Reached Control Levels Research Articles

Induction of genomic instability after an acute whole-body exposure of mice to 56Fe ions

The purpose of this study was to evaluate dose–response relationships for the in vivo induction of micronuclei (MN) as a measure of both initial radiation damage and the induction of genomic instability. These measurements were made in mouse blood erythrocytes as a function of radiation dose, radiation quality, time after irradiation, and the genetic background of exposed individuals. Blood samples were collected from two strains of mouse (CBA/CaJ and C57BL/6J) at different times up to 3 months following a whole-body exposure to various doses of 1 GeV/amu 56Fe ions (0, 0.1, 0.5 and 1.0 Gy, at the dose rate of a 1 Gy/min) or 137Cs gamma rays (0, 0.5, 1.0 and 3.0 Gy, at the dose rate of 0.72 Gy/min). Blood-smear slides were stained with acridine orange (AO). The frequencies of MN were measured in mature normochromatic-erythrocytes (MN-NCEs) and in immature polychromatic-erythrocytes (MN-PCEs). Effects of both types of radiation on erythropoiesis were also evaluated. As a measure of cell progression delay, a dose-dependent decrease in numbers of PCEs was observed at day 2 post-exposure in both strains, regardless of radiation quality. Subsequently, the levels of PCEs increased in all exposed mice, reaching control levels (or higher) by day 7 post-exposure. Further, at day 2 after the exposure, there was no increase in the frequency of MN-PCEs in CBA/CaJ mice exposed to 56Fe ions while the frequency of MN-PCEs elevated as a function of dose in the C57BL/6J mice. At day 4, there was no dose related increase in MN-NCEs in either strain of mouse exposed to 137Cs gamma rays. Additionally, at the early sacrifice times (days 2 and 4), 56Fe ions were slightly more effective (per unit dose) in inducing MN-NCEs than 137Cs gamma rays in CBA/CaJ mice. However, there was no increase in the frequency of MN-NCEs at late times after an acute exposure to either type of radiation. In contrast, both types of radiation induced increased MN-PCEs frequencies in irradiated CBA/CaJ mice, but not C57BL/6J mice, at late times post-exposure. This finding indicates the potential induction of genomic instability in hematopoietic cells of CBA/CaJ mice by both types of radiation. The finding also demonstrates the influence of genetic background on radiation-induced genomic instability in vivo.

Effects of prolonged treatment with cyproterone acetate on hydrolytic enzymes in seminal vesicles of the mouse.

Daily s.c. injections of cyproterone acetate cause a time-related decrease in the weight and beta-glucuronidase activity of seminal vesicles in mouse. Protein content shows an initial increase and subsequently, after 120-180 days of treatment, a decline (reaching control levels). Acid phosphatase activity also decreases but only after long-term treatments. 5 alpha-DHT tends to bring the values of various parameters towards the control range. However, its effects are of a lesser magnitude in animals treated for 90 days with the antiandrogen. It is proposed that the antifertility action of cyproterone acetate is due mainly to its negative influence not only on the epididymis but also on seminal vesicles.

Glucose-lowering, hepatoprotective and hypolipidemic activities of stem bark ofFicus racemosain streptozotocin-induced diabetic Rats

The present study evaluated the antihyperglycemic, hepatoprotective, and hypolipidemic effects of F. racemosa bark powder and aqueous extract in streptozotocin-induced diabetic rats. Streptozotocin-induced diabetic rats (n = 6) were treated with F. racemosa Linn (Moraceae) bark powder (FRP) and aqueous extract (FRAE) for six weeks. Blood glucose was determined every 15 days using a portable glucometer. At the end of the study period, the rats were sacriÞ ced and levels of serum glucose, protein, total cholesterol, triglycerides, AST, ALT, TBARS, and glutathione were determined as indicators of antihyperglycemic, hypolipidemic, and hepatoprotective activities, as well as of antioxidant potential. TBARS and glutathione levels were determined in the liver and the kidneys also. Both the bark powder and aqueous extract of F. racemosa bark caused a signiÞ cant reduction (P ≤ 0.05) in blood glucose (54 and 66% respectively). A signiÞ cant reduction (P ≤ 0.05) was also observed in serum cholesterol and triglyceride levels to the control levels. The aqueous extract was more effective and caused a signiÞ cant reduction (P ≤ 0.05) in TBARS, AST, ALT levels compared to untreated diabetic rats. However, it did not reach control levels. A signiÞ cant increase in glutathione concentrations over the control levels was also observed in rats treated with F. racemosa bark. It is concluded that F. racemosa bark has a signiÞ cant hypolipidemic and hepatoprotective effect besides being a potent antihyperglycemic agent. Key words: Antihyperglycemic, antioxidant effect, Ficus racemosa, hypolipidemic, hepatoprotective.

