 2 g. Percent changes from baseline in low-density lipoprotein (LDL) cholesterol, non-HDL cholesterol, and other lipid measurements at week 24 were determined by analysis of variance, with statistical testing performed separately between the rosuvastatin monotherapy group and each remaining treatment group. Daily doses of rosuvastatin 40 mg reduced LDL and non-HDL cholesterol significantly more than either ER niacin 2 g or rosuvastatin 10 mg/ER niacin 2 g (-48% vs -0.1% and -36% for LDL cholesterol and -49% vs -11% and -38% for non-HDL cholesterol, respectively; p <0.01 for all comparisons); no additional reduction in LDL or non-HDL cholesterol was observed with the combination of rosuvastatin 40 mg/ER niacin 1.0 g (-42% and -47%; p = NS). Triglyceride reductions ranged from -21% (ER niacin monotherapy) to -39% (rosuvastatin 40 mg/ER niacin 1 g), but no observed differences were statistically significant. Compared with rosuvastatin alone, rosuvastatin 10 mg/ER niacin 2 g produced significantly greater increases in HDL cholesterol (11% vs 24%, p <0.001) and apolipoprotein A-I (5% vs 11%, p <0.017). Similar increases in HDL cholesterol and apolipoprotein A-I were noted between the monotherapy groups. Over 24 weeks, rosuvastatin alone was better tolerated than either ER niacin alone or the combinations of rosuvastatin and ER niacin.

The urinary excretion rate (ng/h/1.73 m2) of prostanoids was determined with a capillary gas-liquid chromatographic mass spectrometric method in 19 patients with cystic fibrosis (CF) aged 1-29 years. Patients with CF showed an increased excretion of prostaglandin E2 metabolites (PGE-M) and thromboxane B2 and its metabolites at all ages. An imbalance in the excretion pattern of thromboxane B2 metabolites also suggested a relative impairment of beta-oxidation. There was no increased excretion of dinor-6-keto-PGF1 alpha, indicating normal prostacyclin biosynthesis. No correlation was found to genotype, clinical score, lung function or bacterial colonization but a significant negative relation was found between the main prostanoids in the urine and serum phospholipid levels of essential fatty acids. The results show that, contrary to the generally accepted decrease of prostanoid excretion in essential fatty acid deficiency, patients with CF increase their production parallel to the development of the deficiency. Since prostanoid synthesis is rate limited by arachidonic acid release, our data support a previously presented hypothesis about a pathological regulation of the release of arachidonic acid in CF.

The Mg(2+) cofactor of the F(1)F(0) ATP synthase is required for the asymmetry of the catalytic sites that leads to the differences in affinity for nucleotides. Vanadyl (V(IV)=O)(2+) is a functional surrogate for Mg(2+) in the F(1)-ATPase. The (51)V-hyperfine parameters derived from EPR spectra of VO(2+) bound to specific sites on the enzyme provide a direct probe of the metal ligands at each site. Site-directed mutations of residues that serve as metal ligands were found to cause measurable changes in the (51)V-hyperfine parameters of the bound VO(2+), thereby providing a means by which metal ligands were identified in the functional enzyme in several conformations. At the low-affinity catalytic site comparable to beta(E) in mitochondrial F(1), activation of the chloroplast F(1)-ATPase activity induces a conformational change that inserts the P-loop threonine and catch-loop tyrosine hydroxyl groups into the metal coordination sphere thereby displacing an amino group and the Walker homology B aspartate. Kinetic evidence suggests that coordination of this tyrosine by the metal when the empty site binds substrate may provide an escapement mechanism that allows the gamma subunit to rotate and the conformation of the catalytic sites to change, thereby allowing rotation only when the catalytic sites are filled. In the high-affinity conformation analogous to the beta(DP) site of mitochondrial F(1), the catch-loop tyrosine has been displaced by carboxyl groups from the Walker homology B aspartate and from betaE197 in Chlamydomonas CF(1). Coordination of the metal by these carboxyl groups contributes significantly to the ability of the enzyme to bind the nucleotide with high affinity.

