Effects of polymer-coated metal oxide nanoparticles on goldfish (Carassius auratus L.) neutrophil viability and function.

Exposure effects from polyacrylic acid (PAA) metal-oxide nanoparticles (TiO2, CeO2, Fe2O3, ZnO) on fish neutrophil viability and effector functions (degranulation, respiratory burst, inflammatory gene expression) were investigated using primary kidney goldfish (Carassius auratus L.) neutrophils as a model. Several studies have reported cytotoxic effects of NPs but there are limited reports on their potential to perturb the innate immune system of aquatic organisms. PAA-TiO2 significantly decreased neutrophil viability in a time and dose-dependent manner at all measured time points (0-48 h) and concentrations (0-200 µg/mL). Maximum viability decreased by (mean ± SEM): 67.1 ± 3.3%, 78.4 ± 4.2% and 74.9 ± 5.0% when exposed to 50, 100 and 200 µg/mL for 48 h, respectively. PAA-ZnO also significantly decreased neutrophil viability but only at 48 h exposures at higher concentrations. Neutrophil degranulation increased by approximately 3% after 30 min and by 8% after 4 h when exposed to sublethal doses (10 µg/mL) of PAA-CeO2 or PAA-Fe2O3. All PAA-NPs induced an increase in neutrophil respiratory burst when exposed to 10 µg/mL for 30 and 60 min, however, PAA-Fe2O3 was the only NP where the response was significant. Lastly, NPs altered the expression of a number of pro-inflammatory and immune genes, where PAA-TiO2 most significantly increased the mRNA levels of pro-inflammatory genes (il-1b, ifng) in neutrophils by 3 and 2.5 times, respectively. Together, these data demonstrate that goldfish neutrophils can be negatively affected from exposures to PAA-coated NPs and are functionally responsive to specific core-material properties at sublethal doses. These changes could perturb the innate response and affect the ability of fish to respond to pathogens.

Studies on the resistance/reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts exposed to medium-pressure ultraviolet radiation.

The ex vivo and in vivo reactivation of Giardia muris cysts and Cryptosporidium parvum oocysts after exposure to different doses of ultraviolet (UV) radiation was determined using animal infectivity. The infectivity of UV-treated parasites stored for 1-4 days (G. muris) or 1-17 days (C. parvum) at room temperature in the dark was similar to that of organisms administered immediately after UV treatment, indicating that the parasites did not reactivate ex vivo. In contrast, we observed in vivo reactivation of G. muris in three of seven independent animal infectivity experiments, when parasites were treated with relatively low doses of medium-pressure UV (<25 mJ/cm(2)). Our observations indicate that G. muris cysts and C. parvum oocysts exposed to medium-pressure UV doses of 60 mJ/cm(2) or higher did not exhibit resistance to and/or reactivation following treatment. This suggests that when appropriate doses of UV are used, significant and permanent inactivation of these parasites may be achieved.

Antimicrobial mechanisms of fish phagocytes and their role in host defense.

Phagocytosis is a primitive defense mechanism in all multicellular animals. Phagocytes such as macrophages and neutrophils play an important role in limiting the dissemination of infectious agents, and are responsible for the eventual destruction of phagocytosed pathogens. These cells have evolved elaborate killing mechanisms for destroying pathogens. In addition to their repertoire of degradative enzymes and antimicrobial peptides, macrophages and neutrophils can be activated to produce a number of highly toxic molecules. Production of reactive oxygen and nitrogen intermediates by these cells are potent cytotoxic mechanisms against bacteria and protozoan pathogens. Studies in fish suggest that the biological basis of these inducible killing mechanisms is similar to those described in mammals. More recent work suggest novel roles for regulating these killing responses in fish. In this review, we describe the biological basis of these killing mechanisms and how they are regulated in fish.

Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes.

Trout primary kidney monocyte-like cultures (T-PKM) were generated by incubating head kidney leukocytes in the presence of cell-conditioned medium (CCM). This technique was adapted from procedures that were previously used to cultivate in vitro-derived kidney macrophages (IVDKM) from the goldfish. Flow cytometric analysis of the initial T-PKM cultures, identified three cell sub-populations, but only one of these sub-populations survived extensive cultivation periods (i.e. >8 days) in the presence of CCM. Functionally, reactive oxygen intermediate (ROI) production was detected following stimulation of T-PKM with PMA. However, these cells failed to produce reactive nitrogen intermediates (RNI) in response to immunological stimuli. In contrast, goldfish IVDKM were capable of producing both ROI and RNI. Using the dihydrorhodamine (DHR) assay and flow cytometry, we identified two ROI-producing sub-populations in goldfish IVDKM but only a single ROI-producing sub-population was present after extended cultivation of T-PKM. This T-PKM sub-population was subsequently sorted using the flow cytometer and shown to possess monocyte-like morphology by microscopic and cytometric analysis. Thus, acquisition of antimicrobial functions following cultivation of kidney leukocytes of rainbow trout and goldfish is markedly different, and may be due to the failure of trout monocyte-like cells to undergo a final differentiation step in vitro.