Laser Etching: A Novel Technology to Label Florida Grapefruit

Laser labeling of fruit and vegetables is an alternative means of labeling produce in which a low-energy carbon dioxide laser beam etches the surface and reveals a contrasting underlying layer. These etched surfaces can promote water loss and may increase the number of entry sites for decay-promoting organisms. The long-term effects of laser labeling on produce quality during storage have not been examined. We conducted experiments to measure water loss, peel appearance, and potential decay in laser-labeled grapefruit (Citrus paradisi) during storage. Laser-labeled fruit stored at 10 °C and two relative humidities (i.e., 95% and 65%) for 5 weeks showed no increase in decay compared with nonetched control fruit, suggesting that laser labeling does not facilitate decay. This was confirmed by experiments where Penicillium digitatum spores were coated on fruit surfaces before and after etching. In either case, no decay was observed. In agar plates containing a lawn of P. digitatum spores, the laser etching reduced germination of spores in contact areas. Water loss from etched areas and label appearance were determined during storage. Water loss from waxed etched surfaces reached control levels after 24 h in storage. Label appearance slowly deteriorated during 4 weeks in storage and was proportional to laser energy levels and ambient relative humidity. Waxing the labeled surface reduced water loss by 35% to 94%, depending on the wax formulation used. We concluded that laser labeling provides the grapefruit industry a safe alternative to adhesive sticker labeling without enhancing decay susceptibility.

Laser Etching: A Novel Technology to Label Florida Grapefruit

Laser labeling of fruit and vegetables is an alternative means of labeling produce in which a low-energy carbon dioxide laser beam etches the surface and reveals a contrasting underlying layer. These etched surfaces can promote water loss and may increase the number of entry sites for decay-promoting organisms. The long-term effects of laser labeling on produce quality during storage have not been examined. We conducted experiments to measure water loss, peel appearance, and potential decay in laser-labeled grapefruit (Citrus paradisi) during storage. Laser-labeled fruit stored at 10 °C and two relative humidities (i.e., 95% and 65%) for 5 weeks showed no increase in decay compared with nonetched control fruit, suggesting that laser labeling does not facilitate decay. This was confirmed by experiments where Penicillium digitatum spores were coated on fruit surfaces before and after etching. In either case, no decay was observed. In agar plates containing a lawn of P. digitatum spores, the laser etching reduced germination of spores in contact areas. Water loss from etched areas and label appearance were determined during storage. Water loss from waxed etched surfaces reached control levels after 24 h in storage. Label appearance slowly deteriorated during 4 weeks in storage and was proportional to laser energy levels and ambient relative humidity. Waxing the labeled surface reduced water loss by 35% to 94%, depending on the wax formulation used. We concluded that laser labeling provides the grapefruit industry a safe alternative to adhesive sticker labeling without enhancing decay susceptibility.

Renal organic anion transporter 1 is maldistributed and diminishes in proximal tubule cells but increases in vasculature after ischemia and reperfusion

Renal solute clearances are reduced in ischemic acute kidney injury. However, the mechanisms explaining how solute clearance is impaired have not been clarified. Recently, we reported that cadaveric renal allografts exhibit maldistribution of organic anion transporter 1 (OAT1) in proximal tubule cells after ischemia and reperfusion, resulting in impairment of PAH clearance. In the present study, we characterized renal OAT1 in detail after ischemia-reperfusion using a rat model. We analyzed renal OAT1 using confocal microscopy with a three-dimensional reconstruction of serial optical images, Western blot, and quantitative real-time RT-PCR. OAT1 was distributed to basolateral membranes of proximal tubule cells in controls. With ischemia, OAT1 decreased in basolateral membrane, especially in the lateral membrane domain, and appeared diffusely in cytoplasm. After reperfusion following 60-min ischemia, OAT1 often formed cytoplasmic aggregates. The staining for OAT1 started reappearing in lateral membrane domain 1 h after reperfusion. The basolateral membrane staining was relatively well discernable at 240 h of reperfusion. Of note, a distinct increase in OAT1 expression was noted in vasculature early after ischemia and after reperfusion. The total amount of OAT1 protein expression in the kidney diminished after ischemia-reperfusion in a duration-dependent manner until 72 h, when they began to recover. However, even at 240 h, the amount of OAT1 did not reach control levels. The kidney tissues tended to show a remarkable but transient increase in mRNA expression for OAT1 at 5 min of ischemia. Our findings may provide insights of renal OAT1 in its cellular localization and response during ischemic acute kidney injury and recovery from it.

Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/reperfusion in rats: effect of neuroprotectant simvastatin.

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

O3-01-06: Evidence of vascular recovery after removal of vascular Aβ by passive immunotherapy in PDAPP mice

Background: Cerebral amyloid angiopathy is a common neuropathological feature of Alzheimer’s disease and is characterized by vascular deposition of fibrillar amyloid -protein (VA ). Vascular structural changes are associated with VA deposits, including localized loss of smooth muscle cells (SMC) and changes in extracellular matrix (ECM). Passive immunization with antibodies to the A N-terminus (3D6, aa 1-5) has been shown to effectively reduce amyloid deposition in the vasculature of PDAPP transgenic mice. In the present study we characterized structural changes induced by amyloid on SMC and ECM of PDAPP mouse vessels and assessed the effects of passive immunotherapy. Methods: Mice were immunized weekly for either 3 or 9 months with 1 or 3 mg/Kg of 3D6 antibody. High-resolution, quantitative IHC analyses of vascular components ( -actin for SMC and collagen-IV for ECM) were performed on meningeal vessels from the sagittal sinus, where VA deposition is prominent ( 70% of vessels affected). Microhemorrhage events were monitored by hemosiderin detection or ferritin immunohistochemistry. Results: We found that changes in the vascular wall are invariably associated with VA , and they included both degeneration (decreased thickness) and hyperplasia/hypertrophy (increased thickness) of SMC and ECM. These two contrasting findings were often observed in the same vessel and were not present in wild type animals or PDAPP vessels lacking amyloid. The extreme degrees of thickening and thinning of the SM resulted in a widely variable vascular phenotype in untreated PDAPP mice. Passive immunotherapy restored the pattern of vascular SMC and ECM thicknesses and reduced the phenotypic variability in a doseand time-dependent manner, with the high dose of 3D6 reaching control levels (wild type) at 9 months. Although the incidence of microhemorrhage increased in the 3-month group, it reduced to control levels after 9 months of treatment. Conclusions: Our results suggest that passive immunotherapy allows the recovery of meningeal vessels from amyloid-induced structural changes. Furthermore, the treatment-related increase in microhemorrhage appears to be a transient event that resolves during VA clearance. Mechanisms of repair may be triggered by VA removal, which ultimately lead to recovery from vascular dysfunction. Experiments to assess this possibility are in progress.

Differential changes in mGlu2 and mGlu3 gene expression following pilocarpine-induced status epilepticus: A comparative real-time PCR analysis

Group II metabotropic glutamate (mGlu II) receptors subtype 2 and 3 (mGlu2 and mGlu3) are subtle regulators of neuronal excitability and synaptic plasticity in the hippocampus. In recent years, researchers have investigated the potential neuroprotective and anticonvulsant effects of compounds acting on mGlu II receptors. However, abnormal expression and function of mGlu2 and mGlu3 have been reported in temporal lobe epilepsy, a phenomena that may limit the therapeutic effectiveness of these potentially new antiepileptic drugs. Here, we investigated seizure-induced changes in mGlu2 and mGlu3 mRNA following pilocarpine-inducted status epilepticus (SE) and subsequent epileptogenesis. Relative changes in gene expression were assessed by comparative analysis of quantitative real-time PCR (qrtPCR) by the delta–delta CT method. Pilocarpine-treated and control rats were sacrificed at different periods (24 h, 10 days, one month and more than two months) following SE. Total RNA was isolated from microdissected dentate gyrus and processed for RT-PCR and qrtPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control gene. Analysis of relative quantification (RQ) ratios of mGlu2 and mGlu3 mRNA expression revealed a significant down-regulation of both targets at 24 h after SE. Gene expression partially recovered at 10 days following SE reaching control levels at one month after SE. Two month after SE, mGlu2 mRNA expression was significantly down-regulated to ~ 41% of control expression whereas mGlu3 mRNA was comparable to control levels. Our data indicate that mGlu2 and mGlu3 expression is dynamically down-regulated or selectively enhanced during critical periods of epileptogenesis. Seizure-induced differential dysregulation of mGlu2 and mGlu3 receptors may affect the availability of these molecular targets for therapeutic compounds in epilepsy.

