Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells.

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.

Early treatment of SIV+ macaques with an α4ß7 mAb alters virus distribution and preserves CD4+ T cells in later stages of infection.

Integrin α4ß7 mediates the trafficking of leukocytes, including CD4+ T cells, to lymphoid tissues in the gut. Virus mediated damage to the gut is implicated in HIV and SIV mediated chronic immune activation and leads to irreversible damage to the immune system. We employed an immuno-PET/CT imaging technique to evaluate the impact of an anti-integrin α4ß7 mAb alone or in combination with ART, on the distribution of both SIV infected cells and CD4+ cells in rhesus macaques infected with SIV. We determined that α4ß7 mAb reduced viral antigen in an array of tissues of the lung, spleen, axillary, and inguinal lymph nodes. These sites are not directly linked to α4ß7 mediated homing; however, the most pronounced reduction in viral load was observed in the colon. Despite this reduction, α4ß7 mAb treatment did not prevent an apparent depletion of CD4+ T cells in gut in the acute phase of infection that is characteristic of HIV/SIV infection. However, α4ß7 mAb appeared to facilitate the preservation or restoration of CD4+ T cells in gut tissues at later stages of infection. Since damage to the gut is believed to play a central role in HIV pathogenesis, these results support further evaluation of α4ß7 antagonists in the study and treatment of HIV disease.

Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia.

BACKGROUND: Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). METHODS: We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. RESULTS: The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. CONCLUSIONS: Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

Incidence and description of autoimmune cytopenias during treatment with ibrutinib for chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is frequently complicated by secondary autoimmune cytopenias (AICs). Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase approved for the treatment of relapsed CLL and CLL with del(17p). The effect of ibrutinib treatment on the incidence of AIC is currently unknown. We reviewed medical records of 301 patients treated with ibrutinib, as participants in therapeutic clinical trials at The Ohio State University Comprehensive Cancer Center between July 2010 and July 2014. Subjects were reviewed with respect to past history of AIC, and treatment-emergent AIC cases were identified. Before starting ibrutinib treatment, 26% of patients had experienced AIC. Information was available for a total of 468 patient-years of ibrutinib exposure, during which there were six cases of treatment-emergent AIC. This corresponds to an estimated incidence rate of 13 episodes for every 1000 patient-years of ibrutinib treatment. We further identified 22 patients receiving therapy for AIC at the time ibrutinib was started. Of these 22 patients, 19 were able to discontinue AIC therapy. We found that ibrutinib treatment is associated with a low rate of treatment-emergent AIC. Patients with an existing AIC have been successfully treated with ibrutinib and subsequently discontinued AIC therapy.

Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/LFA-1 and VCAM-1/VLA-4.

The adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressed in atherogenic lesions are thought to regulate monocyte diapedesis. To better understand their specific roles we used function-blocking antibodies and examined in a culture model the morphology, motility, and diapedesis of THP-1 cells interacting with human coronary artery endothelial cells. The number of motile THP-1 cells was reduced only when VCAM-1 or both ICAM-1 and VCAM-1 were blocked. Blockade of ICAM-1 and VCAM-1, either separately or together, reduced to the same degree the distance that THP-1 cells traveled. Diapedesis was reduced only during the simultaneous blockade of both adhesion molecules. Blockade of either ICAM-1 or VCAM-1 inhibited pseudopodia formation, but ICAM-1 blockade induced the formation of filopodia. We suggest that the interactions of endothelial ICAM-1 and VCAM-1 with their ligands differentially regulate distinct steps of diapedesis by modulating the ratio of active and inactive forms of small GTPases such as Rho, Rac, and Cdc42.

Relationship of microtubule organization in lymphocyte to the capping of immunoglobulin.