Products of proteolytic cleavage of transferrin induce nitric oxide response of goldfish macrophages.

Enzymatic cleavage product of transferrin induced the production of nitric oxide (NO) by LPS-stimulated goldfish macrophages. A NO-inducing factor was purified from the supernatants of mitogen-stimulated goldfish kidney leukocytes using fast performance liquid chromatography (FPLC) and the purified proteins analyzed by microcapillary reverse-phase HPLC nano-electrospray tandem mass spectrometry. The proteins were identified as truncated forms of transferrin, having approximate molecular weights (MW) of 33, 35, and 37kDa (kilodaltons). The precursor form (i.e. full-length) of transferrin did not enhance NO production by LPS-stimulated goldfish macrophages, but enzymatic cleavage of this precursor form correlated with enhanced production of NO by goldfish macrophages. Enzymatic cleavage of transferrin was dependent on the presence of stimulated kidney leukocytes and was shown to occur in response to both mixed lymphocyte reactions (MLR) and the mitogenic stimulation of goldfish kidney leukocytes. Time course analysis revealed that 24h after kidney leukocyte MLR or mitogen stimulation, cleaved transferrin products appeared in the supernatants of cultured cells, which was related to the on-set of NO-inducing activity of these preparations. To confirm these findings, bovine transferrin was digested in vitro using protease XXVII. The resulting cleavage products had approximate MW of 33, 35, and 37kDa. When these peptides were subjected to the purification protocols used to purify a NO-inducing factor from goldfish leukocyte supernatants, they were shown to elute to identical fractions. To examine the potential role of fish transferrin in mediating goldfish NO production, carp transferrin was purified from serum and following protease-digestion and purification by FPLC, the truncated proteins were found to elute to similar fractions as bovine transferrin. Furthermore, mitogen-stimulated leukocyte supernatants prepared in the absence of bovine serum (carp serum only) retained NO-inducing activity, indicating that this response was not an artifact of bovine serum components (i.e. bovine transferrin). Anti-bovine and anti-carp transferrin polyclonal antibodies identified the presence of truncated forms of transferrin in the active fractions of FPLC-separated mitogen-stimulated leukocyte supernatants prepared in the presence of bovine or carp serum, respectively. Thus, our results suggest a novel role for fish transferrin as one of the factors that mediates teleost macrophage antimicrobial functions.

Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes.

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.

Coronary MRA: use in assessing anomalies of coronary artery origin.

PURPOSE: The purpose of this work was to describe the use of coronary MR angiography (MRA) in the clinical evaluation of a series of patients with anomalous origin of the coronary arteries suspected on coronary angiography. METHOD: Eight patients underwent coronary MRA to further define variant coronary anatomy that was found on coronary angiography. A 2D segmented k-space gradient echo sequence was used with breath-holding. MRA images were assessed for traversal of an anomalous artery between the aorta and pulmonary artery trunks, which carries the greatest clinical significance. RESULTS: Of six patients with anomalous origin of the right coronary artery on angiography, two were shown by MRA to have an interarterial course of the anomalous vessel. Neither of two left coronary arteries with ectopic origin coursed between the great arteries, although one passed through the septum. CONCLUSION: Coronary MRA is a useful adjunctive technique to angiography in the evaluation of the relationship of anomalous coronary arteries to the great arteries.

Arterial diastolic pressure augmentation by intra-aortic balloon counterpulsation enhances the onset of coronary artery reperfusion by thrombolytic therapy.

