Nucl Med Commun, 2002 Aug, 23(8), 715 - 20 Assessment of inflammatory bowel disease by using two different (99m)Tc leucocyte labelling methods; Cardoso VN et al.; The aim of this study was to retrospectively compare the diagnostic accuracy of (99m)Tc hexamethylpropyleneamine oxime (HMPAO) white blood cell scintigraphy in patients with a suspicion of active inflammatory bowel disease by using two different cell suspension media: leukocyte poor plasma (LPP) and Hanks' balanced salt solution (HBSS) . Leukocytes from 30 patients were labelled using LPP and in 28 cases using HBSS . In the LPP method the leukocytes were resuspended in 0.5 ml cell-free plasma while in the HBSS method the cells were resuspended in 0.5 ml HBSS . Scintigraphic images were obtained at 30 min and 2 h after injection of 185-200 MBq (99m)Tc-HMPAO leukocytes . The leukocyte labelling efficiency was 65.5% and 89.0%, respectively, for the LPP and HBSS methods . There were 22 true positive, seven true negative and one false negative result in the LPP group, while in the HBSS group results were 18, nine and one, respectively . Diagnostic accuracy was similar with both methods although sensitivity was slightly higher in the HBSS group . In summary, these date indicate that leukocyte scintigraphy labelling using HBSS as the resuspension medium should be used as a first option for white blood cell labelling and diagnosis of inflammatory bowel disease. Med Princ Pract, 2002 Apr-Jun, 11(2), 100 - 4 Inhibition of platelet aggregation by rat trophoblasts; Tewari R et al.; OBJECTIVE: To determine the effect of rat trophoblast cell suspensions on adenosine diphosphate (ADP)-induced platelet aggregation . MATERIALS AND METHODS: The trophoblasts were isolated from ectoplacental cones (a preplacental tissue very rich in trophoblasts) developed in rat embryos on day 12 of pregnancy (normal period of gestation: 22-23 days) . Platelet-rich plasma (PRP) was obtained from adult male rats . The trophoblasts were preincubated (37 degrees C, 30 min), suspended in the medium, and then re-incubated with PRP for 3-5 min . RESULTS: 5 x 10(4) and 7 x 10(4) trophoblasts inhibited ADP-induced platelet aggregation by 10 and 18%, respectively . When the trophoblast cell concentration was increased to 1-2 x 10(5) cells, a proaggregatory phenomenon was observed, even in the absence of ADP . However, there was no inhibition of aggregation or promotion of aggregation when fixed trophoblasts or live endometrial stromal cells were incubated with PRP . CONCLUSION: The results indicate that the aggregation inhibition response was cell specific and concentration dependent . A 68-kD protein was detected in the medium when it was conditioned with 5-7 x 10(4) but not with 1-2 x 10(5) trophoblasts . However, the inhibitory or stimulatory effect does not seem to be dependent on the presence of this protein. J Neurosci Res, 2002 May 1, 68(3), 276 - 83 Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain: effects of previous amplification in vitro; Buchet D et al.; We assessed the developmental potential of human telencephalic progenitor cells, with and without previous amplification in vitro, following grafting into the nonlesioned adult mouse CNS . Cell suspensions, shown to contain neuroepithelium-like and neuroblast-like cells, were injected into the subventricular zone (SVZ) and the striatum . These two regions were selected for comparative studies because one, the SVZ, is mitotically active, whereas the other, the striatum, is mitotically inactive . In situ hybridization with a human-specific Alu probe showed that the cells survived for up to 30 weeks in both targets and migrated away from the injection site . Fresh cells continued to proliferate and gave rise to very extended grafts before differentiating into neurons and glia . We further show that, when grown in vitro prior to grafting, human cells acquired new properties: Their proliferation was very limited, and they differentiated more rapidly . This study therefore provides new information about the use of these cells, which are a potential tool for both cellular and gene therapy . Teratog Carcinog Mutagen, 2002, 22(4), 271 - 84 Monitoring urban air particulate matter (fractions PM 2.5 and PM 10) genotoxicity by plant systems and human cells in vitro: a comparative analysis; Poma A et al.; Increased incidence of mortality and sickness due to cardiopulmonary complications has been associated with elevated levels of urban air particles (UAP), with an aerodynamic diameter of 10 microm (PM 10) and 2.5 microm (PM 2.5) . In the present report alternative plant systems and human cells in vitro are associated with human hazard and genotoxic risk assessment of UAP . The genotoxic activities associated with the coarse (PM 10) and the fine fraction (PM 2.5) of airborne particulates have been analyzed by evaluating micronuclei induction and/or sister-chromatid exchange (SCE) using in vitro models of Daucus carota and HS 27 human fibroblast cell suspensions and Zea mays root meristems . Results show variability in the response of the test systems and indicate that the mutagenicity trend in both plant and human cell cultures was directly correlated to the concentration of carbon-rich particles in the fraction of the PM 2.5 airborne particulates . Moreover, in plant tissues, the frequency of micronuclei and SCE was related to an enhancement of the specific activity of the stress-related enzyme peroxidase . Pflugers Arch, 2002 Jun, 444(3), 452 - 6 Epub 2002 Apr 04. Separation of neonatal rat ventricular myocytes and non-myocytes by centrifugal elutriation; Boerma M et al.; The preparation of pure cardiac myocyte cultures from neonatal rats is hampered by the presence of non-myocytes, which can proliferate during culturing, thereby causing a progressive decrease in the proportion of myocytes . In order to obtain myocyte cell suspensions of high purity, a method based on centrifugal elutriation was developed . Cardiac cells, isolated from neonatal rat heart ventricles, were subjected to elutriation using flow rates that increased step-wise from 20 to 80 ml/min . The cell fraction obtained at 80 ml/min consisted of 68-90% myocytes . Still, upon culturing, the remaining non-myocytes proliferate, causing the proportion of myocytes to decrease to 60 +/- 2% at day 5 . A second elutriation protocol was developed in which myocytes and non-myocytes were separated after a period of co-culturing for 4-5 days . By this approach a fibroblast-rich cell fraction (87 +/- 5%) and a myocyte-rich cell fraction (82 +/- 6%) were obtained . In conclusion, centrifugal elutriation creates the opportunity to separate neonatal rat myocytes from non-myocytes, either freshly isolated or after a period of culturing . Particularly, cell separation after a period of culturing ventricular cells offers an advantage to analyse the experimental effects on myocytes and non-myocytes separately. J Biol Chem, 2002 Sep 13, 277(37), 33878 - 83 Epub 2002 Jul 09. Purification and characterization of norcoclaurine synthase . The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants; Samanani N et al.; Norcoclaurine synthase (NCS; EC ) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in benzylisoquinoline alkaloid biosynthesis in plants . NCS was purified 1590-fold to homogeneity from cell suspension cultures of meadow rue (Thalictrum flavum ssp . glaucum) . The purification procedure, which resulted in a 4.2% yield, involved hydrophobic interaction, anion exchange, hydroxyapatite, and gel filtration chromatography . Purified NCS displayed native and denatured molecular masses of approximately 28 and 15 kDa, respectively, suggesting that the enzyme is composed of two subunits . Two-dimensional polyacrylamide gel electrophoresis revealed two major and two minor isoforms with pI values between 5.5 and 6.2 . NCS activity was maximal at pH 6.5 to 7.0 and temperatures between 42 and 55 degrees C and was not affected by divalent cations . The enzyme showed hyperbolic saturation kinetics for 4-HPAA (K(m) = 335 microm) but sigmoidal saturation kinetics for dopamine (Hill coefficient = 1.8) suggesting cooperativity between the dopamine binding sites on each subunit; thus, NCS might play a regulatory, or rate-limiting, role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis . Product inhibition kinetics performed at saturating levels of one substrate and with norlaudanosoline as the inhibitor showed that NCS follows an iso-ordered bi-uni mechanism with 4-HPAA binding before dopamine . NCS activity was highest in soluble protein extracts from roots followed by stems, leaves, and flower buds. Eur J Neurosci, 1991, 3(8), 758 - 763 Expression of Human Neurofilament-light Transgene in Mouse Neurons Transplanted into the Brain of Adult Rats; Vidal-Sanz M et al.; To investigate the expression of nerve cell-specific transgene products in neural transplants, we implanted into the hippocampus of immunosuppressed adult Sprague - Dawley rats cell suspensions obtained from the septal region of the fetal brain of mice that carry the human neurofilament-light (hNF-L) gene . In grafts examined between 3 weeks and 7 months after transplantation, axons and nerve cell somata immunoreacted to antibodies specific to the human NF-L subunit . Thus, the hNF-L protein appears to be a suitable marker of these grafted neurons . Transgenic mice bearing the hNF-L gene may be a convenient source of donor tissue or be used as hosts for neural transplantation studies . Furthermore, the hNF-L promoter/enhancer elements in this transgene may help direct neuronal expression of heterologous genes that could influence nerve cell responses in either the transplant or host tissues. Eur J Neurosci, 1991, 3(9), 905 - 918 Host Brain Regulation of Fetal Locus Coeruleus Neurons Grafted to the Hippocampus in 6-Hydroxydopamine-Treated Rats . An Intracerebral Microdialysis Study; Kalen P et al.; Release properties of intrahippocampal transplants of noradrenergic neurons were monitored by microdialysis in awake and halothane-anaesthetized rats . Fetal locus coeruleus neurons were implanted as a cell suspension into hippocampi deprived of their innate noradrenalin (NA) innervation by intraventricular 6-hydroxydopamine treatment . Dialysis probes of the loop type were implanted into the dorsal hippocampus 1 - 2 days before each experiment, i.e . 7 - 11 months after grafting . Age-matched intact and lesion-only animals served as controls . Microscopic analysis showed a graft-derived tyrosine hydroxylase immunoreactive, presumably noradrenergic, fibre network throughout the dorsal hippocampal formation, surrounding the probe site . The innervation density varied from sub- to supranormal . The grafts restored baseline NA release in the graft-reinnervated hippocampus to near-normal levels both in awake and halothane-anaesthetized animals . Potassium chloride (100 mM) in the perfusion fluid induced a dramatic increase in NA release that was similar in magnitude in the grafted and intact hippocampi . A NA uptake blocker (desipramine) added to the perfusion fluid at 5 microM induced a similar increase in NA output in the grafted and intact hippocampi, and the output was substantially reduced by tetrodotoxin, added at 1 microM in the presence of uptake blockade . Electrical stimulation of the lateral habenular nucleus (15 Hz, 0.5 mA) in halothane-anaesthetized rats induced a significant increase in NA output both in the intact and grafted hippocampi . This effect was abolished by transection of the fasciculus retroflexus, which carries the efferent projections of the habenular complex . Behavioural activation through handling induced a consistent increase in NA release only in the intact animals, but in a few grafted rats (which also responded to habenular stimulation) the NA output was clearly elevated by handling . Forced immobilization induced a significant increase in NA output both in the intact and grafted hippocampi, but in the grafted ones the response was somewhat smaller and more transient . In the same set of animals, swimming in warm water (25 - 30 degrees C) induced a sharp increase in NA output in the intact animals, whereas only one of the grafted rats responded by increased NA output . The results indicate that the locus coeruleus grafts, despite their ectopic location, can become functionally integrated with the host brain, and that the activity of the transplanted noradrenergic neurons can, under some circumstances, be modulated from