We have used a double fluorescence staining procedure to examine the distribution of antitubulin-staining structures in mouse splenic lymphocytes induced to patch and cap surface immunoglobulin by treatment with antiimmunoglobulin antibodies. A well organized network of fibers, extending from a single microtubule organizing center (MTOC) associated with the centriole pair, was detected by antitubulin staining at all stages of the capping process. In lymphocytes possessing as intact network, the cap was formed and internalized over the region of the cytoplasm containing the organizing center, Golgi apparatus, and most other organelles. In contrast, in lymphocytes which had been subjected to a colchicine treatment sufficient to completely disassemble networks prior to cap induction, no relationship was detected by immunofluorescence between the position of the centrioles (a marker for the MTOC) and the location of the surface cap. Electron microscopic observation of colchicine-treated cells, induced to cap by ferritin-conjugated anti-Ig, revealed extensive disorganization of cytoplasmic organelles and exclusion of organelles from the region of underlying the cap. These results indicate a role for the microtubule network of lymphocytes in maintaining cytoplasmic polarity and in specifying the site of Ig cap formation.

The distribution of fibro-fatty atherosclerotic lesions in the aortae of casein- and cholesterol-fed rabbits.

Atherosclerotic lesions were induced in the aortas of 50 rabbits by feeding a semi-purified cholesterol-free casein diet or normal rabbit chow with a low level of added cholesterol for 6 or 10 months. Following perfusion fixation, the aortae from these animals were opened along their length, stained with oil red O and photographed en face. Orifice associated lesions were mapped by measuring radial lesion length at 10 degrees intervals circumferentially around ostia. Histology of these lesions revealed abundant smooth muscle cells surrounded by collagen and elastin in the extracellular matrix, typical of fibrous plaques, as well as oil red O staining lipid and some macrophage derived foam cells. These fibro-fatty lesions were found distal and lateral to ostia, at the same locations as fatty streaks seen in rabbits fed a 2% cholesterol diet for 1 week to 2 months in previous studies. The results of this study show that in moderately hypercholesterolemic rabbits fed an atherogenic diet for 6 to 10 months, advanced atherosclerotic plaques develop in the same location as the fatty streaks seen in short term experiments.

Co-localization of substance P and dopamine beta-hydroxylase with growth-associated protein-43 is lost caudal to a spinal cord transection.

After spinal cord injury, abnormal responses of spinal cord neurons to sensory input lead to conditions such as autonomic dysreflexia, urinary bladder dyssynergia, muscle spasticity and chronic pain syndromes. These responses suggest that the spinal cord undergoes marked reorganization after an injury. In previous studies, we demonstrated changes in individual patterns of immunoreactivity for growth-associated protein-43, dopamine beta-hydroxylase and substance P that suggest growth and/or changes in expression of neurotransmitter enzymes and peptides in the cord caudal to a transection injury. In the present study we determined whether (i) growth-associated protein-43 and dopamine beta-hydroxylase or substance P were co-expressed in the same neurons prior to cord injury, and (ii) these patterns of expression changed after injury. A change in co-localization patterns caudal to an injury would suggest diversity in responses of different populations of spinal neurons. We used double-labelling immunocytochemistry to determine whether either dopamine beta-hydroxylase or substance P was co-localized with growth-associated protein-43 in control rats and in rats one, two or six weeks after spinal cord transection. We focused on the intermediate gray matter, especially the sympathetic intermediolateral cell column. In control rats, fibres travelling in a stereotyped ladder-like pattern in the thoracic gray matter contained growth-associated protein-43 co-localized with dopamine beta-hydroxylase or substance P. In spinal rats, such co-localization was also observed in spinal cord segments rostral to the cord transection. In contrast, caudal to the transection, substance P and growth-associated protein-43 were found in separate reticular networks. Immunoreactivity for dopamine beta-hydroxylase disappeared in fibres during this time, but was clearly present in somata. Immunoreactivity for growth-associated protein-43 was also found in somata, but never co-localized with that for dopamine beta-hydroxylase. These observations demonstrated co-localization of growth-associated protein-43 with dopamine beta-hydroxylase and substance P in descending spinal cord pathways. Caudal to a cord transection, this co-localization was no longer found, although each substance was present either in an abundant neural network or in somata. One population of spinal neurons responded to cord injury by expressing the growth-associated protein, whereas two others changed in the intensity of their expression of neurotransmitter peptides or enzymes or in the abundance of fibres expressing them. Thus, three populations of spinal neurons had distinct responses to cord injury, two of them increasing their potential input to spinal sensory, sympathetic or motor neurons. Such responses would enhance transmission through spinal pathways after cord injury.