BACKGROUND: The early establishment of infarct artery reperfusion by intravenous thrombolytic therapy has improved survival after acute myocardial infarction. Investigations of reperfusion have focused on the effects of specific thrombolytic agents, anticoagulation, and platelet inhibition. However, little attention has been given to the relation of arterial blood pressure to thrombolysis, a factor that probably affects thrombolytic agent delivery to the obstructing thrombus. METHODS AND RESULTS: The effect of arterial diastolic pressure augmentation by intra-aortic balloon counterpulsation (IABP) on reperfusion after intravenous thrombolytic therapy was studied in a canine model. A critical left anterior descending coronary artery stenosis was created by an occluder. Acute thrombosis immediately proximal to the occluder was formed by local injection of a blood and thrombin mixture into a segment of the artery that had intimal damage (groups 1 through 3). Continuous coronary blood flow velocity was measured by an epicardial Doppler probe. Group 1 (n = 7) served as control. Group 2 (n = 6) received an intravenous, front-loaded recombinant tissue-type plasminogen activator (rTPA) regimen (1.25 mg/kg total dose, 15% as bolus, 50% in the first 30 minutes, and 35% for the following 60 minutes). Group 3 (n = 6) received the same rTPA regimen with IABP beginning at the start of rTPA administration. Coronary blood flow velocity, arterial pressure, and heart rate were observed for 150 minutes after the start of thrombolytic therapy. Five animals did not undergo coronary thrombosis (group 4) and had coronary blood flow velocity determined before and after IABP at baseline and after creation of critical stenosis. Mean systolic arterial blood pressure and heart rate were not statistically different between groups. Peak augmented diastolic pressure by IABP was 97.9 +/- 1.3% of systolic pressure in group 3 dogs. Spontaneous reperfusion did not occur in any group 1 dogs. All animals treated with rTPA reperfused. Reperfusion occurred in group 3 (13.1 +/- 2.1 minutes) earlier than in group 2 (39.2 +/- 9.4 minutes, P = .02). Overall duration of arterial patency did not differ between group 2 (81.4 +/- 16.6 minutes) and group 3 (94.9 +/- 15.3 minutes, P = .52). Reocclusions occurred with similar frequency (P = .85) in groups 2 and 3. In group 4, IABP did not increase baseline coronary blood flow velocity. CONCLUSIONS: This study demonstrates that augmentation of diastolic arterial pressure by IABP enhances thrombolysis, leading to faster reperfusion. This effect appears to be unrelated to an increase in coronary blood flow and may be due to an effect of the augmented diastolic blood pressure wave on the obstructing thrombus. These findings suggest that the time to reperfusion by rTPA may be blood pressure dependent. The relation of arterial blood pressure to successful thrombolysis may have important implications for future treatment strategies for myocardial infarction.

Age-related differences in computed tomographic scan measurements.

Seventy-nine healthy men ranging in age from 31 to 87 years underwent a computed tomographic (CT) scan and were administered a neuropsychologic test battery. Midventricular, high ventricular, and supraventricular CT slices were analyzed for each individual. Computerized techniques calculated the percent of fluid volume and the mean CT density for each slice. The mean CT density of a standard tissue sample was also evaluated. The results suggest that fluid volume at the level of the ventricles is fairly stable until individuals are in their 60s, when a dramatic increase occurs. The percent of fluid volume above the level of the ventricles appears to increase slightly the ventricles appears to increase slightly in the 50s and then level off. Whole slice mean CT density numbers decreased in a linear fashion with increasing age, but the mean CT density of a standard tissue sample did not. A discriminant function derived from the CT measures was significantly correlated with a discriminant function derived from the neuropsychologic test battery. Findings based on subjects whose health status has been less carefully screened may differ from those in the present study.

Statistical assessment of perceptual CT scan ratings in patients with Alzheimer type dementia.

Three neuroradiologists perceptually evaluated CT of 24 patients with Alzheimer type dementia and 22 normal control subjects and made a dichotomous judgment for each case (i.e., normal control or Alzheimer type dementia). The mean percentage of patients correctly classified was 83.3%. The neuroradiologists also completed perceptual ratings on each scan. Thirteen regions were rated for atrophy on a scale of 0-4. Discriminant function analyses of several sets of perceptual atrophy ratings (optimized on an exploratory set and evaluated on a test set) showed that the perceptual ratings of temporal lobe regions produced an average accuracy of 88.57%. In contrast, only 74.26% of the cases were correctly classified when the discriminant functions were based on perceptual ratings of midventricular and supraventricular areas. Linear measures of atrophy correctly classified only 65.20% of the subjects. The results suggest that atrophy ratings of brain regions that show the characteristic macroscopic neuropathological changes of Alzheimer disease may be used by neuroradiologists to reach more accurate diagnostic decisions.

Anesthetic management for birth of the "Canton" quadruplets: a multidisciplinary team approach.

This article presents a case study of the successful anesthesia management in the birth of quadruplets at Aultman Hospital in Canton, Ohio. A review of recent literature is also provided. With the increasing use of fertility drugs, multiple births will inevitably cease to be viewed as a rare phenomenon. Anesthesia practitioners will increasingly be presented with the challenge of planning for the management of these patients. The authors believe an anesthetic plan must be integrated into a multidisciplinary team approach to achieve successful management of parturient and neonates.