Treatment with Interleukin-7 Restores Host Defense against Pneumocystis in CD4+ T-Lymphocyte-Depleted Mice.

Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4(+) T lymphocytes are critical for host defense against this infection, but in the absence of CD4(+) T lymphocytes, CD8(+) T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis, and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8(+) T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8(+) T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8(+) central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8(+) T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8(+) T lymphocytes.

De novo biosynthesis of arachidonic acid and 5,11,14-eicosatrienoic acid in the cricket Teleogryllus commodus.

The de novo biosynthesis of 5,11,14-eicosatrienoic acid (5,11,14-20:3), arachidonic acid (20:4(n - 6] and eicosadienoic acid (20:2(n - 6] and the elongation/desaturation of linoleic acid (18:2(n - 6] to 20:4(n - 6) and alpha-linolenic acid (18:3(n - 3] to eicosapentaenoic acid (20:5(n - 3] were demonstrated in adult males of the field cricket Teleogryllus commodus. Sodium [1-14C]acetate, [1-14C]18:2(n - 6) and [1-14C]18:3(n - 3) were injected into adult male crickets and after an incubation period, the testes and remaining tissues were extracted and the methyl esters obtained from the phospholipid and triacylglycerol fractions were analyzed. After 5 days of daily injections of [1-14C]acetate, the methyl esters of the triene and tetraene fatty acids from the testicular phospholipid fraction were purified by AgNO3-TLC and HPLC and analyzed by GLC, radio-HPLC, and radio-GLC of ozonolysis products. The results demonstrate the de novo biosynthesis of 20:2(n - 6), 20:4(n - 6) and an isomer of 20:3(n - 6) with double bonds in the 5,11,14 positions. the elongation/desaturation of 18:2(n - 6) to 20:4(n - 6) and 18:3(n - 3) to 20:5(n - 3) was demonstrated by analysis of the methyl esters derived from the testicular phospholipid fraction by radio-HPLC after injecting crickets with radiolabeled substrates.

Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells.

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.

The effects of phenytoin on the performance of rats in a delayed match-to-place task.

We have shown previously that phenytoin impairs learning in rats in several different behavioral paradigms (Churchill et al., 1998, 2003; Banks et al., 1999). The present study has examined this drug's effects on performance in a delayed match-to-place water maze paradigm developed by Steele and Morris (1999). We find that phenytoin retards performance, but only when the inter-trial interval (ITI) is short (i.e., 15-sec). With longer ITIs (i.e., 20-min, 2-hr), the performance of the phenytoin-treated rats was quite comparable to the controls. We suggest that this pattern of results stems from a disruption of spatial working memory, perhaps due to the effects of the drug on hippocampal function (cf., Churchill et al., 1998, 2003). This disruption is, however, not so profound that consolidation is prevented.

Effective dose of dental CBCT-a meta analysis of published data and additional data for nine CBCT units.

OBJECTIVES: This article analyses dose measurement and effective dose estimation of dental CBCT examinations. Challenges to accurate calculation of dose are discussed and the use of dose-height product (DHP) as an alternative to dose-area product (DAP) is explored. METHODS: The English literature on effective dose was reviewed. Data from these studies together with additional data for nine CBCT units were analysed. Descriptive statistics, ANOVA and paired analysis are used to characterize the data. RESULTS: PubMed and EMBASE searches yielded 519 and 743 publications, respectively, which were reduced to 20 following review. Reported adult effective doses for any protocol ranged from 46 to 1073 µSv for large fields of view (FOVs), 9-560 µSv for medium FOVs and 5-652 µSv for small FOVs. Child effective doses from any protocol ranged from 13 to 769 µSv for large or medium FOVs and 7-521 µSv for small FOVs. Effective doses from standard or default exposure protocols were available for 167 adult and 52 child exposures. Mean adult effective doses grouped by FOV size were 212 µSv (large), 177 µSv (medium) and 84 µSv (small). Mean child doses were 175 µSv (combined large and medium) and 103 µSv (small). Large differences were seen between different CBCT units. Additional low-dose and high-definition protocols available for many units extend the range of doses. DHP was found to reduce average absolute error for calculation of dose by 45% in comparison with DAP. CONCLUSIONS: Large exposure ranges make CBCT doses difficult to generalize. Use of DHP as a metric for estimating effective dose warrants further investigation.