A method for examining the endothelial cytoskeleton in situ using immunofluorescence.

A simple technique is described for producing en face preparations of endothelial cells (EC) from large blood vessels fixed in situ that are suitable for studying the distribution of specific antigens in EC by immunofluorescence. This method has permitted us to examine the distribution of various components of the cytoskeleton, including microtubules (MT), centrioles, and microfilaments (MF) in EC in vivo.

Cellular localization of P-glycoprotein in brain versus gonadal capillaries.

P-glycoprotein, the multidrug resistance protein that actively transports a wide variety of lipophilic substrates out of cancer cells, has recently been described in some normal tissues, including the endothelium of the brain and testes. Here we show that P-glycoprotein is also expressed in ovarian endothelium. In ovarian capillaries, the immunolabeled protein was detected with two monoclonal antibodies to P-glycoprotein. It was shown to be membrane-bound and to transport a known P-glycoprotein substrate. Expression of P-glycoprotein in endothelial cells suggests that this transport protein plays a role in enhancing or restricting vascular permeability to lipophilic molecules. If it does, then its role may be predicted from its site of expression on the luminal or abluminal face of the capillary wall. In the region of the endothelial nucleus, endothelial membranes are sufficiently far apart that they can be distinguished at the light microscopic level. Confocal examination of tissue sections double labeled for P-glycoprotein and nuclei confirmed that, in brain, P-glycoprotein is expressed only on luminal membranes. This location is consistent with its putative role in protecting the neuropil from circulating lipophilic molecules. In both testicular and ovarian endothelium, however, P-glycoprotein is expressed on both luminal and abluminal membranes. This localization suggests that it acts to exclude P-glycoprotein substrates from the endothelial cells themselves.

Usefulness and limitations of FISH to characterize partially cryptic complex chromosome rearrangements.

Interpretation of a complex chromosome rearrangement (CCR) using only G-band analysis is difficult and potentially inaccurate. We present two patients with de novo, partially cryptic, CCRs that illustrate both the value and limitations of using fluorescence in situ hybridization (FISH) whole chromosome paint probes to characterize these types of rearrangements. In a patient referred because of features of Townes-Brocks syndrome, G-band analysis revealed an unbalanced CCR involving 3 chromosomes (2,11 and 16) and at least 4 breakpoints. A more complex rearrangement involving two cryptic insertions and at least 6 breakpoints, however, was detected using whole chromosome paint probes specific for the 3 chromosomes involved in the rearrangement. In this case, FISH studies were essential for accurate characterization of this patient's rearrangement. In a second patient, G-band analysis revealed that a 12-year-old male with obesity, small genitalia, attention deficit disorder, learning disabilities, and behavior problems, carried a CCR involving 4 chromosomes (3, 5, 10 and 13) with 6 breakpoints. This rearrangement seemed unbalanced, with missing terminal 3p26. 2-pter material. Our G-band interpretation of this karyotype was confirmed by FISH using whole chromosome paint probes specific for the involved chromosomes. Although no evidence of the "missing" 3pter material was observed using a chromosome 3 paint, FISH analysis using a chromosome 3p unique telomere probe identified telomeric 3p material on the distal long arm of the derivative 10 chromosome. This case illustrates the limited value of painting probes to detect small rearrangements, especially those involving terminal chromosome regions.

Effects of injection mechanics, pH of infusate and 6-hydroxydopamine on cerebromicrovascular permeability in rats.

