Electrokinetic device design and constraints for use in high conductance solutions.

The quest for new cell-free DNA and exosome biomarker-based molecular diagnostics require fast and efficient sample preparation techniques. Conventional methods for isolating these biomarkers from blood are both time-consuming and laborious. New electrokinetic microarray devices using dielectrophoresis (DEP) to isolate cell-free DNA and exosome biomarkers have now greatly improved the sample preparation process. Nevertheless, these devices still have some limitations when used with high conductance biological fluids, e.g. blood, plasma, and serum. This study demonstrates that electrochemical damage may occur on the platinum electrodes of DEP microarray devices. It further examines two model device designs that include a parallel wire arrangement and a planar array. Effective isolation of fluorescent beads with parallel wires is shown under low-conductance conditions (10-4  S/m), but electrothermal flow overcomes DEP forces under high conductance conditions (>0.1 S/m). Planar devices are shown to be effective under high conductance conditions (∼1 S/m) without the deleterious effects of electrothermal flow. This study provides new insights into design compromises and limitations for producing future electrokinetic devices for better performance with high conductance solutions.

Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

Physicochemical properties of ferumoxytol, a new intravenous iron preparation.

BACKGROUND: Intravenous iron is a critical component of anaemia management. However, currently available preparations have been associated with the release of free iron, a promoter of bacterial growth and oxidative stress. MATERIALS AND METHODS: We determined the molecular weight, dialysability and capacity for free iron release of ferumoxytol, a semi-synthetic carbohydrate-coated superparamagnetic iron oxide nanoparticle. Ferumoxytol was compared with three intravenous iron preparations in clinical use: iron dextran (low molecular weight), sodium ferric gluconate and iron sucrose. Intravenous iron preparations were also incubated in rat, and pooled human sera (at concentrations of 600 microM and 42 microg mL(-1) respectively) from healthy subjects. RESULTS: The molecular weight of ferumoxytol was 731 kDa. The relative order of molecular weight was as follows: ferumoxytol > iron dextran > iron sucrose > sodium ferric gluconate. The least ultrafilterable iron was observed with ferumoxytol and the most with ferric gluconate. The least dialysable free iron was observed with ferumoxytol and the most with ferric gluconate. Incubation of intravenous iron preparations in rat or pooled human sera demonstrated minimal free iron release with ferumoxytol. The order of catalytic iron release as detected by the bleomycin detectable iron assay was as follows: ferumoxytol < iron dextran < iron sucrose < ferric gluconate. A similar trend was observed for the in vivo serum concentration of free iron in rats. CONCLUSIONS: In vitro observations from these experiments suggest that ferumoxytol has a favourable profile in terms of tendency to release free iron, in comparison with currently available intravenous iron preparations.

Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation.

Integrins alpha v beta 3 and alpha v beta 5 both mediate cell adhesion to vitronectin yet trigger different postligand binding events. Integrin alpha v beta 3 is able to induce cell spreading, migration, angiogenesis, and tumor metastasis without additional stimulators, whereas alpha v beta 5 requires exogenous activation of protein kinase C (PKC) to mediate these processes. To investigate this difference, the ability of beta 3 or beta 5 to induce colocalization of intracellular proteins was assessed by immunofluorescence in hamster CS-1 melanoma cells. We found that alpha v beta 5 induced colocalization of talin, alpha-actinin, tensin, and actin very weakly relative to alpha v beta 3. alpha v beta 5 was able to efficiently induce colocalization of focal adhesion kinase (FAK); however, it was unable to increase phosphorylation of FAK on tyrosine. Activation of PKC by adding phorbol ester to alpha v beta 5-expressing cells induced spreading, increased colocalization of alpha-actinin, tensin, vinculin, p130cas and actin, and triggered tyrosine phosphorylation of FAK. Unexpectedly, talin colocalization remained low even after activation of PKC. Treatment of cells with the PKC inhibitor calphostin C inhibited spreading and the colocalization of talin, alpha-actinin, tensin, and actin for both alpha v beta 3 and alpha v beta 5. We conclude that PKC regulates localization of cytoskeletal proteins and phosphorylation of FAK induced by alpha v beta 5. Our results also show that FAK can be localized independent of its phosphorylation and that cells can spread and induce localization of other focal adhesion proteins in the absence of detectable talin.

Conservation of T cell receptor conformation in epidermal gammadelta cells with disrupted primary Vgamma gene usage.

A feature that distinguishes gammadelta T cell subsets from most alphabeta T cells and B cells is the association of expression of single T cell receptor (TCR) gamma and delta variable (V) region gene segments with specific anatomic sites. Mice lacking the TCR Vgamma5 chain normally expressed by most dendritic epidermal T cells were shown to retain a conformational determinant (idiotype) ordinarily expressed exclusively by such Vgamma5+ cells. Conservation by shuffled gammadelta TCR chains of an idiotype associated with a specific anatomic site indicates that for TCRgammadelta, as for immunoglobulin, conformation is associated to a greater extent with the function or development of lymphocyte repertoires than is the use of particular gene segments.

