Martha J. Gentry-Nielsen

Pneumococcal Vaccine Response in Cirrhosis and Liver Transplantation

Cirrhosis is a major risk factor for severe pneumococcal infection, and patients evaluated for liver transplantation routinely receive pneumococcal vaccine. This study followed serologic antibody levels of 45 adults evaluated for transplantation and 13 age-matched control subjects. All received 23-valent pneumococcal polysaccharide vaccine (PPS). Serum anti-PPS levels and antibodies specific for capsular types 3 and 23 were measured by ELISA before and 1 and 6 months after vaccination. Antibody levels for the 25 patients who received transplants also were measured immediately before and 3 months after transplantation. Control subjects had higher IgG responses to the whole vaccine, whereas patients appeared to produce more IgM and IgA. IgA, and possibly IgM levels, also declined faster in patients than in control subjects. All anti-PPS levels were at or below prevaccination baselines by 3 months after transplantation. These data suggest that vaccination with PPS may not be effective for patients during and after liver transplantation.

Pathology of the olfactory epithelium: Smoking and ethanol exposure

Objective: To investigate the effects of tobacco smoke on the olfactory epithelium. Cigarette smoking has been associated with hyposmia; however, the pathophysiology is poorly understood. The sense of smell is mediated by olfactory sensory neurons (OSNs) exposed to the nasal airway, rendering them vulnerable to environmental injury and death. As a consequence, a baseline level of apoptotic OSN death has been demonstrated even in the absence of obvious disease. Dead OSNs are replaced by the mitosis and maturation of progenitors to maintain sufficient numbers of neurons into adult life. Disruption of this balance has been suggested as a common cause for clinical smell loss. This current study will evaluate the effects of tobacco smoke on the olfactory mucosa, with emphasis on changes in the degree of OSN apoptosis.

Pharmacodynamic Activity and Efficacy of Linezolid in a Rat Model of Pneumococcal Pneumonia

ABSTRACT Linezolid is a new oxazolidinone antibiotic with potent activity against gram-positive bacteria, including Streptococcus pneumoniae. The pharmacodynamic activity and in vivo efficacy of linezolid were compared to those of ceftriaxone in an immunocompetent rat model of pneumococcal pneumonia. Rats infected intratracheally with 8 × 107 CFU of a penicillin-sensitive (MIC, 0.032 μg/ml) strain of S. pneumoniae were treated for 5 days beginning 18 h postinfection. Groups of rats were sham treated with oral phosphate-buffered saline or received oral liquid linezolid at 25 or 50 mg/kg of body weight twice a day (b.i.d.) or subcutaneous ceftriaxone at 100 mg/kg once daily. Mortality was monitored for 10 days postinfection; blood culturing was performed on day 1 (pretreatment) and on days 3, 5, and 10 postinfection for the determination of bacteremia. Serum also was collected for the determination of pharmacokinetic and pharmacodynamic parameters at 30 min and at 3, 5, and 12 h (linezolid) or 3, 5, and 24 h (ceftriaxone) postdose. The cumulative mortality rates were 100% for the sham-treated group, 58.3% for the low-dose linezolid group, 8.3% for the high-dose linezolid group, and 0% for the ceftriaxone group. Rats in each of the antibiotic treatment groups had significantly fewer bacteria (P < 0.00001) in their bronchoalveolar lavage fluid (BALF) on day 3 postinfection than sham-treated rats. There also were significantly fewer organisms in the BALF of rats treated with ceftriaxone than in the BALF of rats treated with either dose of linezolid. Oral linezolid at 50 mg/kg b.i.d. therefore was as effective as ceftriaxone in experimental pneumococcal pneumonia, whereas the 25-mg/kg b.i.d. dose was significantly less effective. All pharmacodynamic parameters reflected efficacy and were significantly different for the two dosage regimens of linezolid (P < 0.01). However, the free-fraction pharmacodynamic parameters predictive of outcome were a value of >39% for the percentage of time in the experimental dosing interval during which the linezolid concentration exceeded the MIC and a value of >147 for the ratio of the area under the serum concentration-time curve to the MIC.

