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.

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.

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.

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.

MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice.

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.

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.

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.

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.

alpha5beta1 integrin expression and luminal edge fibronectin matrix assembly by smooth muscle cells after arterial injury.

Fibronectin is secreted from the cell as a soluble protein that must then polymerize to regulate cell function. To elucidate the process of fibronectin matrix assembly in vascular disease, we immunostained sections of balloon-injured rat carotid artery for the fibronectin-binding alpha5beta1 integrin. Whereas alpha5beta1 integrin was not evident in the normal carotid artery, its expression was induced after a vascular injury. By 14 days, the alpha5beta1 integrin was localized exclusively to the less differentiated smooth muscle cells (SMCs) at the luminal surface of the neointima. Platelet-derived growth factor-BB, dominant in neointimal formation, selectively increased the expression of the alpha5beta1 integrin by human SMCs in culture. To track the assembly of fibronectin fibers, fluorescence-labeled soluble fibronectin protomers were added to cultured SMCs and to fresh segments of normal and balloon-injured rat carotid arteries. Fibronectin fiber formation in cultured SMCs could be detected within 10 minutes, and was blocked by an RGD peptide, an anti-beta1 integrin antibody, and an anti-alpha5beta1 integrin antibody, but not by an anti-beta3 integrin antibody. En face confocal microscopy of arterial segments revealed that soluble fibronectin had polymerized on the alpha5beta1 integrin-expressing SMCs of the luminal surface of the injured arterial neointima, but not on the alpha5beta1 integrin-negative neointimal SMCs below this or on the endothelial cells of uninjured arteries. Furthermore, in situ fibronectin assembly by the neointimal SMCs was inhibited by an RGD peptide and by an anti-beta1 integrin antibody. These studies indicate that a subpopulation of SMCs in the repairing artery wall orchestrates integrin-mediated fibronectin assembly.

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.

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.

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.

Human sympathetic preganglionic neurons and motoneurons retrogradely labelled with DiI.

The retrograde tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was used to label sympathetic preganglionic neurons (SPN) and motoneurons (MN) in postmortem human spinal cord. Seven months after microinjection of DiI into the ventral part of spinal thoracic segments T4 and T8, DiI-labelled neurons were identified and analyzed. Cryostat sections of spinal cord were prepared for light microscopy, while vibratome sections were analyzed using confocal microscopy. The majority of retrogradely labelled SPNs were located within the intermediolateral nucleus, with a few labelled dendrites having a mediolateral orientation. SPNs were also located within the nucleus intercalatus, around the central canal and in the lateral funiculus. Cell bodies of retrogradely labelled IML neurons were oval, kite- or spindle-shaped. The soma area of SPNs in T4 was approximately 422.9 +/- 20.9 microm2 with a median diameter of 14 +/- 0.6 microm. MNs in the ventral horn were round or oval in shape and often appeared with a few labelled neurites. The soma area of the MNs in T4 was approximately 842.3 +/- 35.1 microm2, with a median diameter of 18.3 +/- 0.1 microm. The mean values for MN soma area and diameter measurements were significantly greater compared to SPNs. However, no difference was observed between MNs in different segments or between SPNs in the same segments. No retrogradely labelled cells were observed within the dorsal horn. These findings indicate that DiI is a useful method for studying fixed human central nervous system tissue.

Activation of p21-CDC42/Rac-activated kinases by CD28 signaling: p21-activated kinase (PAK) and MEK kinase 1 (MEKK1) may mediate the interplay between CD3 and CD28 signals.

CD28, a T cell costimulatory receptor, provides a signal that induces both optimal proliferation and the production of IL-2 by TCR-activated T cells. We show that the stimulation of CD28 leads to the activation of p21-activated kinase and MEK kinase 1. The same pathway was also stimulated in T cells treated with the cell-permeable ceramide analogue, C2-ceramide. The combined stimulation of either CD3 and CD28 or CD3 concurrently with C2-ceramide largely enhanced the activity of p21-activated kinase and MEK kinase 1. Therefore the Rac1/CDC42-coupled pathway(s) is a candidate that transduces and facilitates cross-talk between the CD28 costimulatory signal and the TCR signal.

Stimulation of CD28 with B7-2 promotes focal adhesion-like cell contacts where Rho family small G proteins accumulate in T cells.