Biomechanical Properties of the Condyle Created by Osteodistraction

A new condyle can be reconstructed by osteodistraction, but the biomechanical properties of the neocondyle remain unknown. This study examined the hypothesis that the biomechanical properties of neocondylar cancellous bone could reach control levels 24 weeks after its creation by osteodistraction. The right mandibular condyles were removed and reconstructed by osteo-distraction in 16 adult goats. Their contralateral condyles served as controls. Microstructural and mechanical properties were examined by microcomputed tomography and mechanical testing. At 24 weeks after distraction, the neocondyle grew larger in size, but the shape and histological features were similar to those of the controls. The cancellous bone of the neocondyle even appeared to be more dense and stiffer in comparison with the control condyle. The results of this study suggest that the neocondyle created by osteodistraction develops nearly normal biomechanical properties for functional loading by 24 weeks after creation.

L-Asparaginase diabetes mellitus in rabbits: differing effects of two different schedules of L-asparaginase administration.

The diabetogenic effect of daily injections of 1000 i.u./kg body wt. E coli L-asparaginase was studied in male New Zealand white rabbits and compared with the diabetogenic effect of a single bolus of 10,000 I.U. E coli L-asparaginase/kg body wt. to determine whether the schedule of administration of the drug altered the diabetic syndrome produced. A daily injection of 1000 i.u. L-asparaginase/kg. body wt. was continued for 30 days. During this time glucose levels in rabbits allowed free access to food rose steadily, reaching levels of 717 +/- 63 mg/dl the day after the last injection. Levels of immunoreactive insulin fell, reaching their nadir, 53 +/- 4 pg/ml (approximately 50% of baseline) at 25 days. Glucose levels declined when therapy was discontinued, but remained significantly above control levels 46 days after insulin injections were stopped. (Glucose levels in L-asparaginase-treated groups vs. those in controls on day 46 after discontinuation: 116 +/- 3 vs. 104 +/- 1 mg/dl; P less than 0.0025.) Levels of immunoreactive insulin rose when therapy ended, reaching control levels 17 days after discontinuation. In contrast, a single bolus injection of 10,000 I.U. L-asparaginase/kg resulted in hyperglycemia with hyperinsulinemia. These data suggest that L-asparaginase can induce either a hypoinsulinemic or a hyperinsulinemic diabetic syndrome depending on the schedule of administration of the L-asparaginase and that a mild abnormality in glucose homeostasis persists after discontinuation of L-asparaginase therapy.

Vestibular compensation after unilateral labyrinthectomy: normal versus cerebellar dysfunctional mice.

Loss of vestibular information from one labyrinth produces marked asymmetries of postural and ocular motor control, which resolve over time. Recent developments in mouse genetic engineering, which allow the generation of transgenic and knockout mutant mice, provide a unique opportunity to bridge the gap between the molecular mechanisms that underlie compensation and behaviour. We compared compensation following unilateral labyrinthectomy in wild-type mice and a cerebellar-dysfunctional mouse (the Lurcher mutant). The Lurcher mutant is characterized by a point mutation in the ionotropic glutamate receptor delta 2 subunit gene that results in loss of all Purkinje cells. To further investigate this question, we characterized vestibular compensation in a strain of mutant mice that completely lack cerebellar Purkinje cells. Static signs resolved within 24 hours in wild-type mice but did not fully resolve in Lurcher mice. Dynamic signs were evaluated by the quantitative analysis of vestibulo-ocular (VOR) and vestibulocollic (VCR) reflexes. The VOR assessed at 0.5 Hz exhibited increasing gain from day 1 to day 5, reaching control levels by day 20 for the wild-type mice. In contrast, Lurcher mutant mice showed significantly less compensation over this same period. VOR compensation in the mutant mice was slightly more robust in response to high acceleration thrusts but again never reached control levels. Similarly, VCR gains showed limited compensation and remained subnormal in mutant mice. Compensation for dynamic signs starts at day 5 after unilateral labyrinthectomy in normal mice. Cerebellar dysfunctional mutant mice do not compensate for static signs and show limited vestibular compensation for dynamic signs only. We conclude that other noncerebellar pathways for vestibular compensation exist, and our findings emphasize the need for these to be further explored.