Pneumolysin-Induced Complement Depletion during Experimental Pneumococcal Bacteremia

ABSTRACT To quantify complement depletion by pneumolysin duringStreptococcus pneumoniae bacteremia, cirrhotic and control rats were infected intravenously with one of three isogenic mutant strains of S. pneumoniae expressing different forms of pneumolysin. Outcome measures included clearance of the organisms from the bloodstream, alterations in 50% serum hemolytic complement (CH50) activity and complement C3 levels during infection, and serum opsonic capacity at 18 h postinfection. Cirrhotic rats had significantly lower CH50 and C3 levels than control rats, both before and after infection. However, initial complement levels did not predict bacterial load after 18 h of infection. Changes in CH50 and C3 levels over the 18-h period correlated with numbers of H+C+ but not H+C− or PLY− organisms in the bloodstream at 18 h postinfection. The sera of cirrhotic rats infected with the H+C+ strain had significantly decreased levels of C3 and showed significantly lower opsonizing activity for S. pneumoniaethan sera from H+C+-infected control rats. These studies suggest that under limiting concentrations of complement, the expression of pneumolysin by pneumococci has a significant, negative effect on serum complement levels and reduces serum opsonic activity.

Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae

BackgroundThe risk of mortality from pneumonia caused by Streptococcus pneumoniae is increased in patients with cirrhosis. However, the specific pneumococcal virulence factors and host immune defects responsible for this finding have not been clearly established. This study used a cirrhotic rat model of pneumococcal pneumonia to identify defect(s) in innate pulmonary defenses in the cirrhotic host and to determine the impact of the pneumococcal toxin pneumolysin on these defenses in the setting of severe cirrhosis.ResultsNo cirrhosis-associated defects in mucociliary clearance of pneumococci were found in these studies, but early intrapulmonary killing of the organisms before the arrival of neutrophils was significantly impaired. This defect was exacerbated by pneumolysin production in cirrhotic but not in control rats. Neutrophil-mediated killing of a particularly virulent type 3 pneumococcal strain also was significantly diminished within the lungs of cirrhotic rats with ascites. Levels of lysozyme and complement component C3 were both significantly reduced in bronchoalveolar lavage fluid from cirrhotic rats. Finally, complement deposition was reduced on the surface of pneumococci recovered from the lungs of cirrhotic rats in comparison to organisms recovered from the lungs of control animals.ConclusionIncreased mortality from pneumococcal pneumonia in this cirrhotic host is related to defects in both early pre-neutrophil- and later neutrophil-mediated pulmonary killing of the organisms. The fact that pneumolysin production impaired pre-neutrophil-mediated pneumococcal killing in cirrhotic but not control rats suggests that pneumolysin may be particularly detrimental to this defense mechanism in the severely cirrhotic host. The decrease in neutrophil-mediated killing of pneumococci within the lungs of the cirrhotic host is related to insufficient deposition of host proteins such as complement C3 on their surfaces. Pneumolysin likely plays a role in complement consumption within the lungs. Our studies, however, were unable to determine whether pneumolysin more negatively impacted this defense mechanism in cirrhotic than in control rats. These findings contribute to our understanding of the defects in innate pulmonary defenses that lead to increased mortality from pneumococcal pneumonia in the severely cirrhotic host. They also suggest that pneumolysin may be a particularly potent pneumococcal virulence factor in the setting of cirrhosis.

A Rat Model to Determine the Biomedical Consequences of Concurrent Ethanol Ingestion and Cigarette Smoke Exposure

BACKGROUND Although scientists have used animal models for years to study the effects of ethanol (EtOH) ingestion on humans, the compounding effect of cigarette smoking has been virtually ignored. Because 80 to 95% of human alcoholics smoke, it is imperative to consider the added effects of smoking when trying to determine the consequences of excessive alcohol ingestion. We therefore have developed a rat model for studying the separate and combined results of smoking and drinking on human health. METHODS Male Sprague-Dawley rats were exposed daily for 12 weeks in whole-body chambers to cigarette smoke (smoke-exposed) or room air (sham-exposed). During the final 5 weeks of exposure, the rats were fed liquid diets that contained 0, 16, 26, or 36% EtOH calories. Smoke exposure was quantified by measurement of carboxyhemoglobin, nicotine, and cotinine levels. Body weights, food consumption, blood EtOH concentrations, and various assessments of liver damage and function also were followed. RESULTS Smoke exposure in this rat model approximates that of a moderate to heavy human smoker. Smoke-exposed rats weighed significantly less and ate less food than sham-exposed controls, but both groups ingested equivalent amounts of EtOH for their body weights and had comparable blood EtOH levels. Liver aspartate and alanine aminotransferase levels remained normal. There was an EtOH-induced decrease in asialoglycoprotein receptor binding, but it was not exacerbated by smoke exposure. Alterations in blood cholesterol levels reflected what has been reported for humans, rising with increasing EtOH ingestion and decreasing with smoke exposure. CONCLUSION Our rat model is relevant to what transpires in the vast majority of alcoholics. Both ethanol ingestion and smoke exposure can be manipulated to mimic light to moderate to heavy levels, making it appropriate for studying the separate and combined biomedical consequences of alcohol abuse and cigarette smoking.