Unless a costimulatory signal is provided, TCR recognition of Ag bound to the MHC is insufficient to induce optimal T cell proliferation or the production of IL-2. Here we show that the stimulation of CD28, a T cell costimulatory receptor, by a specific Ab increases F-actin contents in T cells. The interaction between T cells and B7-2-transfected Chinese hamster ovary cells expressing the CD28 ligand leads to the rearrangement of the actin cytoskelton in the region of cell-cell contact. Within the Rho family of G proteins, Rac1, but not Rho, translocates to the sites of cell-cell contact where Tailin also accumulates. These results indicate that the interaction between B7-2 and CD28 establishes a focal adhesion-like cell contact between T cell and APCs. The results also suggest that CD28 signaling is primarily transduced by a cytoskeletal rearrangement/signaling pathway mediated by the Rho family G proteins.

Dietary fish oil. Influence on lesion regression in the porcine model of atherosclerosis.

We examined the influence of dietary fish oil on lesion regression in a porcine model of atherogenesis. Thirty-two female Yucatan miniature pigs were fed an atherogenic diet for 8 months. A no-regression group (n = 8) was killed to determine the extent of atherosclerosis at 8 months. Three regression groups were switched to normal minipig chow supplemented with either MaxEPA fish oil (FO group, n = 8), a control oil with the ratio of polyunsaturated to monounsaturated to saturated fatty acid matched to that of the fish oil (CO group, n = 8), or no oil supplement (NO group, n = 8) for a further 4 months. Plasma cholesterol levels reached between 15 and 20 mmol/L during the atherogenic phase and returned to normal (2 mmol/L) within 2 months of the beginning of the regression diet. Compared with the NO group, fish oil supplementation during the regression phase caused a decrease in VLDL and HDL cholesterol and an increase in LDL cholesterol. Similarly, the control oil also caused a decrease in VLDL cholesterol; however, in contrast to the FO group, HDL cholesterol increased and LDL cholesterol was unchanged. FO LDL, which had decreased levels of 20:4 (n-6 fatty acid) and increased levels of 18:3, 20:5, and 22:6 (n-3 fatty acids), was shown to be twice as susceptible to copper-mediated oxidation as CO LDL particles. Morphological examination of the major blood vessels revealed a significant reduction in lesion area in the ascending and thoracic aorta as well as the carotid artery after the regression diet; however, there was no significant difference between the fish oil and control oil groups in any of the vessels measured. Therefore, despite increased LDL, decreased HDL, and an increased susceptibility to in vitro oxidation of LDL, fish oil supplementation of a regression diet did not influence lesion regression.

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells.

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

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.

Changes in the distribution of LFA-1, catenins, and F-actin during transendothelial migration of monocytes in culture.

To determine changes in the spatial and temporal distribution of cell-cell adhesion molecules during transendothelial migration of monocytes, we examined an in vitro model system of diapedesis using high resolution laser scanning confocal microscopy. Human arterial endothelial cells were cultured to confluence on coverslips coated with Matrigel and activated with IL-1beta before the addition of monocytic THP-1 cells. Seventy per cent of monocytes transmigrated through the endothelium within one hour. Diapedesis, but not adhesion and spreading, was inhibited 8-fold in co-cultures that contained endothelial cell conditioned medium, suggesting the release of an endothelial derived inhibitor. Double immunofluorescence labeling with antibodies to LFA-1, alpha- and beta-catenin, VE-cadherin and with Texas Red phalloidin, identified a circular transmigration passage in endothelial cell-cell contact regions. This passage was formed by an LFA-1-containing pseudopodium that penetrated between endothelial cells. Apical to the transmigration passage, monocytes remained round in shape, while underneath the endothelium, they spread along the Matrigel. The margins of the transmigration passage contained high levels of LFA-1 and F-actin, suggesting a major role of these molecules during the transmigration process itself. Endothelial adherens junctions, as judged by the presence of VE-cadherin and alpha-catenin adjacent to the passage, remained intact during diapedesis. The presence of catenins at heterotypic contact regions between monocytes and endothelial cells during diapedesis suggested cadherin-mediated interactions between the two cell types. These results reveal dynamic changes in the distribution of adhesion molecules and the actin cytoskeleton during monocyte transendothelial migration in culture.

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.