Delayed poststimulus decrease of phrenic motoneuron output produced by phrenic nerve afferent stimulation.

The immediate effects of phrenic afferent nerve activation on ventilation have been shown to be both excitatory and inhibitory. Long-lasting inhibitory effects on respiratory motoneuron output have been reported after stimulation of afferent nerves from limb muscles. However, whether respiratory muscle afferent nerves can produce this effect is unknown. We therefore hypothesized that activation of phrenic afferent nerves may produce a prolonged decrease of respiratory motoneuron output. Six alpha-chloralose-anesthetized dogs were studied after vagotomy and bilateral carotid sinus nerve section. The dogs were paralyzed, and end-tidal CO2 was controlled by mechanical ventilation. The proximal end of the cut thoracic phrenic nerve was electrically stimulated for 1 min at intensities that produced activation of thin-fiber afferents. The contralateral efferent phrenic integrated electroneurogram (ENG) was recorded. During stimulation, phrenic ENG activity increased. ENG activity was recorded during recovery and reached a peak decrease compared with control of 19 +/- 11% (SD) 9.0 +/- 6 min after stimulation and returned to control after 30 min. A qualitatively similar response was seen after stimulation of the gastrocnemius nerve. We conclude that activation of thin-fiber afferents in the phrenic nerve can produce a delayed and prolonged decrease of respiratory motoneuron output similar to that of limb muscle afferent nerves.

Regional diaphragm shortening measured by sonomicrometry.

Diaphragmatic shortening measured by sonomicrometry has been compared in the two major anatomic segments, costal and crural. Data obtained by videofluoroscopy found a variation in subsegmental shortening within segments (Sprung et al. J. Appl. Physiol. 67: 655-662, 1989). No reproducible pattern of subsegmental shortening has emerged, and the mechanisms leading to this subsegmental variation in shortening are unknown. Therefore, we compared subsegmental shortening in both segments of the diaphragm in seven supine pentobarbital-anesthetized dogs. Seven pairs of sonomicrometer transducers were implanted in the two segments, and subsegmental shortening during spontaneous breathing was measured. To determine potential mechanisms contributing to the variation in shortening, measurements were made during stimulated breathing, after epiphrenic stimulation, and during occluded breaths. We found electrical stimulation at physiological frequencies of 10 and 20 Hz reduced the variation in subsegmental shortening, whereas stimulated breathing did not. Occluded breaths showed a consistent decrease in the amount of shortening, particularly in the dome of the costal diaphragm, compared with shortening in the area of apposition. Comparison of shortening between segments revealed greater crural than costal shortening. We conclude that subsegmental variation in activation can contribute to variation in subsegmental shortening and that the afterload can effect shortening during occluded breaths.

Expiratory muscle activity in the awake and sleeping human during lung inflation and hypercapnia.

Expiratory muscle activity has been shown to occur in awake humans during lung inflation; however, whether this activity is dependent on consciousness is unclear. Therefore we measured abdominal muscle electromyograms (intramuscular electrodes) in 13 subjects studied in the supine position during wakefulness and non-rapid-eye-movement sleep. Lung inflation was produced by nasal continuous positive airway pressure (CPAP). CPAP at 10-15 cmH2O produced phasic expiratory activity in two subjects during wakefulness but produced no activity in any subject during sleep. During sleep, CPAP to 15 cmH2O increased lung volume by 1,260 +/- 215 (SE) ml, but there was no change in minute ventilation. The ventilatory threshold at which phasic abdominal muscle activity was first recorded during hypercapnia was 10.3 +/- 1.1 l/min while awake and 13.8 +/- 1 l/min while asleep (P less than 0.05). Higher lung volumes reduced the threshold for abdominal muscle recruitment during hypercapnia. We conclude that lung inflation alone over the range that we studied does not alter ventilation or produce recruitment of the abdominal muscles in sleeping humans. The internal oblique and transversus abdominis are activated at a lower ventilatory threshold during hypercapnia, and this activation is influenced by state and lung volume.

Canine trachealis muscle shortening and cartilage mechanics.

