Role of heparin and nitric oxide in the cardiac and regional hemodynamic properties of protamine in conscious chronically instrumented dogs

BACKGROUND: Because protamine is administered to reverse heparin, a drug that might itself affect the pharmacologic properties of protamine, this study was designed to assess the properties of protamine alone and in the presence of heparin in conscious dogs. METHODS: Twelve dogs were instrumented to continuously record cardiac and regional hemodynamics. On separate occasions, a dose of protamine (0.5, 1, 3, 5, and 8 mg/kg) was randomly administered either alone or in the presence of heparin (ratio 100 IU/mg). Heparin (300 IU/kg) and protamine (3 mg/kg) were administered in the presence of N-methyl-L-arginine, a specific nitric oxide synthase inhibitor. Identical experiments were performed with protamine (8 mg/kg) in the absence of heparin on a separate occasion. RESULTS: Protamine alone produced limited cardiac and regional changes. In the presence of heparin, protamine produced hypotension at 3, 5, and 8 mg/kg, vasodilatation at 3 and 5 mg/kg, and a more pronounced dose-dependent increase in pulmonary pressure at 3, 5, and 8 mg/kg. Simultaneously, transient carotid vasodilatation at 3 and 5 mg/kg, coronary and hepatic vasodilatation at 3, 5, and 8 mg/kg, as well as a decrease in vertebral vascular resistance were recorded at 1, 3, and 8 mg/kg. Protamine produced an immediate increase followed by a secondary decrease in renal vascular resistance. Protamine-induced secondary pulmonary pressor effects were attenuated. In the presence of heparin, nitric oxide synthase blockade selectively attenuated protamine-induced immediate hypotension, systemic vasodilatation, and coronary, mesenteric, and hepatic vasodilations as well as the decrease in portal blood flow and accentuated the renal vasoconstriction. CONCLUSIONS: The presence of heparin accentuated the decrease in cardiac function induced by protamine as well as its effects on regional circulation. The data provide evidence that the nitric oxide pathway is involved in the systemic and selective regional heparin-protamine-mediated vasodilatation in conscious dogs.     

Effects of norepinephrine on the dog common carotid, coronary and renal arteries: in vitro studies

The effects of norepinephrine were studied on segments of the common carotid, left circumflex and renal arteries of dogs with the endothelium intact and removed. Norepinephrine induced a dose-dependent increase in tension in segments of the renal and carotid artery but did not produce any effect on the coronary artery. Although the absence of endothelium did not affect the magnitude of the norepinephrine-induced vasoconstriction of the renal artery, it elicited a more pronounced response in the carotid artery in which the endothelium was removed. Our data suggest that the vascular response to norepinephrine is dependent upon the specific arterial bed and, secondly, that the endothelium plays a determinant role in the magnitude of the response of the carotid artery.     

Cardiac and Regional Hemodynamic Interactions between Halothane and Nitric Oxide Synthase Activity in Dogs

Background: In vitro, halothane appears to affect the role played by nitric oxide in the regulation of vascular tone and cardiac function. In vivo, the results of the interactions between halothane and the nitric oxide pathway remain controversial. The authors investigated the effects of halothane on the cardiac and regional hemodynamic properties of N-methyl-L-arginine (NMA), a specific nitric oxide synthase inhibitor, in dogs.

Methods: Twenty-five dogs were chronically instrumented. Aortic pressure, the first derivative of left ventricular pressure, cardiac output, heart rate, and carotid, coronary, mesenteric, hepatic, portal and renal blood flows were continuously recorded. N-methyl-L-arginine was infused intravenously at 20 mg/kg over 1 min in awake dogs (n = 11) and in 1.2% halothane-anesthetized dogs (n = 10). As a control group, the remaining four dogs were studied awake and during 1.2% halothane for 2 h in the absence of NMA.

Results: In awake dogs, NMA produced a sustained pressor response (34%) and systemic vasoconstriction (40%) associated with a decrease in cardiac output (16%). Regional circulation changes included an immediate and transient increase in carotid (43%) and coronary (237%) blood flows and a subsequent decrease in carotid blood flow (25%). Hepatic and mesenteric blood flows also decreased, by 43% and 16%, respectively. Except for the coronary circulation, regional vascular resistance increased significantly. Halothane did not affect the pressor response to NMA but did blunt the cardiac output changes. Consequently, the systemic vasoconstriction after nitric oxide synthase inhibition was of shorter duration and of lesser magnitude during halothane anesthesia. Halothane also blunted the carotid, mesenteric, and renal vasoconstriction induced by NMA. Finally, in 1.2% halothane-anesthetized dogs, NMA induced a coronary vasoconstriction.     

Molecular epidemiology and mechanisms of rifampicin resistance in Acinetobacter baumannii isolates from Italy

Use of rifampicin (RIF) in combination with colistin (COL) has been proposed for the treatment of multidrug-resistant Acinetobacter baumannii infections owing to in vitro synergism. The aim of the present study was to evaluate the molecular epidemiology and mechanisms of RIF resistance in 57 clinical isolates of A. baumannii in two tertiary care hospitals in Naples (Italy) from 2006 to 2010. Amongst the collection, 36 isolates showed high RIF minimum inhibitory concentrations (MICs) (256 mg/L to ≥512 mg/L), 16 showed intermediate MICs (8-16 mg/L) and 5 had low MICs (4 mg/L). Of the 36 isolates with elevated RIF MICs, 35 were assigned to sequence type ST2 and 1 to ST78. Amongst the 57 isolates, 35 carried at least one mutation in rpoB, including H535L in 9 isolates and double mutations D525N and P544L in 7 isolates, whilst 22 showed no rpoB mutations. Treatment with the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PAβN) of resistant isolates with no mutations in rpoB and different RIF MICs reduced the MIC by >10-fold and restored the synergism between RIF and COL in time-kill studies, whilst it had no effect on strains carrying rpoB mutations. In conclusion, the emergence of elevated RIF MICs in A. baumannii isolates from our geographical area was mostly caused by mutations in rpoB; low to intermediate RIF MICs were also caused by altered membrane permeability to the drug. The phenomenon was contributed by the selection of two prevalent clones both assigned to ST2 genotype. These data may have implications for the correct identification of cases with A. baumannii infection that would not benefit from addition of RIF to COL.     