the host brain in response to environmental challenges. Eur J Neurosci, 1991 Oct, 3(1), 86 - 101 Efferent Projections to the Host Brain from Intrastriatal Striatal Mouse-to-rat Grafts: Time Course and Tissue-type Specificity as Revealed by a Mouse Specific Neuronal Marker; Wictorin K et al.; The developmental time-course and growth characteristics of efferent graft-to-host projections were studied from mouse fetal striatal grafts (E13 - 14) implanted as a cell suspension into the ibotenate-lesioned striatum of immunosuppressed adult rats . A cell surface monoclonal antibody specific for mouse neurons (M6) was used to identify the donor cells and their projections into the host brain . At 3 - 5 days after implantation, sparse fascicles of M6-positive graft-derived fibres extended for approximately 0.3 - 0.4 mm across the graft - host border into the surrounding host striatum . From the beginning they were selectively orientated in one direction, i.e . caudally along the myelinated fibre bundles of the internal capsule . At 8 days, the graft-derived fibres were more numerous and more densely labelled . They ran in dense fascicles inside the myelinated bundles of the host internal capsule and reached the rostral host globus pallidus, a distance of approximately 1.2 mm from the caudal tip of the graft . Two weeks after grafting, the M6-positive fibre fascicles were clearly seen to branch within the globus pallidus to form terminal-like networks . From this time onwards, the immunoreactivity of the outgrowing fibre fascicles gradually diminished, although small but dense terminal-like networks could be found in the host globus pallidus in most, but not all, of the rats at longer survival times (3 - 15 weeks) . This is consistent with previous work showing that outgrowing axons lose their M6 immunoreactivity as they mature and become myelinated . Control grafts of fetal neocortical and fetal cerebellar tissue were used to assess the tissue-type specificity of the efferent fibre growth . The neocortical implants projected densely up to about 3 mm into the host brain, along the internal capsule and the corpus callosum and into the overlying cortex . By contrast, although the cerebellar grafts survived well, they showed very little efferent fibre growth . Double immunostaining for DARPP-32 and M6 revealed that all M6-positive fibre fascicles extending from the striatal (but not neocortical) grafts also showed DARPP-32 positivity, and thus that it was the DARPP-32-positive regions of the striatal grafts that projected to the host brain . It is concluded that graft-to-host projections, running along and inside host myelinated bundles, are formed from intrastriatal striatal grafts within 1 - 2 weeks of implantation . Grafts of neocortical tissue grew well along the same trajectory, whereas neurons of a type not normally projecting along the internal capsule, i.e . cerebellum, failed to extend axons over any significant distance along this trajectory. Eur J Neurosci, 1991, 3(12), 1330 - 1337 Serotonin Reinnervation of the Suprachiasmatic Nucleus by Intrahypothalamic Fetal Raphe Transplants, with Special Reference to Possible Influences of the Target; Daszuta A et al.; Survival and development of fetal serotonin (5-HT) neurons grafted to various brain areas in adult mammals have been suggested to be under host influences . The aim of this study was to determine whether the suprachiasmatic nucleus of the hypothalamus (SCN), a region receiving a 5-HT input which is one of the densest and the most heavily synaptic in the brain, can actually support the development of transplanted 5-HT neurons . The time course and extent of 5-HT reinnervation were therefore investigated with 5-HT immunocytochemistry in adult rats subjected to intraventricular injection of 5,7-dihydroxytryptamine and subsequent grafting of fetal cell suspension of mesencephalic raphe neurons . The ultrastructural features of the newly formed 5-HT terminal plexa were also examined . Serotonin reinnervation of the SCN remained partial up to 4 months post-transplantation, with no apparent predilection of the reinnervating fibres for any particular portion of the nucleus, thus differing from the normal 5-HT innervation of the SCN both quantitatively and qualitatively . This was in sharp contrast to the 5-HT hyperinnervation observed in neighbouring areas such as the supraoptic nucleus, a structure normally provided with only few 5-HT fibres, and the ventral wall of the third ventricle . The graft-derived 5-HT axons, however, displayed ultrastructural features that did not appear different from those of their normal counterparts; in particular they re-established defined synaptic contacts with the host population . These results may indicate that the mature SCN specifically lacks a trophic factor necessary for the ingrowth of graft-derived 5-HT fibres, or that it represents an inhibitory environment for such an ingrowth . The limited ability of regrowing 5-HT axons to restore a normal density of 5-HT innervation could also be related to the fact that these neurons normally establish a relatively high number of synaptic contacts in the target region. Eur J Neurosci, 1989 May, 1(3), 189 - 195 Host Corticostriatal Fibres Establish Synaptic Connections with Grafted Striatal Neurons in the Ibotenic Acid Lesioned Striatum; Wictorin K et al.; The connections between host corticostriatal afferents and neurons in intrastriatal grafts of foetal striatal tissue have been studied with electron microscopic immunocytochemistry using Phaseolus vulgaris leucoagglutinin (PHA-L) as a label of the host corticostriatal fibres . Adult rats with unilateral ibotenic acid lesions of the head of the caudate putamen received foetal cell suspension grafts from E14-15 rat embryos into the lesioned striatal area . Ten months after transplantation, multiple iontophoretic injections of PHA-L were made into the host frontal cortex . These injections labelled large numbers of corticostriatal fibres which extended across the graft - host border to form a rich axonal network mainly in the peripheral portions of the grafts . At the ultrastructural level a total of 134 PHA-L-labelled terminals were identified to form asymmetric synaptic contacts with neurons within the grafts . Of these contacts, 83% were in contact with dendritic spines, 12% with dendritic shafts, and 5% with small shafts or spines . The synaptic contacts were similar to those identified in intact regions of the host striatum that were spared by the lesion . However, the synapses in the host striatum were almost exclusively in contact with spines (98%) . These results demonstrate that the corticostriatal projection, which constitutes a major source of afferent control in the normal striatum, not only extends axons into the intrastriatal striatal grafts, but also establishes synaptic connections with the implanted neuronal elements. Eur J Neurosci, 1989 Jan, 1(6), 690 - 701 Intrinsic Organization and Connectivity of Intrastriatal Striatal Transplants in Rats as Revealed by DARPP-32 Immunohistochemistry: Specificity of Connections with the Lesioned Host Brain; Wictorin K et al.; Intrastriatal grafts of tissue obtained from the striatal or neocortical primordia of rat fetuses have been studied with respect to their intrinsic organization and connectivity using antibodies to DARPP-32 in combination with acetylcholinesterase (AChE) histochemistry, tyrosine hydroxylase (TH) immunocytochemistry, and anterograde and retrograde axonal tracing techniques . The striatal grafts were characterized by distinct patches of DARPP-32-immunoreactive neurons, which were identical to the densely AChE-positive patches stained in adjacent sections from the same specimens . The non-patch areas possessed only few DARPP-32-positive neurons and contained only sparse AChE-positive fibres . The cortical grafts, by contrast, contained no neurons with clear-cut DARPP-32-positivity and they exhibited a sparse, evenly distributed AChE fibre network, similar to that seen in the non-patch areas of the striatal grafts . The host dopaminergic afferents, as revealed by TH immunostaining, had grown selectively into the DARPP-32-positive patches in the striatal grafts, where they formed a dense terminal network around the DARPP-32-positive cell bodies . The non-patch areas, as well as the cortical grafts, received only sparse TH innervation . By contrast, the host cortical afferents, labelled by Phaseolus vulgaris leucoagglutinin from the host frontal cortex, were seen to extend into both the patch and non-patch areas of the striatal grafts . Transplant neurons projecting into the host brain were labelled by Fluoro-Gold injections into the ipsilateral host globus pallidus . These injections labelled large numbers of medium-sized neurons within the striatal grafts and the vast majority of them (over 85%) were confined to the DARPP-32-positive patches . Similar Fluoro-Gold injections labelled only few graft neurons in the cortical grafts . The results indicate that the striatal grafts are composed of a mixture of striatal and non-striatal tissue, and that the striatal graft compartment selectively establishes afferent and efferent connections with the host nigro-pallidal system . These graft connections demonstrate a remarkable specificity in the formation of graft - host connectivity . The results, moreover, suggest that developmental properties of the grafted striatal primordium are retained and expressed in the implanted cell suspension, and that the neuronal systems of the lesioned adult host brain, at least to some extent, remain responsive to growth regulating mechanisms normally operating during ontogenetic development. Scand J Immunol, 2002 Jul, 56(1), 76 - 84 Separation of human lymphocytes from citrated blood by density gradient (NycoPrep) centrifugation: monocyte depletion depending upon activation of membrane potassium channels; Boyum A et al.; Routine one-step centrifugation procedures (Lymphoprep = LP, Percoll) commonly used for separation of blood cells split the cells into two major fractions . After centrifugation the mononuclear cells (MNC = monocytes and lymphocytes) are located on the top of the separation fluid, whereas erythrocytes and granulocytes have sedimented to the bottom . We now show that a relatively pure lymphocyte suspension can be obtained by one-step centrifugation of citrated blood by using NycoPrep (NP = iohexol), a nonionic X-ray contrast agent . With this gradient medium also the monocytes pass to the bottom, leaving lymphocytes on the top . In parallel separations with LP, which contains Ficoll and a fully dissociated sodium salt of a contrast medium, the results were as usual, i.e . approximately 70-85% lymphocytes and 30-15% monocytes in the top fraction . The monocyte depletion with NP depended upon the use of citrated (ACD) blood and a proper balance of density and osmolality of the gradient medium, and was enhanced by 20 min preincubation with CaCl2 at room temperature . Monocyte depletion could not be obtained with LP . Under optimal conditions (density 1.075 g/ml, osmolality 280-300 mOsm/kg), the monocyte admixture amounted to approximately 1 (0-2)%, in separations with buffy coat samples . For freshly drawn blood, it was necessary to slightly modify the NP solution . The monocyte depletion was counteracted by blockers of K+ channels or by KCl in the cell suspension . Following incubation in NP of Percoll-separated cells, an enhanced release of K+ was observed . The results are interpreted as follows: NP mediates the opening of K+ channels of MNC, which leads to efflux of K+, accompanied with associated anions (Cl-) . This reduces the osmolality inside the cells which therefore expel water to maintain osmotic equilibrium . In this regard it appears that monocytes are more sensitive than lymphocytes, their density therefore increasing more, so that they are able to pass the density barrier otherwise exerted by the gradient medium. J Biomol Screen, 2002 Jun, 7(3), 233 - 46 Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader; Kassack MU et al.; Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties . Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs . Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins . Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector . Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s . Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free {Ca(2+)} on agonist stimulation . Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM) . SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response . Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors . Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist . Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes. Gen Comp Endocrinol, 2002 May, 126(3), 352 - 8 Pineal melatonin secretion, but not ocular melatonin secretion, is sufficient to maintain normal immune responses in Japanese quail (Coturnix coturnix japonica); Moore CB et al.; Reports that plasma melatonin is an important immune regulator in avian species have been rather sparse and contradictory . Also, the primary source of immune-modulating melatonin has yet to be determined in birds . In Japanese quail (Coturnix coturnix japonica), the pineal gland and eyes contribute roughly two thirds and one third of the melatonin found in the blood, respectively . Two experiments were conducted to evaluate melatonin as an immune modulator in Japanese quail and to determine the primary source of immune-modulating melatonin in this species . Experiment 1 was designed to evaluate the involvement of the pineal gland and the eyes in immunocompetence . Each of three groups of quail was assigned a surgical treatment and the cellular and humoral immune responses were determined 8 weeks following surgery . The surgical treatments were pinealectomy (Px), sham pinealectomy (SH-Px), and ocular enucleation (eye removal (Ex)) . Experiment 2 utilized exogenous melatonin as a replacement to reconstitute immune responses in surgically immunocompromised birds . In this experiment, 50.0 microg/ml of melatonin, or diluent only, was provided to Px and SH-Px birds in the drinking water ad libitum . The cellular and humoral immune responses were determined after 8 weeks of melatonin treatment . In both experiments, a cutaneous basophil hypersensitivity reaction to phytohemagglutinin was measured to evaluate the cellular immune response . To evaluate the humoral immune response, primary antibody titers were determined 7 days postintravenous injection with a Chukar red blood cell suspension . Flow cytometric analysis of peripheral blood lymphocytes was performed to determine the relative percentage of CD4(+) and CD8(+) T- and B-lymphocytes in all treatments of Experiment 2 . In Experiment 1, both the SH-Px and Ex surgical treatments produced similar cellular and humoral immune responses, and these responses were significantly greater than those in Px-treated birds . Pinealectomy significantly reduced the cellular and humoral immune responses from SH-Px by 25.8% and 41.3%, respectively . In Experiment 2, Px again resulted in depressed cellular and humoral immune responses . In addition, Px significantly reduced CD8(+) T-lymphocyte numbers compared to SH-Px, while B-lymphocytes remained unchanged . Melatonin administration to Px birds increased the cellular (32.9%) and humoral (30.6%) immune responses to the level of control (SH-Px) birds, although this reconstitution was not due to increased CD8(+) T- or B-lymphocytes . From these data, it was clear that removal of the pineal gland, but not the eyes, reduced cellular and humoral immune responses, which were reconstituted to normal levels by exogenous melatonin . These data suggest that immunodepression is only observed in birds with two thirds of the plasma melatonin removed by pinealectomy . Removal of one third of the plasma melatonin (by ocular enucleation) is not sufficient to reduce cellular and humoral responses in the Japanese quail . (c) 2002 Elsevier Science (USA). Exp Neurol, 2002 Jul, 176(1), 154 - 62 A role for tumor necrosis factor alpha in death of dopaminergic neurons following neural transplantation; Clarke DJ et al.; Poor survival of transplanted dopaminergic (DA) neurons remains a serious obstacle to the success of cell replacement therapy as an alternative to the current treatments for Parkinson's disease (PD) . We have examined the temporal release profile of an inflammatory cytokine, tumor necrosis factor-alpha (TNFalpha), following transplantation of fetal mesencephalic tissue into the rat striatum . The amounts of TNFalpha released in vivo when added to cultures of embryonic DA neurons, significantly reduced the survival of DA neurons in vitro, and this cell death could be prevented by the inclusion of an antibody to the TNFalpha receptor type 1 . Inclusion of this antibody in cell suspensions during transplantation also increased the survival of transplanted fetal DA neurons by approximately 250% . Use of this therapeutic antibody approach may offer significant improvements to neural transplantation as a treatment for PD. Invest Ophthalmol Vis Sci, 2002 Jul, 43(7), 2114 - 21 An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture; Koizumi N et al.; PURPOSE: A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants . In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium . The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium . The new cell-suspension technique was compared with the existing explant method . METHODS: Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM . Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures . The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12) . RESULTS: Both cell-suspension and explant culture methods produced a healthy epithelial cell layer . The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells . In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells . Both types of cultivated epithelium showed positive expression of K3 and K12 keratins . In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells . CONCLUSIONS: Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system . The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium . Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation. Reproduction, 2002 Jul, 124(1), 85 - 94 Isolation and purification of type A spermatogonia from the bovine testis; Izadyar F et al.; The aim of this study was to isolate and purify bovine type A spermatogonia . Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion . During the isolation and purification steps, the viability of cells was determined using live/dead staining . The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens . Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit . The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells . After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient . Finally, fractions containing 65-87% pure type A spermatogonia were obtained . Large and small type A spermatogonia with different numbers and sizes of nucleoli were found . DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens . Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step . In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population . At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cells. J Biol Chem, 2002 Sep 6, 277(36), 32978 - 84 Epub 2002 Jun 27. Two E2F sites in the Arabidopsis MCM3 promoter have different roles in cell cycle activation and meristematic expression; Stevens R et al.; The commitment to DNA replication is a key step in cell division control . The Arabidopsis MCM3 homologue forms part of the mini chromosome maintenance (MCM) complex involved in the initiation of DNA replication at the transition G(1)/S . Consistent with its role at the G(1)/S transition we show that the AtMCM3 gene is transcriptionally regulated at S phase . The 5' region of this gene contains several E2F consensus binding sites, two of which match the human consensus closely and whose roles have been studied here . The identity of the two sequences as E2F binding sites has been confirmed by electrophoretic mobility shift assay analyses . Furthermore the promoter is activated by AtE2F-a and AtDP-a factors in transient expression studies . One of the E2F binding sites is shown to be responsible for the G(2)-specific repression of the promoter in synchronized cell suspension cultures . In contrast, the second E2F binding site has a role in meristem-specific expression in planta as deletion of this site eliminates the expression of a reporter gene in root and apical meristems . Thus two highly similar E2F binding sites in the promoter of the MCM3 gene are responsible for different cell cycle regulation or developmental expression patterns depending on the cellular environment. J Neurosci Methods, 2002 May 30, 117(1), 23 - 31 Evaluation of brain toxicity following near infrared light exposure after indocyanine green dye injection; Keller E et al.; Indocyanine green (ICG) has excellent safety records and is widely used in medical diagnosis . Recently, a new method has been developed to estimate cerebral blood flow (CBF) using ICG in combination with near-infrared spectroscopy (NIRS) . The new technique may be of wide clinical interest, as it is noninvasive and easy to perform at the bedside in stroke patients . Additionally, ICG with the use of specific wavelength lasers is documented to be effective in photodynamic therapy (PDT) . Under normal conditions ICG does not cross the intact blood brain barrier (BBB) . However, in patients with brain injuries where the BBB may be disturbed, ICG could accumulate in brain parenchyma and in combination with NIR-light exposure, phototoxicity could occur . The aim of the present study was to examine the possible toxicity of ICG in combination with NIRS in a specific setting for CBF measurements . In five rats with mannitol induced BBB breakdown no traces of ICG were found during spectrophotometric analysis of the brain cell suspensions . In ten rats with disrupted BBB there were no significant increases of brain temperature or histological signs of brain damage following 1 h NIR-light exposure after ICG injection . The existing literature concerning the application of ICG in combination with NIR light is reviewed. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 1999 Jul, 13(4), 244 - 8 {The experimental study on optimal cell density and formation time of tissue engineered autologous cartilage}; Xia WY et al.; OBJECTIVE: This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application . METHODS: The chondrocytes isolated from mini swines' ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml . The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine . The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density . Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage . RESULTS: The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics . The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week . CONCLUSION: With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time. Biophys J, 2002 Jul, 83(1), 161 - 71 Simulations of molecular diffusion in lattices of cells: insights for NMR of red blood cells; Regan DG et al.; The pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiment, conducted on a suspension of red blood cells (RBC) in a strong magnetic field yields a q-space plot consisting of a series of maxima and minima . This is mathematically analogous to a classical optical diffraction pattern . The method provides a noninvasive and novel means of characterizing cell suspensions that is sensitive to changes in cell shape and packing density . The positions of the features in a q-space plot characterize the rate of exchange across the membrane, cell dimensions, and packing density . A diffusion tensor, containing information regarding the diffusion anisotropy of the system, can also be derived from the PGSE NMR data . In this study, we carried out Monte Carlo simulations of diffusion in suspensions of "virtual" cells that had either biconcave disc (as in RBC) or oblate spheroid geometry . The simulations were performed in a PGSE NMR context thus enabling predictions of q-space and diffusion tensor data . The simulated data were compared with those from real PGSE NMR diffusion experiments on RBC suspensions that had a range of hematocrit values . Methods that facilitate the processing of q-space data were also developed. Metabolism, 2002 Jul, 51(7), 876 - 80 Hepatic gluconeogenic capacity from various precursors in young versus old rats; Sumida KD et al.; Hepatic gluconeogenic capacity was studied in young (4 months of age) and old (24 months of age) male Fischer 344 rats fasted for 24 hours using the isolated hepatocyte technique . Following the isolation of liver cells, the following precursors were added to the cell suspensions and incubated for 30 minutes: lactate (5 mmol/L), pyruvate (5 mmol/L), alanine (5 mmol/L), glutamine (5 mmol/L), oxaloacetate (5 mmol/L), glycerol (5 mmol/L), dihydroxyacetone (10 mmol/L), fructose (10 mmol/L), or saline (no precursor addition) . To confirm that glucose production reflects gluconeogenic capacity, there was significant depletion of hepatic glycogen after the 24-hour fast and minimal alterations in glycogen content once substrates were added . Adjusting the gluconeogenic rates to reflect 100% cell viability resulted in no difference between young and old animals for any substrate used with the sole exception of fructose . The hepatic glucose production from fructose was 34% greater for young versus old animals . The results suggest that following a period of starvation the basal glucose production rates from hepatocytes, incubated with precursors entering the gluconeogenic pathway prior to fructose-6-phosphate, are equivalent in young and old rats . Cell Transplant, 2002, 11(3), 265 - 74 Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain; Zigova T et al.; Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo . These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value . Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells . In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines . To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes . Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF) . Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone . One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes . Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain . Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III beta-tubulin) . These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain. Cell Transplant, 2002, 11(3), 195 - 205 Tauroursodeoxycholic acid improves the survival and function of nigral transplants in a rat model of Parkinson's disease; Duan WM et al.; There is accumulating evidence showing that the majority of cell death in neural grafts results from apoptosis when cells are implanted into the brain . Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated hydrophilic bile acid, has been found to possess antiapoptotic properties . In the present study we have examined whether the supplementation of TUDCA to cell suspensions prior to transplantation can lead to enhanced survival of nigral grafts . We first conducted an in vitro study to examine the effects of TUDCA on the survival of dopamine neurons in serum-free conditions . The number of tyrosine hydroxylase (TH)-positive neurons in the TUDCA-treated cultures was significantly greater than that of control cultures 7 days in vitro . In addition, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the TUDCA-treated cultures was dramatically smaller than that in the control cultures . In the transplantation study, a 50 microM concentration of TUDCA was added to the media when nigral tissue from Sprague-Dawley (SD) rats was trypsinized and dissociated . Two microliters of cell suspension containing TUDCA was then stereotaxically injected into the striatum of adult SD rats subjected to an extensive unilateral 6-hydroxydopamine lesion of the nigrastriatal dopamine pathway . At 2 weeks after transplantation, the rats that received a cell suspension with TUDCA exhibited a significant reduction in amphetamine-induced rotation scores when compared with pretransplantation value . There was a significant increase (approximately threefold) in the number of TH-positive cells in the neural grafts for the TUDCA-treated group when compared with the controls 6 weeks postgrafting . The number of apoptotic cells was much smaller in the graft areas in the TUDCA-treated groups than in the control group 4 days after transplantation . These data demonstrate that pretreatment of the cell suspension with TUDCA can reduce apoptosis and increase the survival of grafted cells, resulting in an improvement of behavioral recovery. Transfus Med, 2002 Jun, 12(3), 173 - 9 Study of five cell salvage machines in coronary artery surgery; Burman JF et al.; We evaluated the effectiveness, ease of use and safety of five machines for blood salvage during coronary artery surgery . All were equally effective in concentrating red cells . We measured haemoglobin, packed cell volume, free haemoglobin, white cells, neutrophil elastase, platelets, thrombin-antithrombin complex (TAT), prothrombin activation peptide F1.2, fibrin degradation product (d-dimers), tissue plasminogen activator (tPA) and heparin in wound blood, in washed cell suspensions and in a unit of bank blood prepared for each patient . All machines were equally safe and easy to use and were equally effective in removing heparin and the physiological components measured . There were no adverse effects on patients . Clotting factors are severely depleted both in salvaged blood, even before washing, and in bank blood . Cell savers are a valuable adjunct to coronary artery surgery, but careful monitoring of coagulation is required when the volumes of either bank blood or salvaged blood are large. Physiol Res, 2002, 51(1), 73 - 8 Functional cross-talk of Ca2+-mobilizing endothelin receptors in C6 glioma cells; Malik R et al.; There are conflicting results concerning the receptor subtype(s) involved in calcium-mediated endothelin signaling in the glial cells . In order to elucidate the role of endothelin A and B receptors in these processes, we have studied the effect of a complex spectrum of endothelin receptor ligands on intracellular calcium concentration changes in proliferating and differentiated C6 rat glioma cells . Cell differentiation was induced by dibutyryl-cAMP and assessed by the glial fibrillar acidic protein content . Intracellular calcium changes were measured in cell suspensions using fluorescent probe Fura-2 . The specific endothelin B receptor agonists sarafotoxin S6c and IRL-1620 did not influence the intracellular calcium concentration . However, calcium changes induced by endothelin-1 and especially by endothelin-3 after the pretreatment of cells with one of these endothelin B receptor specific agonists were significantly enhanced even above the values attained by the highest effective endothelin concentrations alone . Such endothelin B-receptor ligand-induced sensitization of calcium signaling was not observed in differentiated C6 cells . Moreover, endothelin-induced calcium oscillations in differentiated C6 cells were less inhibited by BQ-123 and BQ-788 than in their proliferating counterparts . In conclusion, the specific activation of endothelin B receptor in C6 rat glioma cells does not affect intracellular calcium per se, but probably does so through interaction with the endothelin A receptor . The pattern and/or functional parameters of endothelin receptors in C6 rat glioma cells are modified by cell differentiation. Arch Microbiol, 2002 Jul, 178(1), 53 - 8 Epub 2002 Apr 27. Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes; Kaden J et al.; Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor . It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures . In the present study, L -cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners . Low concentrations of L -cysteine or L -cystine (10-100 microM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine . Cell suspensions of G . sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W . succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups . Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions . The redox potential prevailing in acetate-oxidizing cocultures was -160 to -230 mV . Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine . Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine- N-oxide, anthraquinone-2,6-disulfonate, or ascorbate. Shock, 2002 Jun, 17(6), 508 - 12 Mechanisms of platelet-neutrophil interactions and effects on cell filtration in septic shock; Kirschenbaum LA et al.; ABSTRACT-We examined the mechanisms and the adhesive molecules mediating platelet-neutrophil adhesion in patients with septic shock . Neutrophils, platelets, and platelet poor plasma (NPPP) were isolated from 12 normal volunteers . Platelets and neutrophils were stimulated with platelet poor plasma (SPPP) removed from 12 patients in septic shock . Cell adhesion was assessed by filtration through 5-microm pore filters and by flow cytometry . Blocking monoclonal antibodies were used against the platelet and neutrophil surface receptors glycoprotein complex IIb/IIla, P-selectin, ICAM-2, CD11a, CD11b, and CD18 . The filtration pressure (Pi) of cells suspended in SPPP was significantly greater than that of cells suspended in NPPP (24 +/- 1.0 mmHg vs . 14 +/- 1.0 mmHg; P< 0.05) . The difference between the Pi of cells suspended in SPPP or NPPP (deltaPi SPPP-NPPP) in the presence of monoclonal antibodies anti-CD41, anti-CD62P, abciximab, anti-CD11a, anti-CD11b, and anti-CD18 was significantly less than the APi SPPP-NPPP of cell suspensions without the addition of these monoclonal antibodies (P < 0.01) . The greatest reduction in Pi occurred when platelet receptor P-selectin was blocked simultaneously with the CD11b receptor on the neutrophil as compared to all other single blocking monoclonal antibodies or combinations of monoclonal antibodies . The mean fluorescence of activated platelet CD63-PE binding to neutrophils suspended in SPPP was significantly greater than that of cells suspended in NPPP (780 +/- 130 Ifu vs . 295 +/- 35 Ifu; P < 0.05) . The greatest attenuation in mean fluorescence occurred by blocking the P-selectin receptor on the platelet simultaneously with CD11b receptor on the neutrophil . We conclude that platelet-neutrophil aggregation is increased in septic shock . This aggregation is mediated by the interaction of multiple platelet and neutrophil surface receptors . The platelet receptor P-selectin and the neutrophil receptor CD11b/CD18 appear to play the most important role in these interactions. J Biomed Sci, 2002 May-Jun, 9(3), 246 - 52 Natural plasmid transformation in Escherichia coli; Tsen SD et al.; Although Escherichia coli does not have a natural transformation process, strains of E . coli can incorporate extracellular plasmids into cytoplasm 'naturally' at low frequencies . A standard method was developed in which stationary phase cells were concentrated, mixed with plasmids, and then plated on agar plates with nutrients which allowed cells to grow . Transformed cells could then be selected by harvesting cells and plating again on selective agar plates . Competence developed in the lag phase, but disappeared during exponential growth . As more plasmids were added to the cell suspension, the number of transformants increased, eventually reaching a plateau . Supercoiled monomeric or linear concatemeric DNA could transform cells, while linear monomeric DNA could not . Plasmid transformation was not related to conjugation and was recA-independent . Most of the E . coli strains surveyed had this process . All tested plasmids, except pACYC184, could transform E . coli . Insertion of a DNA fragment containing the ampicillin resistance gene into pACYC184 made the plasmid transformable . By inserting random 20-base-pair oligonucleotides into pACYC184 and selecting for transformable plasmids, a most frequent sequence was identified . This sequence resembled the bacterial interspersed medium repetitive sequence of E . coli, suggesting the existence of a recognition sequence . We conclude that plasmid natural transformation exists in E . coli . Mutat Res, 2002 Jun 27, 518(1), 39 - 45 Collaborative validation study of the in vivo micronucleus test using mouse colonic epithelial cells; Ohyama W et al.; The in vivo micronucleus test using mouse colonic epithelial cells was evaluated as the 11th collaborative study organized by the Collaborative Study Group on the micronucleus test (CSGMT) with three model chemicals that were known to induce chromosome damage in mouse colonic cells . Five laboratories participated in this validation study . All three model chemicals, i.e . 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), N-methyl-N-nitrosourea (MNU), and mitomycin C (MMC), induced micronucleated colonic epithelial cells in a 4-day exposure protocol in all participating laboratories . We confirmed that the present single cell suspension method could be used to detect the model chemicals as micronucleus inducers in mouse colonic epithelial cells . Advantages of this method are that experiments are easy to perform and that intact cells can be analyzed . The present study suggested that the colon micronucleus assay proposed here is useful for mechanistic studies of colon carcinogenesis. Cryobiology, 2002 Feb, 44(1), 46 - 53 Cryopreservation of vascular endothelial cells as isolated cells and as monolayers; Pegg DE; This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads . Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C . The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C . Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min . The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells . Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min . Biosilon microcarrier beads were used for the attached cells . Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C . Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake . The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min . These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment . (c) 2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Jun 21, 294(4), 872 - 8 DNA fragmentation and morphological changes in apoptotic human lymphocytes; Czene S et al.; Cell suspensions enriched in cells at various stages of apoptosis were obtained by separation of irradiated human peripheral blood lymphocytes on density gradients at different post-irradiation times . The state of DNA fragmentation in the cells was determined by comet assay and pulsed field gel electrophoresis . The morphologically distinguishable features of apoptosis such as chromatin condensation and cell shrinkage correlated with discrete stages of DNA fragmentation . It was found that >/=50 kbp fragmentation of DNA occurs already in cells of normal density whereas the subsequent DNA fragmentation onto fragments <50 kbp occurs in parallel with cell shrinkage and simultaneous increase in cell density.The observed stages of DNA fragmentation seem to be separated in time that could allow in case of abortive apoptosis formation of chromosomal aberrations . (c) 2002 Elsevier Science (USA). Physiol Plant, 2002 Mar, 114(3), 395 - 404 Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+ Kawano T, Kawano N, Muto S, Lapeyrie F. Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response . Recently, we have demonstrated that treatment of tobacco (Nicotiana tabacum) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li+, Na+, K+), alkali earth metals (Mg2+, Ca2+) or lanthanides (La3+, Gd3+) . In this study, we tested the effect of extracellular supplementation of Zn2+ and Mn2+ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent . Extracellular supplementation of Zn2+ and Mn2+ inhibited the generation of superoxide in response to addition of salts . Although both Zn2+ and Mn2+ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn2+ simply inhibited total production of superoxide while Zn2+ inhibited the early phase of superoxide production and induced the slow release of superoxide . Roles of Mn2+ and Zn2+ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed. Med Mycol, 2002 Apr, 40(2), 201 - 8 Fungicidal activities of commonly used disinfectants and antifungal pharmaceutical spray preparations against clinical strains of Aspergillus and Candida species; Gupta AK et al.; The antifungal efficacy of commercial chemical disinfectants and pharmaceutical antifungal agents against medically important moulds and yeast species was investigated . Chlorine, phenol, sodium dodecyl sulfate and quaternary ammonium salts were the chemical disinfectants, and bifonazole and terbinafine were the antifungal pharmaceutical products tested against clinical isolates of Aspergillus and Candida species . Fungal inocula were obtained from conidial preparations of two A . ochraceus strains and yeast cells of C . albicans, C . krusei and C . parapsilosis . The antifungal activities were evaluated either by determining the kill rate in a cell suspension media at different contact periods, or by examining the viability and growth on plates sprayed with the active ingredient . Chlorine (1%) was the only disinfectant with the ability to cause a rapid inactivation of all five strains . Phenol (5%) was equally effective against Candida species; however, a number of A . ochraceus conidia were able to survive this treatment for up to 1 h . Benzalkonium chloride (0.5%) and cetrimide (0.5%) were also able to disinfect the three Candida species rapidly; however, these two quaternary ammonium compounds were relatively ineffective against A . ochraceus . In spray experiments, quaternary ammonium compounds had a fungicidal activity against Candida species and were fungistatic against A . ochraceus conidia . All five fungal strains were able to resist 0.5% sodium dodecyl sulfate, present either in the suspension solution or on the sprayed plate . Of the two pharmaceutical antifungal products tested, bifonazole (1%) were essentially ineffective against all five strains . Terbinafine (1%) had a fungicidal activity against A . ochraceus and C . parapsilosis . In suspension experiments, an exposure to 0.01% terbinafine required a contact period of 1 h for a complete inactivation of A . ochraceus conidia and an onset of fungicidal effect on C . parapsilosis yeast cells . Terbinafine was only moderately effective against C . albicans and was completely ineffective against C . krusei. Planta Med, 2002 May, 68(5), 475 - 6 Jasmonic acid stimulates taxane production in cell suspension culture of yew (Taxus x media); Baebler S et al.; Cell lines of Taxus x media Rehd . were established via embryo and callus culture and grown on B5 medium containing 10 microM 2,4-D and 1 microM kinetin . Jasmonic acid (JA) was used to elicit taxane synthesis in a selected yew cell line . 100 microM JA was added 7 days after subculture . JA strongly increased taxane content in the cells and in the medium . At the time of the highest amount in elicited cultures, the enhancement of paclitaxel production was 19-fold and 4-fold in the cells and medium, respectively, compared to controls (non-elicited cultures) . The time course of taxane accumulation in the cells and in the medium and the release of taxanes into the medium were not changed after JA elicitation. Planta Med, 2002 May, 68(5), 420 - 4 Efficient paclitaxel production using protoplasts isolated from cultured cells of Taxus cuspidata; Aoyagi H et al.; Efficient isolation of protoplasts from Taxus cuspidata cultured cells, localization of paclitaxel in the cultured cells, and efficient production of paclitaxel by protoplasts were studied . Hemicellulase, potassium citric acid solution, and degassing treatments were effective in increasing the yield of protoplasts isolated from T . cuspidata cultured cells . Protoplasts yields (3.2 - 6.4 x 10(6) number/g-fresh weight cells) were achieved by combining the various treatments with specific culture and cell phases . It was found that about 30% and 35% of paclitaxel in the cells was located in cell walls and/or between the cell wall and cell membrane (CW) of suspension cells in the growth phase and in the stationary phase, respectively . About 30% and 43% of paclitaxel in the cells was located in CW of the cells grown in solid culture in growth phase and in the stationary phase, respectively . In comparison with cell suspension culture, protoplasts in a static culture and the protoplasts immobilized in agarose gel in shaking culture resulted to about 6 times increase in the extracellular paclitaxel accumulation. Bioorg Med Chem, 2002 Aug, 10(8), 2479 - 83 Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia; Yu B et al.; Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine . The newly discovered enzyme is dependent on NADPH and oxygen . It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide . The CO-effect is reversible with light (450 nm) . The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase . A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined . K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine . Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months . The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme. Folia Histochem Cytobiol, 2002, 40(2), 105 - 6 DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies; Czuba A et al.; The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry . DNA ploidy is an established prognostic factor in many types of cancers . Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread . Investigated specimens were breast cancer FNA biopsy cell suspensions . Measurements were performed using flow cytometry . Material studied comprised 143 cases analyzed in 1999-2000 . We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle . Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases . DNA ploidy was compared to receptor status of the investigated cells . DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors . Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies. Nippon Hinyokika Gakkai Zasshi, 2002 May, 93(4), 532 - 8 {MHC-class I expression on prostate carcinoma and modulation by IFN-gamma}; Naoe M et al.; OBJECTIVES: In this study, we investigated MHC-class I expression on both prostate cancer and normal prostate and compared those data with the number of CD8+ lymphocyte . Secondary, we investigated effect of IFN-gamma to the MHC-class I expression on prostate carcinoma . MATERIALS AND METHODS: Twenty cryo-preserved benign prostate samples (Seven normal prostates and five benign prostatic hypertrophy samples.) and fifteen prostate carcinoma samples were used for immunohistochemistry of CD8 and MHC-class I . Eleven fresh single cell suspensions of prostate carcinoma were used for IFN-gamma study . After 24 hours IFN-gamma stimulation, MHC-I expression was measured by FACs analysis . RESULTS: Interestingly, significant correlation was observed between MHC-class I expression and the CD8+ lymphocyte infiltrate (r = 0.705, P < 0.0001) . After 24 hrs IFN-gamma stimulation, MHC-class I expression was up-regulated in all samples (p < 0.05) . CONCLUSION: Reduced expression of MHC-class I was thought as one of the factor which is related to the reduced degree of TILs (tumor infiltratig lymphocytes) in prostate cancer . IFN-gamma which is secreted mainly from CTL (cytotoxic T lymphocyte) might increase the degree of TIL through up-regulated MHC-class I. Nat Med, 2002 Jul, 8(7), 751 - 5 Epub 2002 Jun 10. A tumor-homing peptide with a targeting specificity related to lymphatic vessels; Laakkonen P et al.; Blood vessels of tumors carry specific markers that are usually angiogenesis-related . We previously used phage-displayed peptide libraries in vivo to identify peptides that home to tumors through the circulation and that specifically bind to the endothelia of tumor blood vessels . Here we devised a phage screening procedure that would favor tumor-homing to targets that are accessible to circulating phage, but are not blood vessels . Screening on MDA-MB-435 breast carcinoma xenografts yielded multiple copies of a phage that displays a cyclic 9-amino-acid peptide, LyP-1 . Homing and binding to tumor-derived cell suspensions indicated that LyP-1 also recognizes an osteosarcoma xenograft, and spontaneous prostate and breast cancers in transgenic mice, but not two other tumor xenografts . Fluorescein-labeled LyP-1 peptide was detected in tumor structures that were positive for three lymphatic endothelial markers and negative for three blood vessel markers . LyP-1 accumulated in the nuclei of the putative lymphatic cells, and in the nuclei of tumor cells . These results suggest that tumor lymphatics carry specific markers and that it may be possible to specifically target therapies into tumor lymphatics. Prostaglandins Leukot Essent Fatty Acids, 2002 Jan, 66(1), 19 - 29 Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma; Leaver HA et al.; The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death . The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue . Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected . Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro . These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used . In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed . Also, there was little evidence of acute inflammatory infiltration in regressing tumours . Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression . Cryo Letters, 2002 Mar-Apr, 23(2), 103 - 12 Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cell suspensions; Jitsuyama Y et al.; The freezing tolerance of asparagus (Asparagus officinalis L.) embryogenic cells, as determined by electrolyte leakage, was increased by the incubation of samples in medium containing 0.8 M sucrose . To elucidate the mechanism involved, we investigated the changes in soluble carbohydrates, cell ultrastructure and proteins accompanying the increase in freezing tolerance following incubation in sugar-rich medium . During sugar incubation, the intracellular sucrose content increased from 67 mol g-1FW to 429 mol g-1FW; it was also metabolized into fructose and glucose, as determined by high-performance liquid chromatography . Microscopy revealed that sugar incubation induced plasmolysis of embryogenic cells and drastic changes in cell ultrastructure with the appearance of rough endoplasmic reticulum (rER) . Furthermore, immunoblotting analysis with anti-dehydrin antiserum revealed that a dehydrin-like protein appeared only when maximal freezing tolerance was induced by sugar incubation . These results suggest that freezing tolerance of asparagus embryogenic cells is increased by a complex mechanism involving notably changes in cell ultrastructure and accumulation of certain sugars and proteins during sugar incubation. IEEE Trans Biomed Eng, 2002 Jun, 49(6), 605 - 12 Dependence of induced transmembrane potential on cell density, arrangement, and cell position inside a cell system; Pavlin M et al.; A nonuniform transmembrane potential (TMP) is induced on a cell membrane exposed to external electric field . If the induced TMP is above the threshold value, cell membrane becomes permeabilized in a reversible process called electropermeabilization . Studying electric potential distribution on the cell membrane gives us an insight into the effects of the electric field on cells and tissues . Since cells are always surrounded by other cells, we studied how their interactions influence the induced TMP . In the first part of our study, we studied dependence of potential distribution on cell arrangement and density in infinite cell suspensions where cells were organized into simple-cubic, body-centered cubic, and face-centered cubic lattice . In the second part of the study, we examined how induced TMP on a cell membrane is dependent on its position inside a three-dimensional cell cluster . Finally, the results for cells inside the cluster were compared to those in infinite lattice . We used numerical analysis for the study, specifically the finite-element method (FEM) . The results for infinite cell suspensions show that the induced TMP depends on both: cell volume fraction and cell arrangement . We established from the results for finite volume cell clusters and layers, that there is no radial dependence of induced TMP for cells inside the cluster. J Biotechnol, 2002 Jul 3, 96(3), 205 - 11 Increased production of human granulocyte-macrophage colony stimulating factor (hGM-CSF) by the addition of stabilizing polymer in plant suspension cultures; Lee JH et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that stimulates the production of granulocytes, macrophages, and white blood cells . Secretion of human GM-CSF from cell suspension cultures of genetically modified tobacco has been facilitated using natural mammalian leader sequences . At the mid-exponential growth phase (day 4 after the initiation of cell suspension culture), GM-CSF was detected in the medium at a maximum concentration of 180 microg l(-1) . However, the secreted GM-CSF was unstable in the medium, and rapidly degraded after day 5 . In order to stabilize the secreted GM-CSF, three stabilizing polymers were tested, polyethylene glycol, polyvinylpyrrolidone and gelatin . Gelatin was the most effective in stabilizing the secreted GM-CSF . Following the addition of 5% (w/v) gelatin, the maximum GM-CSF concentration reached 783 microg l(-1), a 4.6-fold increase over control. Cytotherapy, 2000, 2(3), 187 - 93 Selection and expansion of T cell from untreated patients with CLL: source of cells for immune reconstitution? Husebekk A, Fellowes V, Read EJ, Williams J, Petrus MJ, Gress RE, Fowler DH. BACKGROUND: Lymphocyte-derived malignancies can be treated with combinations of drugs that efficiently eradicate normal and malignant lymphocytes . Lack of T lymphocytes after treatment of B lymphocyte CLL (B-CLL) makes the patients susceptible to serious infections and may limit the benefit of the therapy . The aim of the study was to purify and culture-expand normal T lymphocytes from B-CLL patients prior to therapy . These cells could be frozen and given to the patients in the lymphopenic period post-chemotherapy . METHODS: T lymphocytes were isolated from the mononuclear cell apheresis products from five patients with previously untreated B-CLL . The apheresis products were red-cell depleted by density gradient centrifugation . B-lymphocyte purging was performed by incubating with MAbs to four different B-cell epitopes, followed by magnetic-bead depletion . One round of negative selection removed >90% of the B lymphocytes . The T-lymphocyte enriched cell suspension was cultured for 10/11 days in the presence of IL-2 and the anti-T cell receptor Ab OKT3 . In addition, in some cultures anti-CD22 ricin immunotoxin was added . RESULTS: T cells from CLL patients expanded 4.7-21-fold over a 10/11 days culture interval . After culture, CLL cells could no longer be identified by flow cytometric evaluation . The cultured T lymphocytes were predominantly CD8(+), and were capable of lysing autologous CLL cells through a fas-dependent mechanism . DISCUSSION: Selection and expansion of T lymphocytes by this method may represent a strategy for enhancing immunity in the lymphopenic period following CLL treatment. Biosci Biotechnol Biochem, 2002 Apr, 66(4), 902 - 5 New medium composition for high betacyanin production by a cell suspension culture of table beet (Beta vulgaris L.); Akita T et al.; The effect of a revised Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.) . The effects of a high iron concentration and low concentration of zinc on betacyanin production were not cumulative . The composition of the new revised medium for high betacyanin production was established by reducing the concentration of inorganic nitrogen (30 mM), modifying the ratio of ammonium to nitrate (1:14), reducing the concentration of zinc (0.0003 mM), and removing copper and cobalt . The revised LS medium enabled the maximum betacyanin yield of 550 mg/l to be obtained from a 14-day culture . This medium promoted the betacyanin production in three types of cell line differing in the betacyanin productivity . The betacyanin productivity (40 mg/l x day) was higher than that quoted in any other previous reports. Phytochemistry, 2002 Jun, 60(3), 289 - 93 Jasmonic acid-induced hypericin production in cell suspension cultures of Hypericum perforatum L . (St . John's wort); Walker TS et al.; Hypericum perforatum L . (St . John's wort) is an herbal remedy widely used in the treatment of mild to moderate depression . Hypericin, a photosensitive napthodianthrone, is believed to be the compound responsible for reversing the depression symptoms . In this study, novel in vitro cell culture systems of H . perforatum were used to monitor the effect of elicitation on cell growth and production of hypericin . A dramatic increase in cell growth and hypericin production was observed after exposure to jasmonic acid (JA) . However, other elicitors such as salicylic acid (SA) and fungal cell wall elicitors failed to show any stimulatory effect on either cell growth or hypericin production . Cell cultures treated with JA and incubated in the dark showed increased growth and hypericin production as compared to the cultures grown under light conditions . Jasmonate induction in dark conditions played an important role in growth and hypericin production in cell suspension cultures, to our knowledge an undocumented observation. Phytochemistry, 2002 Jun, 60(3), 263 - 7 Origin of two isoprenoid units in a lavandulyl moiety of sophoraflavanone G from Sophora flavescens cultured cells; Yamamoto H et al.; Cell suspension cultures of Sophora flavescens produced large amounts of sophoraflavanone G, an 8-lavandulylated flavanone and lupalbigenin, a 6,3'-di-dimethylallylated isoflavone, by the simultaneous addition of cork tissues and methyl jasmonate . The labeling pattern of the isoprene units resulting after administration of {1-13C} glucose into the cell cultures in the presence of the above additives revealed that two isoprene units in the lavandulyl group of sophoraflavanone G and two dimethylallyl groups of lupalbigenin were biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway. Arch Dermatol Res, 2002 May, 294(3), 135 - 42 Epub 2002 Apr 19. The suitability of cells from different tissues for use in tissue-engineered skin substitutes; van den Bogaerdt AJ et al.; Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment . The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution . Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds . We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer . In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time . Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected . Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days . In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028) . In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively) . The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029) . Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells . The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices . We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue . Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts. Plant J, 2002 May, 30(4), 431 - 46 A custom microarray analysis of gene expression during programmed cell death in Arabidopsis thaliana; Swidzinski JA et al.; Programmed cell death (PCD) is a form of cellular suicide requiring active gene expression, and occurs in both animals and plants . While the cascade of events and the genes that control PCD have been extensively studied in animals, we remain largely ignorant about the similar process in plant cells . Many of the key proteins of animal cell death such as the Bcl-2 family and the caspase family of proteases do not appear to be conserved in plants, suggesting that plants may employ unique mechanisms to execute PCD . To identify genetic elements of PCD in plants, we monitored changes in transcript levels of approximately 100 selected genes during cell death in an Arabidopsis cell suspension culture using a cDNA microarray . PCD was induced in the cell cultures by two independent means (heat treatment or by allowing the cultures to senesce) to allow the distinction to be drawn between changes in gene expression that are related to PCD and those that are specific to a particular treatment . We argue that genes whose expression is altered during PCD induced by two different means may be generally involved in all types of PCD . We show that certain oxidative stress-related genes, including CSD1, CSD3, and GPX, in addition to cysteine proteinases, some transcription factors, and HR-related genes may serve as markers of a core plant cell death programme . Additionally we observe a down-regulation of the mitochondrial adenine nucleotide transporter and suggest that this may be an early event in the execution of plant PCD. Artif Cells Blood Substit Immobil Biotechnol, 2002 Mar, 30(2), 127 - 36 Ultrasound-induced physiological changes in cultured cells of Petunia hybrida; Bohm H et al.; Petunia hybrida cell suspension cultures were exposed to ultrasonic standing wave fields at 2.43 MHz for 40 min with mean sound pressures (within homogenous sound fields) varying from 0 (control) to ca . 