Canine trachealis muscle will shorten by 70% of resting length when maximally stimulated in vitro. In contrast, trachealis muscle will shorten by only 30-40% when stimulated in vivo. To examine the possibility that an elastic load applied by the tracheal cartilage contributes to the in vivo limitation of shortening, single pairs of sonomicrometry crystals were inserted into the trachealis muscle at the level of the fifth cartilage ring in five dogs. The segment containing the crystals was then excised and mounted on a tension-testing apparatus. Points on the active length-tension curve and the passive length-tension relation of the cartilage only were determined. The preload applied to the muscle before contraction varied from 10 to 40 g (mean 21 +/- 4 g). The afterload applied by the cartilage during trachealis contraction ranged from 13 to 56 g (30 +/- 6 g). The calculated elastic afterloads were substantial and appeared to be sufficient to explain the degree of shortening observed in four of the seven rings; in the remaining three rings, the limitation of shortening was greater than would be expected from the elastic load provided by the cartilage. Additional sources of loading and/or additional mechanisms may contribute to limited in situ shortening. In summary, tracheal cartilage applies a preload and an elastic afterload to the trachealis that are substantial and contribute to the limitation of trachealis muscle shortening in vivo.

Effect of vagal stimulation and parenteral acetylcholine on canine trachealis muscle shortening.

Canine trachealis smooth muscle shortening (TMS) in response to vagal nerve stimulation is approximately 30%, far less than the 70% predicted from in vitro studies. We hypothesized that in vivo airway smooth muscle activation during vagal stimulation may be submaximal, and in this study we wished to determine TMS during maximal activation. TMS was studied in 12 alpha-chloralose-anesthetized dogs during vagal stimulation, systemic acetylcholine injection, and local acetylcholine injection. Bilateral vagal stimulation produced TMS of 26 +/- 5% (SE) length at functional residual capacity (LFRC). Maximal TMS during systemic injection of acetylcholine was 28 +/- 12% LFRC but may have been limited by delivery of acetylcholine to the muscle because asystole occurred at higher concentrations. TMS was greatest during local injection of acetylcholine (48 +/- 7% LFRC). There was a greater increase in pulmonary resistance and decrease in dynamic compliance during systemic acetylcholine injection than during vagal stimulation. We conclude that bilateral vagal nerve stimulation does not maximally activate trachealis smooth muscle but that the maximal shortening achieved with local injection of acetylcholine is still less than isotonic shortening in vitro. These data suggest that maximal shortening in vivo is limited by the afterload provided by the tracheal cartilaginous rings.

Decrease in left ventricular contractility after tumor necrosis factor-alpha infusion in dogs.

Whether systolic contractility or diastolic compliance changes soon after tumor necrosis factor-alpha (TNF-alpha) exposure is not known. Accordingly, we measured hemodynamics, left ventricular contractility using the slope of the end-systolic pressure-volume relationship, and diastolic pressure-volume relationships in six control dogs and in six dogs receiving 60 micrograms.kg-1.h-1 i.v. of TNF-alpha. Mean aortic pressure decreased by 22% 1 h after TNF-alpha infusion and remained decreased (P < 0.05). Cardiac output increased by 19% 1 h after TNF-alpha infusion and remained significantly greater than control values (P < 0.05). Left ventricular contractility decreased by 23% (P < 0.05) 1 h after TNF-alpha infusion and decreased by 52% (P < 0.01) 5 h after TNF-alpha infusion. The diastolic pressure-volume relationship did not change in the TNF-alpha group or the control group. Ejection fraction did not change after TNF-alpha infusion despite the decrease in contractility because afterload decreased. We conclude that TNF-alpha is important in causing the hypotensive, hyperdynamic circulation of sepsis. The new finding that left ventricular contractility is decreased shortly after TNF-alpha infusion suggests that TNF-alpha, or another mediator released very soon after TNF-alpha, is an important myocardial depressant factor.

Protective effects of immunoactive peptide, FK565 against systemic and local infections with herpes simplex virus and murine cytomegalovirus and respiratory tract infection with influenza virus in mice.