1. The receptive field properties, laminar distribution and afferent connectivity of cells in area 18 of the cat are described. 2. Testing with both moving and stationary stimuli revealed three main receptive field types which have been termed S, C and B, respectively (cf. Henry, 1977; Henry, Lund & Harvey, 1978). All three classes may show end-zone inhibition and units exhibiting this property have been designated SH, CH and BH. 3. S cells can be divided into spatially separate lights and/or dark edge response regions when tested with moving edges and usually have separate ON and/or OFF areas when tested with stationary flashing stimuli. They are the most commonly encountered cell type in area 18 and occur most frequently in laminae IIIb, IVa and VI. 4. Both C and B cells have spatially coincident light and dark edge response regions and give mixed ON and OFF discharges when tested with stationary flashing stimuli. Compared to B cells however, C cells have large receptive fields, they are broadly tuned for stimulus orientation and generally have a relatively high rate of spontaneous activity. C cells are more common than B cells and are encountered most often in laminae IVb and V. 5. Electrical stimulation of the optic chiasm (OX) and optic radiation (OR) was used to examine the afferent connectivity of parastriate neurons. Cells driven from both OX and OR have been divided into two main groups and it is argued that group 1 cells are directly, and group 2 cells are indirectly, excited by rapidly conducting afferent fibres. Group 1 cells are found most often in laminae IIIb, IVa, IVb and VI, and their distribution closely follows the anatomically defined laminar disposition of geniculocortical afferent terminals. Group 2 neurones predominate in laminae II-IIIa, IIIA and V. 6. The majority of S and SH cells are directly driven, whereas most C and CH cells have OX and OR latencies suggestive of indirect activation by thalamic afferents. 7. The intrinsic organization and possible functional role of area 18 is discussed in the light of these results.

This study illustrates the diversity of feeding responses of individually polyphagous southern armyworms, Spodoptera eridania, to plants with differing allelochemics. In spite of the near optimal leaf water and nitrogen contents of the young foliage, it is apparent that vastly different larval growth performance results from dill, lima bean, and cabbage. Cabbage is the poorest food (as measured by larval growth rates and metabolic costs of processing the plant biomass). Unlike the case with certain other plant species or cultivars that are costly to process, with cabbage, S. eridania does not compensate for low efficiencies (E.C.D.'s) with increased consumption rates (R.C.R.'s). Biochemical or physiological reasons for this inability are unknown.A sequence of foods (changed each 18-24 h) apparently did not add sufficient stress upon the MFO system to be detected in the respiratory expenditures of S. eridania larvae, in spite of the fact that dill is known to contain insecticidal and synergistic chemicals (Lichtenstein et al. 1974). The larval growth performances and metabolic expenditures in these sequences were intermediate between the best food (dill) and the worse (cabbage). Significant differences were observed however between the sequential switching sequences, perhaps indicating that particular periods during the instar are especially more sensitive to certain allelochemics. Actual respiratory costs of the lima bean-cabbage-dill (i.e. B-C-D) sequence were 40-50% higher than observed for the other two sequences and more than 50% higher than the theoretical metabolic costs based on the proportions actually eaten and known costs associated with each food.This study and a related one (Scriber 1981a) illustrate how consumption rates, feeding efficiences, and larval growth of Spodoptera eridania are not species, population, or even individual characteristics, (cf. Fox and Morrow 1981), but instead depend largely upon variations in plant allelochemics and plant nutritional quality (Wolfson 1978; Scriber, 1981 b; Scriber and Slansky 1981). More significantly they illustrate that the food consumed in earlier instars (Scriber 1981 a) as well as the food consumed earlier in an instar can be a major influence upon the observed armyworm growth performances under a given set of environmental conditions.

OBJECTIVE

Arsenic trioxide (ATO), an effective therapeutic agent for acute promyelocytic leukaemia, can cause sudden cardiac death due to long QT syndrome (LQTS). The present study was designed to determine whether ATO could induce cardiac fibrosis and explore whether cardiac fibroblasts (CFs) are involved in the development of LQTS by ATO.

RESULTS

ATO treatment of guinea pigs caused substantial interstitial myocardial fibrosis and LQTS, which was accompanied by an increase in transforming growth factor β1(TGF-β1) secretion and a decrease in ether-à-go-go-related gene (HERG) and inward rectifying potassium channel (I(K1)) subunit Kir2.1 protein levels. ATO promoted collagen production and TGF-β1 expression and secretion in cultured CFs. Whole-cell patch clamp and western blotting showed that treatment with TGF-β1 markedly reduced HERG and I(K1) current densities and downregulated HERG and Kir2.1 protein expression in HEK293 cells stably transfected with the human recombinant HERG channel and in cardiomyocytes (CMs). These changes were completely reversed by treatment with the protein kinase A (PKA) antagonist, H89. CM and CF co-cultures showed that ATO significantly increased TGF-β1 levels in the culture medium, whereas markedly reduced HERG and Kir2.1 protein levels were observed in CMs compared with ATO-treated CMs not co-cultured with CFs. Finally, in vivo administration of LY364947, a pharmacological antagonist of TGF-β signalling, dramatically prevented interstitial fibrosis and LQTS and abolished aberrant expression of TGF-β1, HERG, and Kir2.1 in ATO-treated guinea pigs.