1.1 MPa . Mean (+/- s.d.; n =6-9) cell viability was reduced to 87+/-10% at 0.6 MPa and to 59 +/- 23% at 1.1 MPa, compared to an initial control value of 92 +/- 6% (P <0.05) . Mean (n = 3) cell alkaline phosphatase concentration increased linearly with sound pressure from a control value of 0.006+/-0.001 to 0.02+/-0.01 Sigma-Units microg(-1) protein at 1.1 MPa (P<0.05) . Similarly, mean cell catalase activity increased from a control value of 0.020 +/- 0.003 to 0.026 +/- 0.008 arbitrary units at 1.1 MPa . In contrast, mean cellular lactate dehydrogenase concentration was unchanged . These observations indicate that cellular repair processes associated with increased alkaline phosphatase activity might be triggered by physical cell damage caused by ultrasound . The observed increase in catalase activity suggests increasing production of free radicals and other sonochemicals, which warrants further study . The absence of changes in lactate dehydrogenase indicates that there was no major damage to respiratory pathways or to overall cellular integrity. Adv Exp Med Biol, 2002, 503, 107 - 14 Intestinal absorption of colostral lymphoid cells in newborn animals; Tuboly S et al.; Intestinal absorption of colostral lymphoid cells was studied in 23 piglets of four sows and 17 lambs of ewes . From the colostrum and blood of the dams the lymphoid cells were isolated with Ficoll-Paque and labelled with technetium (Na99mTcO4) . In the 7th hour after birth, 10 ml volume of the cell suspensions (piglets: 10(7) cells, lambs: 5 x 10(7) cells) were injected, following laparotomy, directly into the stomach or into the jejunum, or through a nasooesophageal tube . Cryostat sections of duodenum, jejunum and lymph node samples of animals were examined by autoradiography . It was found that lymphoid cells present in the colostrum of a piglet and lamb of their own mother were absorbed from the digestive tract and, via the lymphatic vessel, were transported to the mesenteric lypmph nodes . Electron microscopy revealed that absorption takes place intercellularly . Colostral cells of sows other than a piglet's own mother (allogeneic cells), the lymphoid cells isolated from the blood and heat-treated colostral lymphoid cells were not absorbed . The immunization of ewes and their lambs by tetanus anatoxin demonstrate, that the absorbed lymphoid cells remain immunologically active, and may transfer immune information to the lambs. J Exp Bot, 2002 Jun, 53(373), 1397 - 409 Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region; Gollop R et al.; Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape . DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway . The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway . In the present study the promoter of the grape dfr gene was cloned . Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs . The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits . The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems . Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits . The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits . Light, calcium and sucrose induced the dfr gene expression . In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light . The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene . The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways. J Vet Med Sci, 2002 Apr, 64(4), 381 - 3 Homogeneous cell suspension of Malassezia pachydermatis obtained with an ultrasonic homogenizer; Murai T et al.; It is difficult to produce homogeneous cell suspensions of Malassezia pachydermatis, since yeast cells paste up and form many clumps . However, homogeneous fungal suspensions are required for susceptibility examinations and biochemical analyses . Although several types of trials have been carried out using glass homogenizers and many types of agents to obtain homogeneous fungal suspension . They have not yielded good results . We therefore attempted to use an ultrasonic homogenizer to separate clumps of yeast cells into separate individual cells . We succeeded in this fashion in producing homogeneous cell suspensions of M . pachydermatis . These results indicate that an ultrasonic homogenizer can be used to prepare homogeneous fungal suspensions of M . pachydermatis. Cancer Immunol Immunother, 2002 Jun, 51(4), 229 - 34 Epub 2002 Mar 29. Light chain myeloma plasma cells induce a strong cell-mediated immune response mainly directed against the monoclonal light chain determinants in a murine experimental model; Galea HR et al.; An important goal for the treatment of human B cell malignancies lies in the induction of an active immune response directed against the tumoral clone, and more particularly against antigenic determinants of the monoclonal immunoglobulin (Ig) . The presence of idiotype-reactive T-cells in patients with multiple myeloma has been previously reported, and strategies to increase their responsiveness towards the malignant plasma cells are being actively explored . Light chain (LC) myeloma is often an aggressive form of the disease, regarding which antitumoral immunity has not yet been studied . Here, we investigated in an experimental murine model a secreted monoclonal LC that may behave as a strong tumor antigen after immunization of animals with mitomycin C-treated malignant plasma cells producing monoclonal Ig . Non-dividing plasma cells were utilized as a cell suspension to immunize the mice intraperitoneally (i.p.) . The immunized mice produced anti-Ig LC antibodies, mounted a cell-mediated response mainly directed against the monoclonal LC determinants, and survived tumor challenge with significant frequency . These results suggest that plasma cells are capable of presenting antigenic determinants derived from a secreted monoclonal LC in an MHC class I context, and of predominantly inducing a monoclonal Ig-specific T-cell response which can contribute to tumor rejection. Clin Cancer Res, 2002 May, 8(5), 1085 - 91 Detection of circulating cytokeratin-positive cells in the blood of breast cancer patients using immunomagnetic enrichment and digital microscopy; Witzig TE et al.; PURPOSE: To examine the feasibility for identifying and enumerating cytokeratin positive (CK+) cells in the peripheral blood of breast cancer patients . EXPERIMENTAL DESIGN: Blood specimens from 34 normal donors (negative controls), 15 samples to which carcinoma cells were added (positive controls), and 84 breast cancer patients {27 node-negative (N-), 29 node-positive (N+), and 28 metastatic} were studied . RBCs were lysed with ammonium chloride and the resulting cell suspension incubated with anti-EpCAM-conjugated immunomagnetic beads for carcinoma cell enrichment . Immunomagnetically selected cells were placed on slides; stained for CKs 8, 18, and 19; and evaluated with an automated digital microscopy system that rapidly scanned the slide and collected images of cells meeting predefined staining and cytomorphological criteria . A montage of the CK+ cells was reviewed to confirm tumor cell morphology . RESULTS: Eighteen specimens (9 normal, 2 N-, 4 N+, and 3 metastatic) were excluded because of poor cytomorphology or staining artifact . All 15 of the positive controls {95% confidence interval (CI), 78-100%} and none of the 25 negative controls (95% CI, 0-14%) demonstrated CK+ cells . Twenty-one of the 75 (28%; 95% CI, 18-40%) samples from breast cancer patients demonstrated CK+ cells including 76% of patients with metastatic disease (95% CI, 55-91%), 8% with N+ disease (95% CI, 1-26%), and none of those with N- disease (95% CI, 0-14) . The mean number of CK+ cells detected in the 21 CK+ patients was 18.4 (range, 1-120) . CONCLUSIONS: Breast carcinoma cells can be detected in the blood from a significant fraction of metastatic breast cancer patients using immunomagnetic cell enrichment and digital microscopy . The incidence of CK+ cells was low in those with resected N+ disease (at most 26%) and those with resected N- breast cancer (at most 14%) . This technique could be used in large prospective studies of patients with breast cancer to learn whether the detection of rare carcinoma cells is a useful predictive or prognostic factor. Mol Cell Probes, 2002 Feb, 16(1), 49 - 56 In situ detection of Bartonella henselae cells; Hercik K et al.; A new in situ hybridization technique was developed for identification of Bartonella henselae cells in cell suspension or in tissue sections . Use of highly specific probes labeled with either fluorescein or digoxigenin allows discrimination between B . henselae and closely related B . quintana cells . No cross-hybridization with other Bartonella or non-Bartonella species was observed . Besides its specificity it showed higher sensitivity as compared to PCR based detection methods . Moreover, its application allows direct observation of B . henselae in infected tissues. Biotechnol Bioeng, 2002 Jun 30, 78(7), 731 - 40 Sustained hydrogen photoproduction by Chlamydomonas reinhardtii: Effects of culture parameters; Kosourov S et al.; The green alga, Chlamydomonas reinhardtii, is capable of sustained H(2) photoproduction when grown under sulfur-deprived conditions . This phenomenon is a result of the partial deactivation of photosynthetic O(2)-evolution activity in response to sulfur deprivation . At these reduced rates of water-oxidation, oxidative respiration under continuous illumination can establish an anaerobic environment in the culture . After 10-15 hours of anaerobiosis, sulfur-deprived algal cells induce a reversible hydrogenase and start to evolve H(2) gas in the light . Using a computer-monitored photobioreactor system, we investigated the behavior of sulfur-deprived algae and found that: (1) the cultures transition through five consecutive phases: an aerobic phase, an O(2)-consumption phase, an anaerobic phase, a H(2)-production phase and a termination phase; (2) synchronization of cell division during pre-growth with 14:10 h light:dark cycles leads to earlier establishment of anaerobiosis in the cultures and to earlier onset of the H(2)-production phase; (3) re-addition of small quantities of sulfate (12.5-50 microM MgSO(4), final concentration) to either synchronized or unsynchronized cell suspensions results in an initial increase in culture density, a higher initial specific rate of H(2) production, an increase in the length of the H(2)-production phase, and an increase in the total amount of H(2) produced; and (4) increases in the culture optical density in the presence of 50 microM sulfate result in a decrease in the initial specific rates of H(2) production and in an earlier start of the H(2)-production phase with unsynchronized cells . We suggest that the effects of sulfur re-addition on H(2) production, up to an optimal concentration, are due to an increase in the residual water-oxidation activity of the algal cells . We also demonstrate that, in principle, cells synchronized by growth under light:dark cycles can be used in an outdoor H(2)-production system without loss of efficiency compared to cultures that up until now have been pre-grown under continuous light conditions . Eur J Immunol, 1975 Oct, 5(10), 705 - 10 Suppression of immune responses to sheep red cell antigens in rats preimmunized with heterophilic antigen: cellular events; Hellering I et al.; Depression of humoral immune response to sheep red cells (SRC) has been observed in rats previously immunized with heterophilic (Forssman) antigen . Analogous phenomena were observed by assay of splenic plaque-forming cells obtained from rats immunized in vivo with heterophilic antigen (HA) and challenged with SRC . Isophilic immune responses were almost completely suppressed . Kinetics of the primary and secondary immune responses were analyzed . Intervals of two to four days between administration of HA and SRC yielded maximal suppression . Primary immune responses to SRC were determined in cultures of splenic cell suspensions derived from rats which had been either injected in vivo with HA or received no treatment . Under these conditions, too, administration of HA interfered with the antibody responses to SRC . Both adherent and nonadherent cells caused inhibition, with this property appearing earlier in adherent than in nonadherent cells . A hypothesis, capable of explaining the observations made in this study and compatible with similar phenomena described in other systems, has been proposed according to which after immunization with HA, SRC are diverted to HA-stimulated cells which are not equipped to deal with the antigen complex represented by SRC. Eur J Immunol, 1975 Oct, 5(10), 694 - 8 Immunoglobulin-bearing lymphocytes of the chicken . I . Heavy chain immunoglobulin commitment and organ distribution; Hudson L; When purified anti-immunoglobulin light chain antibodies were used in indirect immunofluorescence or labeled with 125I for autoradiographic staining, a similar percentage of Ig-bearing lymphocytes were detected by both techniques in lymphoid cell suspensions from the thymus or blood of 8-14-week-old chickens . However, a larger proportion of Ig positive lymphocytes were detected in suspensions of bursal cells by the more sensitive autoradiographic method, suggesting a lower surface density of Ig: perhaps on newly differentiated stem cells . In thymus and spleen