The protective effects of FK565 against systemic infections with herpes simplex virus (HSV) and murine cytomegalovirus (MCMV), respiratory tract infection with influenza virus and zosteriform rash with HSV investigated in mice. FK565 showed excellent protective activities against systemic infections with both acyclovir (ACV)-sensitive and -resistant HSV at intravenous and subcutaneous doses of 0.1 and 1 mg/kg and oral dose of 1 mg/kg. FK565 showed superior protective activities at subcutaneous doses of 0.01 and 0.1 mg/kg compared to ACV at subcutaneous dose of 15 mg/kg against MCMV infection. In respiratory tract infection with influenza virus, FK565 showed potent protective effects at intravenous, subcutaneous and oral doses of 0.001 to 1 mg/kg. FK565 markedly inhibited zosteriform spread of HSV on the flank skin at an intravenous dose of 0.1 mg/kg and the mice given FK565 survived longer than the control mice. The peritoneal exudate cells from FK565-treated mice suppressed the growth of HSV in mouse embryo fibroblast more strongly than those from the control mice, although FK565 had no direct antiviral activity against HSV. These findings suggest that FK565 enhanced the host defense ability against viral infections by nonspecific activation of macrophages.

Inhibitory effect of FK-565 alone and in combination with zidovudine on retroviral infection by Friend leukemia virus in mice.

The effects of route and starting time of administration on FK-565 inhibition of splenomegaly by Friend leukemia virus (FLV) were studied in mice, and the concomitant effect of FK-565 in allowing reduction of zidovudine dosage was estimated. FK-565 inhibited splenomegaly in intravenous and oral doses of 0.01 to 1 mg/kg, but time of initial dosing had little effect on this inhibition. When 0.01 or 1 mg/kg of FK-565 was given intravenously with intraperitoneal doses of 0.63, 2.5, 10 and 40m g/kg of zidovudine, the inhibition rate of splenomegaly at all doses was markedly and dose-dependently higher than when either drug was given alone, and the concomitant use of FK-565 with zidovudine enabled a 16-fold reduction of the dose of zidovudine. The survival rate and survival time after infection with massive amounts of FLV were higher when FK-565 1 mg/kg and zidovudine 20 mg/kg were given in combination than when either drug was given alone. Inhibition of FLV splenomegaly was reflected in the prolonged survival time of the infected mice.

In vivo antibacterial activity of FK041, a new orally active cephalosporin.

The therapeutic activities of orally administered FK041 were evaluated in mouse models of systemic and local infections with a variety of bacteria and were compared with those of cefdinir (CFDN) and cefditoren pivoxil (CDTR-PI). FK041 exhibited potent therapeutic activity against lethal systemic infections induced by intraperitoneally inoculated Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae with 50% effective doses (ED50) in the range of 0.20 to 0.36 mg/kg and was more active than CFDN and CDTR-PI. This result correlated well with its in vitro activity. The therapeutic effects of FK041 and reference drugs on murine local infections were evaluated in an in vivo pharmacokinetic model simulating human plasma concentrations for oral administration of 50 mg, 100 mg, and 200 mg. Against murine subcutaneous abscess induced by S. aureus, FK041 was as effective as CFDN and significantly more effective than CDTR-PI in reducing the number of recoverable viable bacteria in the skin at the infection sites. The efficacy of FK041 against murine pneumonia with H. influenzae was comparable to that of CDTR-PI and was superior to that of CFDN in reducing viable bacteria activity in the lungs. These results strongly suggest that FK041 has potential for clinical use against various bacterial infections.

Immunoactive peptides, FK-156 and FK-565. I. Enhancement of host resistance to microbial infection in mice.

The protective effect of an immunoactive peptide, D-lactoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(L)-glycine (FK-156) and a related compound, heptanoyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine (FK-565) was determined in mice with various kinds of microbial infections. FK-156 and FK-565 were given to mice either subcutaneously or orally before challenge. The drugs enhanced significantly the defense of mice against acute systemic infections induced by various extracellular and facultative intracellular organisms, and subcutaneous abscess by Staphylococcus aureus. The protective effect of these drugs against Escherichia coli infection differed considerably depending on the route of administration; FK-156 was only effective by the parenteral route; however, FK-565 was effective by both parenteral and oral routes. After subcutaneous dosing with FK-156, the enhancement of host defense of mice against E. coli infection was more rapid than against Listeria infection. The enhancing effects of FK-156 and FK-565 on host defense of mice against pseudomonal infection was more potent than other immunoactive drugs.