A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

BackgroundPhagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria.ResultsThe viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides.ConclusionThis assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

The impact of ethanol and tobacco smoke on intranasal epithelium in the rat.

Background Investigations have shown the influence of ethanol and tobacco smoke on olfaction, epithelial metaplasia, and cancer formation in the head and neck. Analysis of ethanol and tobacco smoke-induced histopathological mucosal changes in the upper respiratory tract may provide important insight into the pathophysiology of secondary olfactory dysfunction. Methods Three groups of laboratory rats were experimentally exposed to either ethanol, tobacco smoke, or both, with a control group having no such exposure. Results Compared with controls, histopathological analysis of nasal mucosa in exposed rats revealed a decrease in the length of olfactory epithelium, especially in the rats exposed to both ethanol and tobacco smoke. Structural changes included loss of cilia and metaplasia. Conclusion The histological changes noted in rats after ethanol and tobacco smoke exposure, if relevant to human physiology, could explain the decreased olfactory ability seen in patients who use these products.

Use of rat models to mimic alterations in iron homeostasis during human alcohol abuse and cirrhosis

With alcoholism, there are marked disturbances in iron homeostasis that are linked to alterations in serum transferrin and ferritin concentrations. This study identifies rat models of alcohol abuse that closely mimic these disturbances. Male rats were placed in one of the following three protocols: (1) pair-feeding of liquid diets for 1-8 weeks; (2) agar-block feeding for 8 weeks; or (3) generation of cirrhosis with CCl(4). Serum samples were analyzed for ferritin, transferrin, and iron levels, and the transferrin iron saturation and ferritin/transferrin ratios were calculated. Liver iron concentrations were also determined. Serum transferrin levels were elevated in animals fed alcohol for 8 weeks in pair-feeding and agar-block feeding protocols, but reduced in rats with cirrhosis. Serum ferritin concentration was reduced in rats fed ethanol in the liquid diet, but increased in rats consuming ethanol in agar blocks, in rats pair-fed the liquid control diet, and in rats with cirrhosis. This finding was mirrored by liver nonheme iron concentrations in all experimental groups, but not in the corresponding control groups. Serum iron levels were significantly elevated only in rats fed the liquid control diet. There was a progressive decrease in transferrin iron saturation and ferritin/transferrin ratios for animals fed ethanol in the liquid diet, but not when ethanol was ingested from agar blocks. The development of cirrhosis resulted in elevated liver iron concentrations and doubled ferritin/transferrin ratios. It is concluded that these models may be used to study disturbances in iron homeostasis that occur during alcohol abuse and the (subsequent) development of liver disease.

Relaxin receptor expression in hepatic stellate cells and in cirrhotic rat liver tissue

Abstract: Relaxin has antifibrotic effects on the hepatic stellate cells (HSCs) responsible for collagen deposition in cirrhosis. The expression of relaxin receptors LGR7 and LGR8 in HSCs and liver disease was examined. Activated and quiescent HSCs expressed LGR7, whereas only activated HSCs expressed LGR8. Relaxin, relaxin‐3, or InsL3 treatment increased cAMP, suggesting activation of both receptors. LGR8 and LGR7 were present in cirrhotic rat liver, but were undetectable in normal liver. In conclusion, both LGR7 and LGR8 are expressed in activated HSCs and cirrhotic liver, suggesting that relaxin, InsL3, or relaxin‐3 may be useful in the treatment of hepatic fibrosis.