The effects of altering the rate, manner and vehicle used for intracerebral injection upon microvascular permeability were studied in Sprague-Dawley rats employing horseradish peroxidase histochemistry. The volume of vehicle delivered and the site of intracerebral injection were kept constant. In comparison to continuous infusion, vascular permeability was significantly greater following manual (intermittent) injections; however, no differences were found when the infusion rate was decreased 10-fold. Use of a buffered vehicle (Hanks' balanced salt solution) with pH adjusted to 7.4, in contrast to the more commonly used non-buffered vehicle (saline-ascorbate), resulted in significant reductions in permeability. The apparent influence of the agent 6-hydroxydopamine (6-OHDA) on changes in vascular permeability was found to vary depending on the type and pH of the vehicle used for injection. Significantly greater permeability resulted with saline-ascorbate (pH 3.1) as the vehicle when compared to Hanks' balanced salt solution (pH 7.4). Changes in vascular permeability can therefore be produced by varying mechanical and vehicular factors which, in the case of 6-OHDA, far outweigh previously reported permeability changes specifically attributed to this neurotoxin.

Vascularization and microvascular permeability in solid versus cell-suspension embryonic neural grafts.

Vascularization and microvascular permeability were assessed in a comparative study of solid (organized) and cell-suspension (dissociated) fetal nigral grafts implanted in the dopamine-deprived striatum of adult rats. Both graft types were analyzed by chromogen detection of intravenously injected horseradish peroxidase (HRP), which outlined vessel walls, and, in cases in which the blood-brain barrier was compromised, permeated the graft and host parenchyma. Survival of graft-derived dopaminergic cells was assessed using tyrosine hydroxylase (TH) immunocytochemistry. Glial reactivity to cell-suspension grafts was similarly assessed with an antibody directed against glial fibrillary acidic protein. Morphometry revealed significantly higher microvessel density in the cell-suspension grafts (p < 0.001), which effectively equaled that found in the contralateral striatum despite rather prominent surrounding glial reactivity. Capillaries in the cell-suspension grafts were not permeable to blood-borne HRP at postimplantation study times of 7, 14, and 30 days whereas, in the solid grafts, permeability in some cases could be detected for up to 30 days. Large numbers of cells immunoreactive for TH were seen in cell-suspension grafts; in contrast, few if any were found in the majority of solid transplants. The multiple-fragment solid graft implant model used clinically compares poorly with the cell-suspension model because it lacks consistency in early revascularization and shows a greater (albeit temporary) tendency for blood-brain barrier dysfunction. Delayed and inadequate vascularization of the solid graft is likely to account for graft failure more often than in the cell-suspension graft. Similarly, a certain critical number of specific grafted cells are required to achieve sufficient expression to bring about a favorable response in the disabled host, and this expression appears to be achieved less consistently with the solid implant technique.

Transendothelial migration of monocytes in rat aorta: distribution of F-actin, alpha-catnin, LFA-1, and PECAM-1.

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or alpha-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent endothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by alpha-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis at the cell surface in the parts of the monocyte located above the endothelium. PECAM-1 was enriched near the endothelial cell-cell junctions, but was not detected in parts that spread beneath the endothelium. Our results suggest a major role for LFA-1 during diapedesis and reveal dynamic changes in the distribution of PECAM-1, the actin cytoskeleton, and alpha-catenin during monocyte diapedesis in situ.

A comparison of three horseshoeing styles on the kinetics of breakover in sound horses.