Immunoactive peptides, FK-156 and FK-565. II. Restoration of host resistance to microbial infection in immunosuppressed mice.

The immunoactive peptides, FK-156 and its analogue, FK-565 were evaluated in various models of mice immunosuppressed with cyclophosphamide, hydrocortisone, mitomycin C, carrageenan and tumor cells. Treatment with FK-156 (subcutaneous) and FK-565 (oral) markedly restored host defense ability against microbial infection. The therapeutic effect of ticarcillin or gentamicin alone against pseudomonal infection in cyclophosphamide- and hydrocortisone-treated mice and tumor-bearing mice was much lower than in normal mice. The therapeutic effect of these antibiotics against pseudomonal infection in immunosuppressed mice was enhanced markedly by combined use with FK-156. The killing ability of macrophages and polymorphonuclear leukocytes of the immunosuppressed mice was also markedly enhanced by dosing with FK-156.

In vivo antibacterial activity of FK482, a new orally active cephalosporin.

The therapeutic activities of orally administered FK482 were compared with those of reference antibiotics against systemic and local infections with a variety of bacteria in mice and rabbits. In systemic infections in mice, oral FK482 was almost as effective as oral cefaclor (CCL) and more effective than oral cephalexin (CEX) against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis infections. However, FK482 afforded superior protective activity when given subcutaneously against E. coli infection in mice, and this activity was more potent than that of subcutaneously given CCL. In comparison with CCL, the reason that the in vivo activity of orally given FK482 against mouse systemic infections was weaker than had been anticipated from its potent in vitro activity was due to its poor oral absorption in mice. In local infections in rabbits, a species in which FK482 was better absorbed than in mice, FK482 was more effective than CCL, CEX or amoxicillin (AMPC). Against pneumonia induced by S. aureus or Streptococcus pyogenes, FK482 was as effective as AMPC and more effective than CCL in reducing the number of viable bacteria in the lungs of infected rabbits. In the oral treatment of experimental ascending pyelonephritis in rabbits, FK482 was superior to CCL and AMPC against methicillin-resistant S. aureus infection, as effective as AMPC and more effective than CCL against Enterococcus faecalis infection, and as effective as cefixime (CFIX) and more effective than CCL and AMPC against E. coli infection in reducing the number of viable bacteria in the kidneys and urine.

In vivo antibacterial activity of FK037, a novel parenteral broad-spectrum cephalosporin.

FK037 has potent therapeutic activity against lethal systemic infections and experimental local infections due to a wide variety of Gram-positive and Gram-negative bacteria such as staphylococci, Streptococcus pneumoniae, Enterobacteriaceae and Pseudomonas aeruginosa in mice. In murine systemic infections, FK037 was the most effective of the cephalosporins and imipenem tested against highly methicillin-resistant Staphylococcus aureus (H-MRSA). It was more effective than ceftazidime against selected strains of S. aureus and Enterobacteriaceae, except Serratia marcescens and P. aeruginosa against which FK037 was as effective as ceftazidime and was as effective as cefpirome against all organisms tested, except MRSA and P. aeruginosa against which FK037 was more effective than cefpirome. These results correlated well with its in vitro activity. In murine local infections, with few exceptions, FK037 was more effective than ceftazidime and cefpirome against Klebsiella pneumonia in ED50 values and against methicillin-sensitive S. aureus (MSSA) subcutaneous abscess, pyelonephritis with Staphylococcus epidermidis, E. coli and P. aeruginosa, intrauterine infections with S. aureus and E. coli in reducing the number of viable bacteria in the abscess, kidneys and uterus. It is noteworthy that the therapeutic effects of FK037 were more potent than had been anticipated from its in vitro activity against local infections with staphylococci and P. aeruginosa when compared with ceftazidime or cefpirome. In addition, the therapeutic effects of FK037 were equipotent or superior to those of cefpirome and ceftazidime against pneumonia due to MSSA, K. pneumoniae and P. aeruginosa in reducing the number of viable bacteria in the lungs in mice using an in vivo pharmacokinetic model simulating human plasma concentrations after drip infusion of usual clinical doses (0.25 to 1.0 g for MSSA, 0.063 to 0.125 g for K. pneumoniae and 1.0 to 2.0 g for P. aeruginosa). FK037 induced an in vivo post-antibiotic effect (PAE) of 3.4 hours against a thigh infection with MSSA in neutropenic mice. These results strongly suggest that it has potential for clinical use against various infections due to bacteria which include staphylococci and P. aeruginosa.