Regulation of cutaneous malignancy by gammadelta T cells.

The localization of gammadelta T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking gammadelta cells are highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, major histocompatibility complex-related molecules structurally resembling human MICA. Each of these is a ligand for NKG2d, a receptor expressed by cytolytic T cells and natural killer (NK) cells. In vitro, skin-associated NKG2d+ gammadelta cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2d engagement. Thus, local T cells may use evolutionarily conserved proteins to negatively regulate malignancy.

Insulin-like growth factor receptor cooperates with integrin alpha v beta 5 to promote tumor cell dissemination in vivo.

Tumor cell interactions with adhesion proteins and growth factors likely contribute to the metastatic cascade. Evidence is provided that insulin or insulin-like growth factor-mediated signals cooperate with the commonly expressed integrin alpha v beta 5 to promote spontaneous pulmonary metastasis of multiple tumor cell types in both the chick embryo and severe combined immune deficiency mouse/human chimeric models. Expression of alpha v beta 5 in tumor cells promoted their adhesion to vitronectin in vitro. However, cell motility required cytokine stimulation, which caused redistribution of alpha-actinin to membrane-adhesive sites containing alpha v beta 5. Significantly, ligation of alpha v beta 5 and cytokine receptors were both required for spontaneous pulmonary metastasis of multiple tumor types even though it was not necessary for primary tumor growth. Thus, tumor cell metastasis can be regulated by a functional cooperation between cytokine signaling events and the adhesion receptor alpha v beta 5 in a manner independent of tumor cell growth. These findings provide evidence that integrin ligation, in conjunction with cytokine activation, plays an important role in the dissemination of malignant tumor cells.

Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.

Managing potential drug-drug interactions between gastric acid-reducing agents and antiretroviral therapy: experience from a large HIV-positive cohort.

Drug-drug interactions between antiretroviral therapy and other drugs are well described. Gastric acid-reducing agents are one such class. However, few data exist regarding the frequency of and indications for prescription, nor risk assessment in the setting of an HIV cohort receiving antiretroviral therapy. To assess prevalence of prescription of gastric acid-reducing agents and drug-drug interaction within a UK HIV cohort, we reviewed patient records for the whole cohort, assessing demographic data, frequency and reason for prescription of gastric acid-reducing therapy. Furthermore, we noted potential drug-drug interaction and whether risk had been documented and mitigated. Of 701 patients on antiretroviral therapy, 67 (9.6%) were prescribed gastric acid-reducing therapy. Of these, the majority (59/67 [88.1%]) were prescribed proton pump inhibitors. We identified four potential drug-drug interactions, which were appropriately managed by temporally separating the administration of gastric acid-reducing agent and antiretroviral therapy, and all four of these patients remained virally suppressed. Gastric acid-reducing therapy, in particular proton pump inhibitor therapy, appears common in patients prescribed antiretroviral therapy. Whilst there remains a paucity of published data, our findings are comparable to those in other European cohorts. Pharmacovigilance of drug-drug interactions in HIV-positive patients is vital. Education of patients and staff, and accurate data-gathering tools, will enhance patient safety.

The c-Abl tyrosine kinase contributes to the transient activation of MAP kinase in cells plated on fibronectin.

Previous work showed that integrin stimulation triggers activation of the c-Abl tyrosine kinase and its transient localization to focal adhesions. We now report that plating cells on fibronectin triggers association of Grb2 with c-Abl, suggesting possible involvement of c-Abl with integrin activation of the MAP kinase pathway. Expression of a kinase-defective c-Abl specifically inhibited the transient induction of Erk2 activity following cell adhesion. Together with the known ability of activated, oncogenic forms of c-Abl to activate Ras and the MAP kinase pathway, these data suggest that c-Abl contributes to the integrin induction of MAP kinase activity.

Clinical significance of perfusion defects by thallium-201 single photon emission tomography following oral dipyridamole early after coronary angioplasty.

The clinical significance of myocardial perfusion defects present early after angiographically successful percutaneous transluminal coronary angioplasty was assessed in 53 patients using thallium-201 single photon emission computed tomography combined with pharmacologic vasodilation induced by a large dose (300 mg) of orally administered dipyridamole. Myocardial tomographic images were obtained at a mean of 20 +/- 6 h (SD) before and 2.9 +/- 2.7 days after angioplasty. Before angioplasty, 15 (28%) of the 53 patients developed angina after dipyridamole administration, in contrast to only 3 (7.5%) of 40 patients after angioplasty (p less than 0.001). The mean percent luminal area stenosis decreased from 93 +/- 6% before angioplasty to 34 +/- 17% after angioplasty (p less than 0.001). Myocardial perfusion defects, present in 49 (93%) of the 53 patients before angioplasty, were reversible in 44 patients (83%), all of whom underwent dilation of arteries supplying the ischemic areas. After angioplasty, 26 (65%) of 40 patients had no ischemic defects, whereas 14 (35%) of the patients still had an ischemic defect in the vascular territory of the dilated artery. After a mean follow-up period of 21.7 months, 13 (33%) of 39 patients developed restenosis, 10 of whom had an ischemic defect early after angioplasty. Restenosis developed in 10 (71%) of 14 patients with an ischemic defect after angioplasty, but in only 3 (11.5%) of the patients without an ischemic defect (p = 0.007). In conclusion, thallium-201 tomography after oral dipyridamole affords convenient assessment of the physiologic significance of coronary stenosis present before angioplasty and the residual stenosis after angioplasty.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of myocardial perfusion in humans by contrast echocardiography. I. Evaluation of regional coronary reserve by peak contrast intensity.

