Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Enhanced involvement of endothelin in the haemodynamic sequelae of endotoxaemia in conscious, hypertensive, transgenic ((mRen-2)27) rats

1. Age-matched (3-4 months old) male, heterozygous, hypertensive, transgenic ((mRen-2)27) rats (abbreviated to TG rats) and the normotensive control animals (homozygous, Hannover Sprague-Dawley rats (abbreviated to SD rats), were chronically instrumented for the assessment of regional haemodynamic responses to continuous lipopolysaccharide (LPS) infusion (150 microg kg(-1) h(-1), i.v.) 2. The early (1-2 h) hypotension in SD rats (-11+/-3 mmHg; n=7) was significantly less than that in TG rats (-35+/-3 mmHg; n=8), but by 24 h mean arterial blood pressure (MAP) in both strains of rat was not different from the pre-LPS value (SD rats: baseline, 108+/-3 mmHg; 24 h LPS, 112+/-4 mmHg; TG rats: baseline, 171+/-2 mmHg; 24 h LPS, 169+/-3 mmHg). At this stage in the SD rats there was a renal vasodilatation (delta vascular conductance, 29+/-10 [kHz mmHg(-1)]10(3)) but not in TG rats (delta vascular conductance 2+/-3[kHz mmHg(-1)]10(3)). 3. Co-infusion of LPS and the non-selective endothelin receptor antagonist, SB 209670 (600 microg kg(-1) bolus, 600 microg kg(-1) h(-1)) between 24 and 31 h in SD rats caused a fall in MAP of 16+/-2 mmHg accompanied by hindquarters vasodilatation (delta vascular conductance 11+/-3 (kHz mmHg(-1))10(3)). In TG rats, under the same conditions, the fall in MAP was -60+/-6 mmHg, and there were renal, mesenteric and hindquarters vasodilatations (delta vascular conductance, 23+/-5, 32+/-7, and 14+/-4 (kHz mmHg(-1))10(3), respectively). All effects, except the hindquarters vasodilatation, were greater in TG than in SD rats. 4. In TG rats infused with LPS alone for 31 h, between 24 and 31 h the fall in MAP was -17+/-4 mmHg, and the changes in renal, mesenteric and hindquarters vascular conductances were 5+/-3, -4+/-5, and 12+/-4 (kHz mmHg(-1)10(3), respectively. 5. Administration of the angiotensin (AT1)-receptor antagonist, losartan (10 mg kg(-1), i.v.) following co-infusion of LPS and SB 209670 between 24 and 31 h caused similar falls in MAP in SD and TG rats (-12+/-3 and -14+/-4 mmHg, respectively). 6. These results, together with previous findings, are consistent with a relative enhancement of the contribution of endothelin to the maintenance of cardiovascular status in endotoxaemic TG rats, particularly through a mesenteric vasoconstrictor action.     

Small wire external fixation of high energy tibial plateau fractures

Open plate osteosynthesis for high energy tibial plateau fractures with dissociation between the metaphysis and diaphysis has been plagued with frequent soft tissue complications. The Harbor-University of California at Los Angeles Medical Center's experience with small wire external fixation supplemented by limited internal fixation is examined. This alternative method of adequate stable fixation offers the advantage of minimal soft tissue compromise. Twenty-four patients with Schatzker Type VI tibial fractures were treated with small wire external fixation. Supplementary limited internal fixation was used with percutaneous screws in 10 patients and with open reduction in one patient. Sixteen patients had isolated fractures, and eight others suffered multiple injuries. Minimum followup was 12 months. All fractures healed. Complications included one septic knee, two infections at screw sites, and one 10 degrees knee flexion contracture. One knee had Grade 3 radiographic arthrosis, five had Grade 2, 10 had Grade 1, and eight showed no arthrosis. The outcomes (Knee Society clinical rating system) of this study compare favorably with outcomes described in reports published previously for this type of fracture, despite inclusion of eight multiply injured patients. This technique preserves the goals of early range of motion and stable fixation for these devastating injuries, while decreasing the observed major wound complications and nonunion rates. However, longer followup may reveal higher arthrosis rates, specifically in those fractures that were not anatomically reduced.     

Study of the non-steady state electrogenic transport of sodium ions by Na+,K(+)-ATPase by the capacitance measurement method

The goal of work was to investigate the electrogenic transport of Na ions by Na+,K(+)-ATPase in membrane fragments absorbed on a planar bilayer lipid membrane. The photorelease of ATP from an inactive precursor, caged ATP, induced a transient current and changes in the net system capacitance measured during the application of an alternating voltage. The increments of capacitance (delta c) decreased with the increase in the frequency of the applied voltage. The characteristic frequency F0 of the steepest slope of the curve significantly decreased in solutions with high ionic strength (either NaCl or choline chloride), in which Na+ transport is decelerated. The value of delta c correlated with the total charge delta q transported across the membrane. The capacitance increments decreased when the Na+ concentration in solution decreased. At a concentration below 2 mM the increment became negative. The increase in membrane capacitance can be attributed to the charge relaxation process inside the protein, as discovered in the cells by other methods. The characteristic frequency F0 depends on the time constants of charge redistribution. The nonlinear dependences of delta c on delta q were explained by a voltage bias across the membrane fragments resulting from pumping. The potentials corresponding to the maximum capacitance change were similar to the midpoint potentials of the equilibrium charge distribution and depended on the Na+ concentrations in solution. The model enabled also the determination of the total capacitance of the active region of a lipid membrane with the adsorbed protein containing membrane fragments.     