Simulation of human plasma levels of beta-lactams in mice by multiple dosing and the relationship between the therapeutic efficacy and pharmacodynamic parameters.

A mathematical multiple dosing model was designed so that human plasma concentration-versus-time curves of beta-lactams are reproduced in mouse plasma. The pharmacokinetic parameters of FK037, a new injective cephalosporin, in volunteers and in the mice model were 6,966 and 6,894 ml, respectively, for Vc, 2.592 and 2.698/h for alpha, 0.2875 and 0.3027/h for beta, and 0.9079 and 1.0506 for K21. Therefore, real pharmacokinetics of humans were reproduced in mice by this method. The 8-hour therapeutic efficacy (the decrease of the viable counts in the lung) against pneumonia with Staphylococcus aureus and Pseudomonas aeruginosa in mice was well correlated with the time above MIC value, but not with AUC, Cmax or AUC above MIC. These results indicate that this model was valuable to evaluate the beta-lactam antibiotics for predicting their clinical efficacy and that the time above MIC is an important factor in selecting beta-lactam agents and determining dosage in pulmonary infection.

In vitro and in vivo transferrable beta-lactam resistance due to a new plasmid-mediated oxyiminocephalosporinase from a clinical isolate of Proteus mirabilis.

A new plasmid-mediated beta-lactamase (FPM-1) with an isoelectric point of 7.2 and a molecular weight of 26,000 was found in a cefuroxime-resistant clinical isolate of Proteus mirabilis strain 6003. FPM-1 can be classified as a type I oxyimino-cephalosporinase on the basis of its substrate specificity and inhibition pattern by clavulanic acid etc., and its conferred resistance on both the strain and transconjugants against most oxyme-type cephalosporins as well as the older ones but not against cefamycins and a few exceptional oxyme-type cephalosporins such as ceftizoxime, ceftazidime and cefixime. In a murine systemic infection model, only these FPM-1-stable drugs exhibited protective activity against the FPM-1-producing P. mirabilis 6003 similar to that against a nonproducing derivative strain. The FPM-1-mediated cefuroxime resistance in P. mirabilis 6003 was transferred to co-infected Escherichia coli 7004 at frequencies between 3.8 x 10(-3) and 4.0 x 10(-2) in a murine ascending urinary tract infection model. In the same infection model due to the FPM-1-producing E. coli transconjugant, FPM-1-stable cefixime was significantly more effective than FPM-1-labile cefteram pivoxil, although both drugs had similar therapeutic effect against its FPM-1-nonproducing counterpart strain.

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli.

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The Km values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg2+, p-chloromercuribenzoate, and diisopropylfluorophosphate.

Isolation and properties of tumor-derived endothelial cells from rat KMT-17 fibrosarcoma.

Rat KMT-17 fibrosarcoma-derived endothelial cells were isolated by Percoll gradient centrifugation with an attaching-speed separation technique, and their properties in culture were examined. The primary cultured tumor-derived endothelial cells (TEC) showed angiotensin-converting enzyme activity, positivity for Factor VIII-related antigen staining, and typical capillary-like formation on Matrigel. The primary cultured TEC monolayer showed greater permeability than normal tissue-derived endothelial cell (aorta, vena cava and epididymal fat capillary) monolayers on FITC-dextran diffusion (molecular weight 70,000). Leukocyte adhesion to TEC was reduced compared to that to fat-derived capillary endothelial cells. These characteristics resembled those of tumor vascular endothelium, and were observed both in the primary and first-passage cell cultures, but not in the fourth-passage cell cultures. Our findings indicate that primary or subcultured TEC are applicable for studies of the physiological characteristics of tumor endothelial cells.