CONCLUSIONS

ATO-induced TGF-β1 secretion from CFs aggravates QT prolongation, suggesting that modulation of TGF-β signalling may provide a novel strategy for the treatment of drug-induced LQTS.

In this review we analyzed the pharmacokinetic basis for high dose treatment with antibiotics of patients with cystic fibrosis. Both our results and those from other well designed pharmacokinetic studies do not support the view that low blood levels of antibacterials are a common feature of CF. We were unable to detect a decrease in absorption, nor could we find evidence for enhanced elimination of antibacterials in CF. Both these factors have been considered responsible for reducing the plasma (and tissue) levels of antibiotics. Most recent studies on kidney function are in agreement with these findings, since neither inulin nor creatinine clearance differ between CF-patients and healthy volunteers. In contrast to previous discussion, the volume of distribution (Vdss) was not elevated for any compound. The rational of weight correction of volume terms like Vdss or total clearance has never been clearly demonstrated and should therefore not be used without prior proof of relevance. Since the variability of pharmacokinetic parameters of antibiotics in CF-patients may be considerable, we suggest that a dose increase of 20-30% may be justified, but cannot agree with two to fourfold increases in dosage as previously proposed and applied in many CF-centers. Until more findings become available for non-adult CF-patients, these conclusions are only valid for adult CF-patients.

The in-vitro investigation of secretory responses of submandibular tissues from three cystic fibrosis (CF) patients and four control subjects showed that responses to a beta-adrenergic stimulus (isoproterenol) were much poorer in CF cells than in control cells. The beta-adrenergic secretory responses of the CF cells (as measured by amylase and mucin secretion) were increased in the presence of 3-isobutyl-l-methyl xanthine, a cyclic nucleotide phosphodiesterase inhibitor. Perhaps an alteration in a regulator of cyclic adenosine monophosphate and Ca2+ metabolism in CF cells is responsible for the decrease in beta-adrenergic function. This proposal would account for the defective regulation of protein secretion, Cl- transport, and Ca2+ homoeostasis in CF exocrine cells and thus might be directly related to the genetic defect.

BACKGROUND

Despite the common practice of clopidogrel loading for coronary stenting, the time dependence and degree of platelet inhibition after this therapy are not well defined. We sought to establish an optimal clopidogrel dosing regimen for sustained platelet inhibition in stented patients.

RESULTS

Platelets were assessed by conventional aggregation with 5 micromol/L adenosine diphosphate (ADP), 1 microg/mL collagen (COLL), and 750 micromol/L arachidonic acid; whole blood aggregation by 1 microg/mL collagen (WBA); shear-induced closure time (CT); contractile force (CF); and expression of 9 surface receptors by flow cytometry in 100 patients undergoing elective stent placement without glycoprotein (GP) IIb/IIIa receptor antagonists. Blood was obtained at baseline and serially over 5 days poststenting after different clopidogrel loading regimens: 300 mg 24 hours before (Group A), 12 hours before (Group B), 3 to 6 hours before (Group C), and 75 mg at the time of intervention (Group D). Before stenting, ADP, COLL, CT, and WBA were reduced by clopidogrel loading (P <.05). CF was not affected by clopidogrel. Before stenting, GP IIb/IIIa expression increased in groups A through C (P <.05), whereas PECAM-1 and CD107a were reduced (P <.05). At 2 hours and 2 days poststenting, platelets, in general, exhibited an increase in activity that was most inhibited by clopidogrel loading. Clopidogrel inhibited GP Ib, platelet/endothelial cell adhesion molecule-1, CD 107a, CD 151, and GP IIb/IIIa expression at day 5 poststenting.

CONCLUSIONS

A 300 mg clopidogrel load given 3 to 24 hours before stenting inhibits platelets at the time of the procedure and reduces poststent activity more than a 75 mg dose given at the time of the procedure. The inhibition of adhesive molecule expression may also contribute an antithrombotic effect. Poststent activation of platelets may warrant higher periprocedural dosing.

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

BACKGROUND

Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease.

METHODS

Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing.

RESULTS

Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found.

CONCLUSIONS

To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.