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.     

Mode of action of lufenuron on larval cat fleas (Siphonaptera: Pulicidae)

Adult cat fleas, Ctenocephalides felis (Bouché), were fed suboptimal in vitro concentrations of lufenuron in blood to allow hatching of flea larvae for cytological study. At concentrations of 0.125, 0.25, and 0.5 ppm, larval hatch was 64, 15, and 4%, respectively. Larvae hatching from eggs laid by adults fed lufenuron at concentrations of 0.025, 0.08, or 0.125 ppm did not differ significantly from the control. However, many larvae from the 0.08-ppm group and higher concentrations died during the 1st instar. Examination of these larvae revealed that they were dying from desiccation caused by bleeding from microscopic lesions in the cuticle or the inability to complete the molt to the next instar. Electron micrographs showed that lufenuron often disrupted formation of the endocuticle resulting in the deposition of an amorphous mass of randomly oriented chitin microfibrils. Other larvae formed normal endocuticle but were unable to digest the old endocuticle or produce new procuticle after apolysis. Failure of larvae to digest old cuticle or form new cuticle was caused by degeneration of the epidermal cells needed for the synthesis of molting fluid and chitin.     

Identification and purification of diphosphoinositol pentakisphosphate kinase, which synthesizes the inositol pyrophosphate bis(diphospho)inositol tetrakisphosphate

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) were recently identified as inositol phosphates which possess pyrophosphate bonds. The molecular mechanisms that regulate the cellular levels of these compounds are not yet characterized. To pursue this question, we have previously purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants [Voglmaier, S. M., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4305-4310]. We now report the identification and purification of another novel kinase, diphosphoinositol pentakisphosphate (PP-IP5) kinase, which uses PP-IP5 as a substrate to form bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) in soluble fractions of rat forebrain. The purified protein, a monomer of 56 kDa, displays high affinity (Km = 0.7 microM) and selectivity for PP-IP5 as a substrate. The purified enzyme also can transfer a phosphate from bis-PP-IP4 to ADP to form ATP. This ATP synthase activity is an indication of the high phosphoryl group transfer potential of bis-PP-IP4 and may represent a physiological role for PP-IP5 and bis-PP-IP4.     

The influence of antibodies to TNF-alpha and IL-1beta on haemodynamic responses to the cytokines, and to lipopolysaccharide, in conscious rats

Male, Long Evans rats (350-450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-alpha and human recombinant interleukin-1 (IL-1)beta, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-alpha and IL-1beta on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-alpha and IL-1beta had any influence on the responses to lipopolysaccharide (LPS). TNF-alpha (10, 100 and 250 microg kg(-1), in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum delta, +101+/-9 beats min(-1)) and modest hypotension (maximum delta, -10+/-2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum delta, +6+/-6, +27+/-9, and +61+/-12% at 10, 100 and 250 microg kg(-1), respectively). IL-1beta (1, 10, and 100 microg kg(-1) in separate groups, n = 8, 8 and 9, respectively) evoked changes similar to those of TNF-alpha (maximum delta heart rate, +69+/-15 beats min(-1); maximum delta mean blood pressure, -14+/-2 mmHg; maximum delta hindquarters vascular conductance, +49+/-17%), but with no clear dose-dependency. TNF-alpha (250 microg kg(-1)) and IL-1beta (10 microg kg(-1)) together caused tachycardia (maximum delta, +76+/-15 beats min(-1)) and hypotension (maximum A, -24+/-2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52+/-6%, +23+/-8%, and +52+/-11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum delta, -50+/-3% and -58+/-3%, respectively). Although bolus injection of LPS (3.5 mg kg(-1)) caused an initial hypotension (maximum delta, -27+/-11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-alpha and IL-1beta or after bolus injection of LPS, the secondary reduction in blood pressure was similar (-16+/-2 and -13+/-3 mmHg, respectively), but in the former group the tachycardia (+117+/-14 beats min(-1)) and increase in hindquarters vascular conductance (+99+/-21%) were greater than after bolus injection of LPS (+54+/-16 beats min ' and +439%, respectively). Pretreatment with antibodies to TNF-alpha and IL-1beta (300 mg kg(-1)) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h. Pretreatment with antibodies to TNF-alpha and IL-1beta before infusion of LPS (150 microg kg(-1) h(-1) for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion. the results indicate that although co-administration of TNF-alpha and IL-1beta can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.     

Giant-cell interstitial pneumonia in a gas station worker

Giant-cell interstitial Pneumonia (GIP) is a very uncommon respiratory disease. The majority of cases of GIP are caused by exposure to cobalt, tungsten and other hard metals. In this report, we describe GIP in a patient who worked in gas station and dealt in propane gas vessels. He presented with clinical features of chronic interstitial lung disease and underwent an open lung biopsy that showed DIP-like reaction with large numbers of intra-alveolar macrophages and numerous large, multinucleated histiocytes which were admixed with the macrophages. Analysis of lung tissue for hard metals was done. Cobalt was the main component of detected hard metals. Corticosteroid therapy was started and he recovered fully.