Influence of hypertension on MAC of halothane in rats

This study was designed to assess the relationship between MAC and hypertension. To this purpose, MAC of halothane was determined in fully inbred spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Because MAC determination was performed in animals whose lungs were mechanically ventilated, the adequacy of the ventilation was initially established in 20 rats equally divided into SHR and WKY, and instrumented with catheters in the abdominal aorta. Subsequently, MAC of halothane was determined in 40 rats equally divided into SHR and WKY, including those instrumented. There were no differences in MAC of halothane between SHR (n = 20) and WKY (n = 20) (1.08 +/- 0.02% vs. 1.11 +/- 0.02%). Subgroup analysis indicated that MAC of halothane was not affected by the presence of an arterial catheter in the abdominal aorta (SHR 1.09 +/- 0.06% vs. 1.08 +/- 0.02%; WKY 1.15 +/- 0.04% vs. 1.08 +/- 0.02%). The authors' data provide experimental evidence that MAC is not affected by either chronic hypertension or limited instrumentation.     

Molecular epidemiology of high-level aminoglycoside-resistant enterococci isolated from patients in a university hospital in southern Italy

OBJECTIVES: We evaluated the genetic and molecular basis of high-level resistance to gentamicin and amikacin in 91 clinical isolates of Enterococcus faecalis and Enterococcus faecium in a university hospital in southern Italy from 1987 to 2003. METHODS: Antibiotic susceptibility was evaluated by disc diffusion and microdilution methods. Genotyping was performed by PFGE and dendrogram analysis. Aminoglycoside resistance genes were analysed by multiplex PCR. Aminoglycoside resistance gene transfer was performed by filter mating. RESULTS: In our strain collection, 44% of E. faecalis and 52% of E. faecium were high-level-resistant to gentamicin. Fifty-two PFGE profiles were identified for E. faecalis and 15 for E. faecium. Although the majority of PFGE patterns were single isolates, four patterns (two for E. faecalis and two for E. faecium) were isolated each in 8 and 4, and 6 and 4 different patients, respectively. The aac(6')-Ie-aph(2')-Ia gene was responsible for high-level resistance to gentamicin and amikacin in E. faecalis and E. faecium; the aph(2')-Id gene responsible for resistance to gentamicin was also isolated in E. faecium; the aph(3')-IIIa and ant(4')-Ia genes responsible for resistance to amikacin were also isolated in E. faecalis and E. faecium. High-level resistance to gentamicin, along with the aac(6')-Ie-aph(2')-Ia gene, was transferred at a frequency of about 10(-5) to 10(-8) per recipient cell in 14 of 17 E. faecalis and 3 of 4 E. faecium different genotypes. CONCLUSIONS: The spread of the aac(6')-Ie-aph(2')-Ia gene was responsible for high-level resistance to gentamicin and amikacin among enterococci isolated from patients in our geographical area.     

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus

Listeria monocytogenes spends most of its intracellular lifecycle in the cytosol of the infected eucaryotic cells. Withinthis cellular compartment originates the endogenous pathwayof antigen processing and presentation. We thus assumed thatrecombinant L. monocytogenes expressing an heterologous protein,the nucleoprotein of the lymphocytic choriomeningltis virus(LCMV), should be able to induce antigen-specific CD8⁺ T cellsin vivo. The LCMV nucleoprotein gene was inserted in phase withthe sequence coding for the putative signal sequence of thehemolysin of L. monocytogenes in order to target its secretioninto the cytosol of the infected cell. The ability of this recombinantbacterium to induce LCMV-reactive CD8⁺ T cells was then monitoredin BALB/c mice. The immune status of the immunized BALB/c micewas studied on the seventh day after a single i.v.injectionof a sublethal dose of the recombinant bacteria: (i) cytotoxicCD8⁺ T cells were detected in liver; (ii) using in vitro re-stimulationwith PMA and ionomycin, secondary cytotoxic CD8⁺ T cells weredetected in spleen; (iii) an early inflammatory reaction dependenton the presence of CD8⁺ T cells occurred in the footpad afterintraplantar inoculation of live LCMV; and (iv) mice were protectedagainst an otherwise lethal intracerebral LCMV challenge; theprotection was accompanied by elimination of the virus. Whenthe immune status of the immunized hosts was monitored for alonger period post-immunization, the balance between immuneprotectiosn and immunopathology described for the anti-LCMVimmune responses was observed; two phases of protection weredetected, flanking a transitory phase of exacerbation of thelymphocytic choriomeningitis disease (weeks 2–5). Takentogether, these results indicate the feasibility of using attenuatedL. monocytogenes as a model of a live vector to induce in vivoCD8-ependent immune responses against intracellular pathogens.