suspensions, the proportions of Ig positive lymphocytes carrying mu and gamma-chains were roughly equal, whereas in the B cell populations of the bursa and blood, cells carrying surface gamma-chains predominated . IgA-bearing lymphocytes were only a minor population (< 5%) in lymphocyte suspensions prepared from the thymus, bursa, blood and spleen of adult chickens, but formed almost 50% of the Ig-bearing lymphocytes in the caecal tonsils. J Asian Nat Prod Res, 2002 Mar, 4(1), 63 - 7 High yield formation of o-aminobenzoic acid-7-O-beta-D-(beta-1,6-O-d-glucopyranosyl)-glucopyranosyl ester in cell suspension cultures of Solanum mammosum; Hartanti L et al.; Cell suspension cultures of Solanum mammosum cultivated in modified Murashige & Skoog media could synthesize o-aminobenzoic acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester from o-amino benzoic acid with a yield of about 20% dry weight in 7 days . The maximum production of o-aminobenzoic acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester was 31.8% on dry weight basis. Immunology, 2002 Apr, 105(4), 391 - 8 Diacylglycerol kinase alpha activity promotes survival of CD4+ 8+ double positive cells during thymocyte development; Outram SV et al.; The diacylglycerol kinases (DGK) form a family of isoenzymes that catalyse the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), both powerful second messengers in the cell . DGKalpha is expressed in brain, peripheral T cells and thymocytes and has been shown to translocate to the nuclear matrix upon T-cell receptor (TCR) engagement . Here, we show that high level expression of DGKalpha is induced following a signal transmitted through the pre-TCR and the protein tyrosine kinase, lck . Activity of DGKalpha contributes to survival in CD4+ 8+ (DP) thymocytes as pharmacological inhibition of DGK activity results in death of this cell population both in cell suspension and thymic explants . DGKalpha promotes survival in these thymocytes through a Bcl-regulated pathway . A consequence of inhibition of DGKalpha is the specific down-regulation of Bcl-xl, whereas in transgenic mice that over-express Bcl-2, death induced by the inhibitor is partially blocked . Thus we report a novel activity of DGKalpha in survival of thymocytes immediately after entry into the DP stage in development. Eur J Biochem, 2002 Apr, 269(8), 2204 - 13 Heterologous expression of a Rauvolfia cDNA encoding strictosidine glucosidase, a biosynthetic key to over 2000 monoterpenoid indole alkaloids; Gerasimenko I et al.; Strictosidine glucosidase (SG) is an enzyme that catalyses the second step in the biosynthesis of various classes of monoterpenoid indole alkaloids . Based on the comparison of cDNA sequences of SG from Catharanthus roseus and raucaffricine glucosidase (RG) from Rauvolfia serpentina, primers for RT-PCR were designed and the cDNA encoding SG was cloned from R . serpentina cell suspension cultures . The active enzyme was expressed in Escherichia coli and purified to homogeneity . Analysis of its deduced amino-acid sequence assigned the SG from R . serpentina to family 1 of glycosyl hydrolases . In contrast to the SG from C . roseus, the enzyme from R . serpentina is predicted to lack an uncleavable N-terminal signal sequence, which is believed to direct proteins to the endoplasmic reticulum . The temperature and pH optimum, enzyme kinetic parameters and substrate specificity of the heterologously expressed SG were studied and compared to those of the C . roseus enzyme, revealing some differences between the two glucosidases . In vitro deglucosylation of strictosidine by R . serpentina SG proceeds by the same mechanism as has been shown for the C . roseus enzyme preparation . The reaction gives rise to the end product cathenamine and involves 4,21-dehydrocorynantheine aldehyde as an intermediate . The enzymatic hydrolysis of dolichantoside (Nbeta-methylstrictosidine) leads to several products . One of them was identified as a new compound, 3-isocorreantine A . From the data it can be concluded that the divergence of the biosynthetic pathways leading to different classes of indole alkaloids formed in R . serpentina and C . roseus cell suspension cultures occurs at a later stage than strictosidine deglucosylation. Photodermatol Photoimmunol Photomed, 2002 Feb, 18(1), 36 - 41 Extracorporeal photochemotherapy for secondary chronic progressive multiple sclerosis: a pilot study; Besnier DP et al.; BACKGROUND/PURPOSE: Extracorporeal photochemotherapy (ECP) has been proposed for the treatment of various auto- and allo-immune reactions . However, a standard ECP regimen did not significantly alter the course of chronic progressive multiple sclerosis (MS) . We tested whether an intensive ECP treatment can affect the course of secondary chronic progressive form of MS . METHODS: Five patients free of immunosuppression were included . Soluble 8-MOP was added ex vivo to a mononuclear cell suspension obtained in a cell separator . This cellular suspension was then irradiated using an UVA irradiator and re-infused into the patient . ECP was performed once a week for 6 weeks and then, depending on clinical evaluation, for a maximum of 6 months, with 2-year follow-up after treatment discontinuation . Scoring was performed with the Kurzke scale and EDSS by a single independent neurologist . RESULTS: One patient was excluded because of recurrent attacks at the very beginning of treatment . Four patients completed the study: one exhibited clinical improvement and three remained stable during the first 6 months of treatment . However, all experienced relapse or worsening of the disease after discontinuation of ECP treatment . CONCLUSION: Our intensive ECP treatment only transiently alters the course of the severe secondary chronic progressive form of MS, with rebound after treatment discontinuation. ANZ J Surg, 2002 Apr, 72(4), 254 - 7 Tumour implantation following laparoscopy using different insufflation gases; Gupta A et al.; BACKGROUND: Because of the possibility of intraperitoneal seeding and port-site recurrences following laparoscopic surgery, the role of laparoscopy in cancer surgery remains controversial . Previous experimental studies have suggested that chemical, metabolic and immunological changes following carbon dioxide (CO2) insufflation may be responsible for this phenomenon . Earlier experimental studies done by the University of Adelaide Department of Surgery have also shown that helium insufflation is associated with none of the adverse changes brought about by CO2 insufflation . Helium insufflation is also associated with lower rates of intra-abdominal tumour spread . The aim of this study was to determine whether these identified benefits apply to inert gases in general . METHODS: Twenty-four Dark Agouti rats were randomized to undergo laparoscopy with 40 min insufflation using one of the following four gases (six rats in each group); CO2, helium, argon and nitrogen . A tumour cell suspension was injected into the abdominal cavity at the beginning of laparoscopy . The rats were killed 7 days after surgery, and the peritoneal cavity and port sites were examined for the presence of tumour . RESULTS: Rats undergoing helium insufflation, had the least number of port-site recurrences and the least amount of intraperitoneal tumour spread . Argon and nitrogen pneumoperitoneum were associated with a large number of port-site recurrences and widespread tumour seeding . The effect of CO2 insufflation was intermediate . CONCLUSION: The choice of insufflation gas influences the incidence of port-site metastases and the degree of intraperitoneal tumour spread following laparoscopic cancer surgery . The reduced port-site recurrences and intraperitoneal spread that followed helium pneumoperitoneum is likely to be a unique property of this gas rather than a property of inert gases in general. Pflugers Arch, 2002 May, 444(1-2), 186 - 92 Epub 2002 Jan 31. Calcium and voltage-dependent alterations of cell volume in neuroblastomaxglioma hybrid NG108-15 cells; Bostel S et al.; Intracellular calcium ({Ca2+}(i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how {Ca2+}(i) controls cell volume . {Ca2+}(i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin . The increase in {Ca2+}(i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution . Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution . In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling . Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid . The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA . Under all these conditions, the membrane input resistance was decreased . Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current . Taken together, these results suggest that, if cell volume is closely dependent on {Ca2+}(i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential. Ann Hematol, 2002 Apr, 81(4), 224 - 7 Epub 2002 Feb 28. Lymphomatous polyp of mantle cell type in the duodenum complicated by gastric cancer: a case of trisomy 3 and t(11;14)(q13;q32); Tsuchiyama J et al.; We experienced a rare case of a lymphomatous polyp of mantle cell type forming a polypoid mass lesion in the duodenum bulbous together with advanced gastric cancer . A total gastrectomy was performed, and the specimen revealed atypical small- to medium-sized lymphoid cells with indented nuclei, which infiltrated the Peyer's patch and formed a nodular mass in the lamina propria and submucosa of the duodenum . The lymphoma cells also infiltrated the lymphoid follicle of the gastric mucosa, spleen, and regional lymph node with a typical mantle zone pattern . Flow cytometric analysis of the single cells of the lymph node and immunohistochemistry of a paraffin-embedded specimen revealed that the lymphoma cells expressed surface CD5, CD19, CD20, and nuclear cyclin D1 . Chromosomal analysis of this single cell suspension revealed that these lymphoma cells have trisomy 3 in conjunction with t(11;14)(q13;q32), which is frequently seen in mucosa-associated lymphoid tissue lymphomas (MALToma) in the stomach and is also reported in mantle cell lymphoma as a secondary genetic alteration . Our report suggests that trisomy 3 may be a common chromosomal abnormality in lymphomatous polyps of mantle cell type. Scand J Immunol, 2002 May, 55(5), 470 - 7 An established immune response against ovalbumin is suppressed by a transferable serum factor produced after ovalbumin feeding: a role of CD25+ regulatory cells; Karlsson MR et al.; We have previously demonstrated that rats fed ovalbumin (OVA) develop a tolerogenic activity in serum, which upon transfer induces tolerance to OVA and suppression of the immune response to a bystander antigen . Here, we have extended these studies and analysed if the tolerogenic activity in serum could suppress an established immune response in the recipients . Rats were immunized with OVA, 4 and 1 week prior to the transfer of serum from either OVA-fed or control animals . Rats that received serum from OVA-fed donors had significantly lower delayed-type hypersensitivity (DTH) reaction against OVA 1 week after the serum transfer compared with the controls, and the levels of immunoglobulin (IgG) anti-OVA antibodies were significantly lower 2 and 4 weeks after serum transfer . Monomeric OVA in amounts corresponding to the OVA transferred with serum did not induce the reduction of DTH response or IgG anti-OVA antibody levels . In vitro, the proliferation of OVA-stimulated spleen cells, taken from recipients of tolerogenic serum, was significantly lower compared with spleen cells from the controls . The in vitro suppression seemed to be mediated by a population of CD25+ cells, because the removal of such cells from OVA-stimulated spleen cell suspensions resulted in increased proliferation in cultures from rats receiving tolerogenic serum . Our results showed that the tolerogenic serum factor can suppress an established immune response in recipient animals, possibly through induction of regulatory CD25+ cells . Whether this capacity might be used to influence chronic inflammatory conditions needs to be investigated. Pulm Pharmacol Ther, 2002, 15(1), 25 - 33 Leukotoxin-activated human pulmonary artery endothelial cell produces nitric oxide and superoxide anion; Okamura S et al.; To provide evidence that pulmonary endothelial cells exposed to 9,10-epoxy-12-octadecenoate (Lx) produce nitric oxide (NO) and superoxide anion (O(2)(*-), we measured NO production, using a NO chemiluminescence analyzer, and nitric