Evaluation of pearl millet and flaxseed effects on egg production and n-3 fatty acid content

1. A 6-week trial was conducted to evaluate the effects of pearl millet (PM) as a replacement for maize, in combination with flaxseed (FS), on productivity, egg trait parameters and egg n-3 fatty acid (FA) content in White Leghorn hens. 2. Six diet treatments were used: a control (CTL, maize–soybean meal based) diet, and diets containing 0, 2, 4, 8 or 12% FS, in which all maize was replaced by PM (PM-0, PM-2, PM-4, PM-8 and PM-12, respectively). All diets were isocaloric and isonitrogenous and met NRC (1994) requirements. Eight cage replicates per treatment were used (three hens per cage). At the end of each week, three eggs were randomly collected from each cage to measure egg trait parameters and yolks were separated, pooled and lyophilised for FA determination. Body weights and feed consumption were recorded weekly. Egg production (number of eggs and egg mass produced) was recorded daily. At the end of the experiment, all hens were killed to determine liver haemorrhage score. 3. Egg traits and flock performance parameters were not different among treatments except at week 4, when birds on the PM-12 diet produced smaller eggs than hens on the PM-0 and PM-2 diets. Yolk pigmentation scores were lower for the PM-0 and PM-2 diets (1·60 ± 0·24 and 1·80 ± 0·20, respectively), increased with higher inclusions of FS (2·75 ± 0·47 for PM-12 diet) but did not reach control levels (6·00 ± 0·01). Liver haemorrhage scores were not affected by dietary treatment. Hens given the PM-8 and PM-12 diets produced eggs with n-3 FA content higher than required to be considered as n-3 FA enriched eggs, and had a lower n-6/n-3 FA acid ratio than eggs of hens consuming CTL or the PM based diets with lower FS supplementation. 4. These results suggest that PM can be used to substitute for maize in the diets of layers and may reduce the amount of FS needed to obtain n-3 FA enriched eggs.

Neurologic Signs of Ciguatera Disease: Evidence of their Persistence

Ciguatoxins exert their effect on the voltage-sensitive sodium channels of the cellular membranes of all excitable tissues. This effect confers to ciguatera disease (CD) its neurologic hallmarks. A prospective study among French Polynesian adults over a two-month period was conducted to characterize and determine the persistence of neurologic symptoms of CD. We compared 47 patients with CD with 125 controls. In the acute phase of the disease, patients had mainly sensory disturbances as detected by an hypoesthesia on the palm of the hand and poorer sway performance compared with controls. Follow-up two months showed improvement of sway performance that eventually reaching control levels. However, for light-touch threshold, even if we observed a decrease threshold towards normal values, more than 50% of patients did not reach normal values 60 days after disease onset. Our results support the existence of neurologic impairments of CD and suggest their persistence for at least two months after onset.

CAPS-1 and CAPS-2 Are Essential Synaptic Vesicle Priming Proteins

Before transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca(2+) levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca(2+) triggered release of transmitters.

Downregulation of prolactin-releasing peptide gene expression in the hypothalamus and brainstem of diabetic rats

We investigated the prolactin-releasing peptide (PrRP) mRNA levels in the hypothalamus and brainstem of streptozotocin (STZ)-induced diabetic rats and fa/ fa Zucker diabetic rats, using in situ hybridization histochemistry. PrRP mRNA levels in the hypothalamus and brainstem of STZ-induced diabetic rats were significantly reduced in comparison with those of control rats. PrRP mRNA levels in the diabetic rats were reversed by both insulin and leptin. PrRP mRNA levels in the fa/ fa diabetic rats were significantly reduced in comparison with those of Fa/ ? rats. PrRP mRNA levels in the fa/ fa diabetic rats were significantly increased by insulin-treatment, but did not reach control levels in the Fa/ ? rats. We also investigated the effect of restraint stress on PrRP mRNA levels in STZ-induced diabetic rats. The PrRP mRNA levels in the control and the STZ-induced diabetic rats increased significantly after restraint stress. The diabetic condition and insulin-treatment may affect the regulation of PrRP gene expression via leptin and other factors, such as plasma glucose level. The diabetic condition may not impair the role of PrRP as a stress mediator.