X-ray microprobe analysis of the retina and RPE in sheep with ovine ceroid-lipofuscinosis.

Ovine ceroid-lipofuscinosis (OCL) is one animal model for the human condition, and because autofluorescent lipopigments are prominent in the brain and eye, it may also prove useful as a model for aging. For example, a progressive decline in electrical recording from brain and retina are observed in both aging and OCL. Samples of retinal and retinal pigment epithelial (RPE) tissues were obtained from a young control. 2 animals with OCL and a normal aged sheep. Specimens were cryo-fractured and examined by scanning electron microscopy/x-ray microanalysis. Measurements made of 6 individual cells in the ganglion layer of OCL specimens, the remainder of the retina, and RPE showed age-related changes in zinc, iron, and copper which were associated with lipopigment accumulation in the RPE. There was marked decrease in phosphate, sulfur, and manganese levels, as photoreceptor cells and their outer segments are lost in the disease process. This is the first report of metal analysis in the retina and RPE in a disease entity, and as a function of normal aging.

Gastrointestinal side effects of intravenous erythromycin: incidence and reduction with prolonged infusion time and glycopyrrolate pretreatment.

OBJECTIVE: To determine the frequency of gastrointestinal toxicity due to intravenous (IV) erythromycin and to attempt to decrease this toxicity by prolonging the infusion time of erythromycin and/or pretreating with the peripheral anticholinergic, glycopyrrolate 0.1 mg IV. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: General medical wards of a tertiary medical center. PATIENTS: A total of 51 hospitalized patients 18 years of age or older who were prescribed IV erythromycin lactobionate (EMLB) 500 mg every 6 hours by their attending physicians. INTERVENTIONS: Each of eight consecutive infusions of EMLB was randomly assigned to one of four groups: control--30-minute infusion/placebo pretreatment; 60/P--60-minute infusion/placebo pretreatment; 30/G--30-minute infusion/glycopyrrolate pretreatment; and 60/G--60-minute infusion/glycopyrrolate pretreatment. MAIN OUTCOME MEASURES: Each infusion was accompanied by a questionnaire in which patients rated the magnitude of nausea and vomiting on a scale of 1 (no toxicity) to 9 (severe toxicity). Scores for both nausea and vomiting were added together for a total toxicity score ranging from 2 to 18. A total score of greater than 8 was defined as clinically important. RESULTS: The 51 patients received a total of 356 infusions with gastrointestinal toxicity occurring in 27 of 51 (53%) patients. Among patients under the age of 40, 22 of 33 (67%) experienced toxicity compared with only five of 18 patients (28%) over the age of 40 (p = 0.018). Clinically important toxicity was seen in 19 of 51 patients (37%), including five who withdrew during the study because of severe nausea and vomiting. In this group, the combination of a 60-minute erythromycin infusion and glycopyrrolate pretreatment decreased clinically important toxicity by 79% from 47% to 10%, a statistically and clinically significant 37% (95% CI, 14% to 60%) difference (p = 0.007). CONCLUSIONS: Gastrointestinal toxicity associated with the IV infusion of erythromycin is common and is more likely to occur in younger patients. A 1-hour infusion of erythromycin combined with pretreatment with glycopyrrolate, 0.1 mg IV, is effective in reducing this toxicity.

Ultrastructural changes in the aqueous outflow apparatus of beagles with inherited glaucoma.

Spontaneous glaucoma in the beagle was exhibited after 6 months of age by elevated intraocular pressures and open iridocorneal angles followed by secondary changes. In order to appreciate alterations of the aqueous outflow apparatus in dogs with this autosomal recessive disorder, the eyes of beagles with inherited glaucoma at ages 1 day through 34 months were examined by light, scanning and transmission electron microscopy. Developmentally, no notable differences were observed between normal and preglaucomatous outflow channels through 7 months of age. In 12-month-old glaucomatous chamber angles clustered basement membrane-like material was found scattered throughout the outer corneoscleral trabecular meshwork. In this region elastin-like fibers appeared to be more numerous and arranged less regularly than age-matched normal eyes. Occasional trabecular cells within the corneoscleral trabecular meshwork possessed small clusters of serrated, opaque rods within their cytoplasm. In the older glaucomatous dogs these changes were more generalized and extensive throughout the entire corneoscleral trabecular meshwork. In some individual eyes the anterior chamber angles were observed to be narrow both clinically and histologically. These outflow apparatuses were additionally characterized by compressed, less organized trabeculae with a concomitant build-up of extracellular materials. No correlation was found between the shallowness of the iridocorneal angle and increase in intraocular pressure. Primary glaucoma in the beagle during its earlier phases compared more positively to open-angle glaucoma in man than any of the other spontaneous types in animals.