A variety of horseshoe designs are believed to 'ease' breakover, or the unloading of the foot once the heels leave the ground. In this study, conventional toe-clip shoes, quarter-clip shoes, fitted to the white line at the toe, and Natural Balance horseshoes were fitted to the front feet of 9 sound Irish Draught-cross type horses. Forceplate and video motion analyses were undertaken during trot locomotion to determine the moment arm of the ground reaction force on the distal interphalangeal (DIP) joint, the peak DIP joint moment and the peak compressive force on the navicular bone. DIP joint moment arm during breakover was reduced with both Natural Balance (mean +/- s.d. 77 +/- 7 mm) and quarter-clip shoes (78 +/- 9 mm) compared to the toe-clip shoes (86 +/- 6 mm) (P<0.01). Peak DIP joint moment was not significantly different (175 +/- 37,171 +/- 38 and 175 +/- 31 Nmm/kg, in Natural Balance, quarter-clip and toe-clip shoes, respectively) and neither was peak force on the navicular bone (5.52 +/- 1.52, 5.79 +/- 1.53 and 6.14 +/- 1.47 N/kg, respectively). Breakover duration (heel off to toe off) was not significantly reduced by the Natural Balance shoes (39 +/- 6 ms) or the quarter-clip shoes (40 +/- 6 ms) compared to toe-clip shoes (42 +/- 9 ms). This study has demonstrated that the use of Natural Balance shoes reduces the moment arm of the ground reaction force (GRF) during breakover but does not reduce the peak DIP joint moment or the force on the navicular bone.

Violence at work: personal and organizational outcomes.

To date, little empirical research has examined the personal and organizational outcomes associated with exposure to workplace violence. On the basis of data from 194 bank tellers, the authors evaluated, and supported, a model suggesting that fear of future violence mediates the relationships between exposure to workplace violence and negative outcomes. Specifically, exposure to workplace violence predicted fear of future violence that, in turn, predicted psychological well-being, somatic symptoms, and intent to leave the organization. These effects emerged after controlling for self-report bias. The mediating role of fear was supported, and implications for future research and practice are discussed.

Transition management as an intervention for survivor syndrome.

In today's health care environment of merged organizations, downsizing and restructuring, employees can be experiencing a debilitating syndrome called "layoff survivor syndrome." This syndrome can have a crippling effect on workers and organizations as employees struggle to adapt to the changed working environment. This article represents my self-reflection as a nursing unit manager who personally experienced survivor sickness and witnessed its impact on the unit staff that I was leading at the time. The work of Noer (1993) is explored to clarify the syndrome and describe how the nursing staff and I manifested the syndrome. The writings of Bridges (1991), Brockner (1992) and Noer (1993) provide timely and relevant insights into managing the impact of layoffs and downsizing on those left behind to carry on. Noer (1993) sees the adaptation to the change as the ability to make the psychological shift from the old business paradigm that perpetuated codependency to the new business paradigm of fostering empowered employees. Bridges (1991) takes us a step further in making this psychological shift to adapt to the new work environment by providing a three phase process he calls transitions. The works of these three authors hold an important message for organizations and employees working in environments that abound with constant change.

Obinutuzumab for the treatment of chronic lymphocytic leukemia.

Obinutuzumab is a novel therapeutic anti-CD20 monoclonal antibody recently approved by the United States Food and Drug Administration (FDA) for use in combination with chlorambucil as first-line treatment of chronic lymphocytic leukemia (CLL). It is distinguished from other anti-B-lymphocyte antigen CD20 (anti-CD20) therapeutic antibodies in current clinical use by its type II properties and glycoengineered Fc region. In vitro these unique properties translate into higher rates of antibody-dependent cytotoxicity and direct cell death compared to rituximab, and obinutuzumab demonstrates improved efficacy in human lymphoma xenograft models and whole blood lymphocyte depletion assays. FDA approval was based upon results from a randomized phase III trial comparing treatment with single-agent chlorambucil to the combination of chlorambucil and either rituximab or obinutuzu-mab. The obinutuzumab arm resulted in higher rates of complete remission and significant improvements in progression-free survival versus either comparator regimen. The majority of patients in the obinutuzumab and chlorambucil arm finished all six planned treatment cycles, and therapy was well tolerated. Toxicities of obinutuzumab are similar to those of other anti-CD20 antibodies, although infusion-related reactions and neutropenia appear to be more common. This trial establishes chemoimmunotherapy with obinutuzumab and chlorambucil as an attractive treatment option for CLL patients, particularly those with comorbid medical illnesses or advanced age. Obinutuzumab remains under study in combination with both chemotherapy and novel agents for CLL and non-Hodgkin's lymphoma, where it is expected to find additional clinical applications.