Modulation of multidrug resistance protein (MRP1/ABCC1) expression: a novel physiological role for ouabain

Besides being a (Na(+),K(+))-ATPase inhibitor, high doses of the hormone ouabain have also been reported to modulate both the expression and activity of proteins belonging to the ATP binding cassette family of transporters, such as ABCC7 (CFTR), ABCB1 (P-glycoprotein), and ABCC1 (MRP1). Although these proteins are present in the kidney, only ABCB1 has a putative physiological role in this organ, secreting endobiotics and xenobiotics. In the present work, we studied the relationship between ouabain and ABCC1 expression and function, aiming to establish a physiological role for ouabain. It was observed that prolonged (24 h) but not short (30 min) incubation with 1 nmol/L or higher ouabain concentrations decreased the expression of ABCC1 protein and induced its mRNA expression. This decrease was rapidly reversible, reaching control levels after incubation of cells in ouabain-free medium for 3 h, denoting a hormonal action. Moreover, concentrations equal or higher than 100 nmol/L ouabain also induced impairment of ABCC1 activity, increasing the accumulation of carboxyfluorescein diacetate, an ABCC1 fluorescent substrate. Because ouabain is now accepted as an endogenous hormone, our results suggest that ABCC1 is regulated by hormones related to body volume control, which may have implications for the treatment of hypertensive cancer patients. Moreover, providing ABCC1 is expressed in several other tissues, such as brain, testis, and the immune system, and is related to the transport of glutathione, it is possible that ouabain release may control a number of functions within these organs and tissues by modulating both the expression and the activity of ABCC1.

Eimeria bovis infection enhances adhesion of peripheral blood mononuclear cells to and their transmigration through an infected bovine endothelial cell monolayer in vitro

The first schizogony of Eimeria bovis takes place in lymphatic endothelial cells of the ileum, resulting in the formation of macroschizonts within 2-3 weeks. In this study, we analyse early cellular immune responses to infected host cells on the basis of peripheral blood mononuclear cell (PBMC) adhesion on and transmigration through infected bovine umbilical vein endothelial cell (BUVEC) monolayers. Adhesion of PBMC was upregulated by an E. bovis infection. Most marked effects were observed 1 day p.i.; thereafter, PBMC adhesion declined reaching control levels from day 8 p.i. onward. CD8(+) T cells adhered more frequently to infected BUVEC (42%) than CD4(+) T cells (25%). About one third of attached PBMC were represented by gammadelta-TCR(+) T cells. Adhesion of T cells was not restricted to parasitised host cells, but occurred almost equally on non-infected BUVEC within the same monolayer. Furthermore, we found moderately enhanced levels of PBMC transmigration through infected BUVEC monolayers, in particular on day 2 p.i. The data presented here suggest that E. bovis infection of BUVEC induces endothelial cell-derived proinflammatory reactions, which appear suitable for the initiation of both adaptive and innate immune responses.

Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility

Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility.

Acceleratedin vitrodifferentiation of blood monocytes into dendritic cells in human sepsis

Summary Sepsis-induced immune depression is characterized by infection susceptibility and monocyte early deactivation. Because monocytes are precursors for dendritic cells (DC), alterations in their differentiation into DC may contribute to defective immune responses in septic patients. We therefore investigated the ability of monocytes to differentiate into functional DC in vitro in patients undergoing surgery for peritonitis. Monocytes from 20 patients collected immediately after surgery (D0), at week 1 and at weeks 3-4 and from 11 control donors were differentiated into immature DC. We determined the phenotype of monocytes and derived DC, and analysed the ability of DC to respond to microbial products and to elicit T cell responses in a mixed leucocyte reaction (MLR). We show that, although monocytes from septic patients were deactivated with decreased responses to lipopolysaccharide (LPS) and peptidoglycan and low human leucocyte antigen D-related (HLA-DR) expression, they expressed the co-stimulatory molecule CD80, CD40 and CCR7. Monocytes collected from patients at D0 and week 1 differentiated faster into DC with early loss of CD14 expression. Expression of HLA-DR increased dramatically in culture to reach control levels, as did responses of DC to LPS and peptidoglycan. However, although patient and control immature DC had similar abilities to induce T cell proliferation in MLR, maturation of DC derived from patients did not increase T cell responses. These results show that circulating monocytes from septic patients express markers of activation and/or differentiation despite functional deactivation, and differentiate rapidly into phenotypically normal DC. These DC fail, however, to increase their T cell activation abilities upon maturation.