Preparation of a uveitogenic peptide by chymotryptic digestion of bovine S-antigen.

Limited proteolysis of bovine S-antigen with alpha-chymotrypsin resulted in the accumulation of three peptides of MW 24,000, 16,000, and 12,000 daltons, respectively. By ELISA (enzyme-linked immunosorbent assay), MW 24,000 peptide was found to react with anti-S antibodies, but the other two peptides did not react with the antibodies under the assay conditions. The reactive peptide was separated from the smaller peptides by gel filtration on Sephadex G-75 and Sephadex G-50. When the MW 24,000 peptide was injected into Lewis rats, severe to mild uveitis was produced in all injected animals. The results indicate that the pathogenic determinant is on the MW 24,000 peptide.

Analysis of eye lens-specific genes in congenital hereditary cataracts and microphthalmia of the miniature schnauzer dog.

The congenital hereditary cataracts and microphthalmia in the miniature schnauzer dog are inherited by an autosomal recessive mode. To understand the genetic basis of these diseases, the authors purified and analyzed leukocyte deoxyribonucleic acid (DNA) from affected and normal animals using a candidate gene approach. Because the genes that encode the lens-specific proteins, specifically, alpha, beta, and gamma crystallins and the membrane protein (MP26), are known to maintain the structure and function of the lens, the authors used complimentary DNA (cDNA) fragments that corresponded to the above genes to search for the mutations at their loci in the affected animals. They found no evidence of the gene deletion and rearrangement in any of the five loci. In addition, the hybridizable sequences of the dog DNA to the specific probes for the human chromosome 4 and 18 loci, which are reported to be involved in the abnormality of the human eye, seem to be unaffected. These data support the notion that the hereditary cataracts and microphthalmia in the dog may be associated with genes other than those reported for several animal systems.

Effects of phacoemulsification and extracapsular lens removal on corneal thickness and endothelial cell density in the dog.

Twenty-one dogs were used to study the effects of phacoemulsification and extracapsular lens removal on corneal endothelium and corneal thickness. Specular microscopy revealed preoperative cell morphology and cell densities similar to man. After surgery endothelial cell counts significantly decreased 22% (central) and 13% (periphery) with phacoemulsification and 34% (central) and 31% (periphery) with extracapsular lens removal. Corneal thickness increased with both procedures. Endothelial cell pathology included cellular enlargement, increased pleomorphism, and focal cellular degeneration. Phacoemulsification time did not correlate with the degree of cell loss. Dog corneal endothelium responds to surgical trauma in a manner similar to man and maintains a functional monolayer via cellular enlargement and migration. The dog, therefore, represents a good animal model for the study of corneal endothelial disease in man.

Prostaglandin biosynthesis by fat body from the tobacco hornworm, Manduca sexta.

We describe prostaglandin (PG) biosynthesis by microsomal-enriched preparations of fat body from larvae of the tobacco hornworm Manduca sexta. Four major PGs were synthesized under most experimental conditions, PGA2, PGE2, PGD2 and PGF2 alpha. PGA2, was the predominant product under most conditions. Unlike mammals, in which PGA2, is generally thought to arise from non-enzymatic rearrangements of PGE2, the fat body preparations did not convert exogenous PGE2 into PGA2. These findings suggest that PGA2 is an important fat body product that is synthesized by a route that does not involve PGE2. The PG synthase activity and the overall profile of PG synthesis were sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal PG biosynthesis was observed with 1 mg of microsomal-rich protein, incubated at 30 degrees C for 1-2 min. The fat body preparations is sensitive to two non-steroidal anti-inflammatory drugs, indomethacin and naproxen, both of which inhibited PG synthesis at low dosages.

Incorporation of polyunsaturated fatty acids into phospholipids of hemocytes from the tobacco hornworm, Manduca sexta.

We examined the incorporation of four radioactive fatty acids, 18:1n-9, 18:2n-6, 20:4n-6 and 20:5n-3, into cellular lipids of hemocytes from tobacco hornworms, Manduca sexta. Most of the radioactivity associated with 18:1n-9 was recovered from triacylglycerols (TGs), and the radioactivity associated with 18:2n-6 was heavily incorporated into phospholipids (PLs) and TGs. Most of the radioactivity associated with the two eicosanoid-precursor polyunsaturated fatty acids (PUFAs), 20:4n-6 and 20:5n-3, was incorporated into PLs. The incorporated fatty acids were redistributed among the lipid classes during 2 h incubations. The two C20 PUFAs were moved from PLs to TGs. While 18:2n-6 underwent little change, 18:1n-9 was redistributed from TGs to PLs. Within PLs, each of the fatty acids were incorporated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PtG) and phosphatidylserine/inositol (PS/PI). The incorporation patterns changed over time, indicating that the incorporated fatty acids were redistributed among the four PL fractions. The radioactivity associated with 18:1n-9 was mostly recovered from the sn-1 position of PC (59%) and PE (83%). Most of the radioactivity associated with 18:2n-6 was found in the sn-2 position of PC (88%) and PE (67%). Over 90% of the radioactivity associated with 20:5n-3 was recovered from the sn-2 position of PC and PE. Incorporation of 20:4n-6 differed from 20:5n-3 because more radioactivity was recovered from the sn-2 position of PC (93%) than PE (69%). These findings are in line with the general background of lipid biochemistry, from which incorporation of 20:4n-6 into PE marks a notable departure: 31% of the radioactivity associated with this acid was recovered from the sn-1 position of PE. These findings indicate that hemocytes from the tobacco hornworm elaborate a fatty acid incorporation system, which exhibits specificity with respect to fatty acid structure and lipid class.

What can we learn from prostaglandins and related eicosanoids in insects?

Eicosanoids are oxygenated metabolites of three C20 polyunsaturated fatty acids (20:3n-6, 20:4n-6, and 20:5n-3). While eicosanoids are very well known in mammalian systems, mostly due to their pharmaceutical interest, there is increasing recognition of the significance of these compounds in insects and other invertebrates. In this paper we consider four major concepts emerging from work on eicosanoids in invertebrates. First, the biological significance of eicosanoids extends far beyond their physiological and pathophysiological actions in human and veterinary medicine. Second, we can greatly improve our understanding of eicosanoids in insects by integrating our work on insects into ongoing studies of other invertebrates. Third, some eicosanoid actions may be fundamental to animals. Fourth, the biochemistry of eicosanoids in insects and other invertebrates can differ from expectations based on the mammalian background. Finally, we point to an uncharted frontier in insect studies--the biochemical mechanisms of eicosanoid action--by drawing attention to some of the work on eicosanoid receptors in mammalian systems.

Prostaglandin biosynthesis by salivary glands isolated from the lone star tick, Amblyomma americanum.

Salivary glands separated from internal tissues of the lone star tick, Amblyomma americanum, are competent to synthesize prostaglandins (PGs). Using an in vitro PG biosynthesis assay four major PGs, namely, PGA2/PGB2, PGD2, PGE2, and PGF2 alpha were synthesized. Under standard assay conditions PGA2/PGB2 was the predominant product. Salivary tissues as well as non-salivary internal tissues were capable of PG biosynthesis. We observed that storing ticks at -80 degrees C for 3 months resulted in reduced PG biosynthesis. This indicates that the tick preparation, unlike comparable mammalian preparations, is not stable to freezing. Cyclooxygenase inhibitors, indomethacin (> 10 microns) and naproxen (> 15 microns), completely inhibited PG biosynthesis. These results demonstrate the presence of a PG biosynthetic system in salivary glands and other internal tissues of the lone star ticks.

Eicosanoid biosynthesis by hemocytes from the tobacco hornworm, Manduca sexta.

We describe eicosanoid biosynthesis by microsomal-enriched preparations of hemocytes from larvae of the tobacco hornworm Manduca sexta. Four major prostaglandins, PGA2, PGE2, PGD2, and PGF2 alpha, and a lipoxygenase product that co-chromatographed with 15-hydroxyeicosatetraenoic acid (HETE) were synthesized under most conditions. The HETE's fraction was the predominant product. Eicosanoid biosynthesis was sensitive to experimental conditions, including incubation time, temperature, and protein concentration. Optimal biosynthesis was observed with 1.5 mg of microsomal-enriched protein, incubated at 30 degrees C for 2 min. The hemocyte preparation is sensitive to low dosages of naproxin and esculetin. As in mammals, most lipoxygenase activity (87%) was localized in the cytosolic fraction of hemocytes. Unlike mammals, in which PGH synthase is associated with intracellular membranes, the hemocytic activity was detected in microsomal (59%), cytosolic (35%) and mitochondrial fractions (5%).

Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum.

We report on prostaglandin (PG) biosynthesis in the lone star tick, Amblyomma americanum. In vitro preparations of whole female ticks and internal tissues were competent to biosynthesize four PGs: PGA2/PGB2, PGD2, PGE2, PGF2 alpha. PGA2/PGB2 was the major product under optimal conditions. PG biosynthesis by whole tick and internal tissues were sensitive to incubation conditions including, protein concentration, time, temperature, pH, and presence of a co-factor cocktail composed of reduced glutathione, hydroquinone, and hemoglobin. Under standard assay conditions, 2 mg/ml protein were incubated at pH 8.0 for 2 min at 32 degrees C. PG biosynthesis was inhibited by indomethacin, a potent cyclooxygenase inhibitor in mammalian systems. Internal tissue preparations were fractionated into cytosolic and microsomal preparations by ultracentrifugation. PG biosynthetic activity was detected in both fractions. The subcellular distribution of PG biosynthetic activity in ticks is similar to other invertebrates, but quite different from mammals, in which PG biosynthetic activity is almost exclusively localized in the microsomal fractions. PGH synthase-2 was detected in the microsomal fraction on western blot analysis. These results suggest that the lone star tick is competent to biosynthesize PGs. These compounds may contribute to the success of tick feeding ecology by attenuating the defense responses of vertebrate hosts during lengthy feeding periods.

Arachidonic acid and prostaglandin E2 in Malpighian tubules of female yellow fever mosquitoes.

The fatty acid compositions of Malpighian tubules from adult females of the mosquito Aedes aegypti were determined for total lipids, phospholipids, triacylglycerols and three phospholipid fractions, namely phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol/phosphatidylserine (PI/PS). The prostaglandin precursor arachidonic acid (20:4n-6) occurred in total lipids and phospholipids, but not triacylglycerols. Within phospholipids, nearly all of the 20:4n-6 was detected in PC, with only traces in PE, and none was detected in PI/PS. Isolated Malpighian tubules incorporated exogenous radioactive 20:4n-6 into tissue phospholipids and diacylglycerols, with most of the radioactivity recovered in diacylglycerol. These data indicate selective incorporation of 20:4n-6 into tissue lipids. PGE2 was detected in Malpighian tubule whole mounts by immunohistochemical staining. These findings support the idea that prostaglandins are physiologically active in mosquito Malpighian tubules.

High-flow gas insufflation to facilitate MIDCABG: effects on coronary endothelium.

BACKGROUND: During less invasive coronary bypass operations on the beating heart, as well as conventional operations using continuous warm cardioplegia, a precise anastomosis is facilitated by a bloodless field. To maintain a clear field, many surgeons use high-flow gas insufflation. However, the potentially damaging effects of gas insufflation on coronary endothelium have not been elucidated. METHODS: Seven pigs underwent median sternotomy. Between two coronary occluders, an arteriotomy in the mid left anterior descending coronary artery (LAD) was performed. In the experimental group (n = 5), the operative field was kept clear by exposing the arteriotomy to a catheter-directed stream of carbon dioxide at 15 L/min. In the control group (n = 2), the arteriotomy was left open to room air. After 20 minutes, the segments of LAD exposed to carbon dioxide or room air, and the unexposed proximal LAD and right coronary artery, were processed, sectioned, and stained together. A murine anti-human tie-2 monoclonal antibody was used to identify endothelium. RESULTS: All unexposed LAD and right coronary artery segments and all LAD segments exposed only to room air demonstrated normal, contiguous staining of endothelium with the murine anti-human tie-2 monoclonal antibody. In contrast, all LAD segments exposed to high-flow carbon dioxide gas insufflation demonstrated near-complete loss of endothelium. CONCLUSIONS: These data demonstrate that high-flow carbon dioxide gas insufflation denudes the coronary artery of its endothelium. This exposes blood elements to the subendothelium and promotes clotting, and endothelial loss may promote smooth muscle cell migration and proliferation. These events set the stage for early and late graft failure.