Myocardial contrast echocardiography was performed during coronary angiography with 2 ml of sonicated meglumine diatrizoate sodium 76% (meglumine) in 40 patients (ranging in age from 25 to 79 years) before and 10 to 15 s after intracoronary injection of papaverine, 8 mg into the right coronary artery (n = 43) and 10 mg into the left (n = 46). The same protocol was repeated in 17 patients 5 to 10 min after completion of coronary angioplasty. In 13 patients with normal coronary angiograms, peak contrast intensity corrected for background myocardial intensity was measured in 36 regions and was found to increase after papaverine from 36 +/- 16 to 55 +/- 22 U (p less than 0.001). In contrast, in the 27 patients with angiographic evidence of coronary artery disease, peak intensity in 64 regions remained unchanged after papaverine (35 +/- 22 versus 36 +/- 23 U). An increase in peak intensity greater than or equal to 10 U was 80% sensitive and 92% specific for coronary artery disease. After successful coronary angioplasty, peak intensity in the involved regions improved significantly (p less than 0.001) during baseline contrast injections (from 32 +/- 16 to 50 +/- 25 U) as well as in the postpapaverine contrast injections (from 30 +/- 12 to 60 +/- 26 U). In conclusion, measurement of peak contrast intensity after intracoronary injections of sonicated meglumine provides a relative index of myocardial perfusion that allows assessment of regional coronary reserve in patients with coronary artery disease. This may be of particular value in evaluating the immediate effects of coronary angioplasty on myocardial perfusion.

Follow-up of adolescents treated in a psychiatric hospital. Predictors of outcome.

In the context of a long-term follow-up of 60 hospitalized adolescent psychiatric patients (26 months to four years after discharge), we examined correlates of outcome. The variables were as follows: (1) severity of psychopathology; (2) onset of symptomatology (process or reactive); (3) type of hospital treatment termination; (4) continuation of individual psychotherapy following discharge; (5) physically destructive behavior before hospitalization; and (6) energy level. Follow-up was by personal clinical interview exploring the expatient's current living conditions, peer relationships, current psychopathology and drug or alcohol use, legal difficulties, academic and work functioning, subjective contentment, and plans for the future. An evaluation was also obtained from parents and/or spouse. Statistical analyses of the interrelationships among the predictor variables and outcome demonstrated the importance of a multivariate approach.

A blood analogue for the experimental study of flow-related thrombosis at prosthetic heart valves.

It is shown that a flowing mixture of unpasteurised cow's milk, rennet and calcium chloride will deposit solid clot in vitro in the neighbourhood of certain kinds of flow obstruction and hydrodynamic disturbance particularly associated with deposition and thrombus formation in a similar stream of blood. The milk and blood deposits are similar in physical structure, both on a microscopic and a gross scales. For established kinds of artificial heart valves, the growth sequence and developed patterns of milk clotting obtained in vitro with pulsatile flows at physiological rates closely resemble patterns of thrombus reported clinically for valves of the same type in vivo. It appears that these phenomena, for which a tentative explanation is outlined, can form the basis of a convenient and rapid experimental method for evaluating in vitro the effects of design changes on the thrombogenicity of artificial heart valves and other cardiovascular devices in vivo. The milk system also shows promise as an aid in fundamental experimental studies of mechanisms of flow-related thrombosis.

Immunobiology of mouse dendritic epidermal T cells: a decade later, some answers, but still more questions.

Over the past decade, overwhelming evidence has accumulated in many species, most notably in mice, that epithelial sites such as skin, intestine, and reproductive tract are populated with relatively discrete subsets of gamma delta cells. Such studies have identified several distinguishing and, in some cases, unique features of the dendritic epidermal T cells (DETC) populating the skin of all normal mice: homogeneous V5-J1-C gamma 1/V1-D2-J2-C delta T-cell receptors devoid of junctional diversity, apparent tissue restriction in adult mice to the skin, an important role for active hair growth in their localization and/or proliferation in the skin, and a capacity to recognize an antigen expressed on stressed epidermal cells. These properties have led to the hypothesis that DETC play distinctive roles in cutaneous immune surveillance and/or immunoregulation via recognition of a common self-antigen expressed by adjacent cells under various potentially harmful circumstances. Despite substantive advances in our knowledge about gamma delta cells in general (e.g., recent evidence that their manner of antigen recognition may be fundamentally different from that used by conventional alpha beta T cells) and about epithelial-specific subsets such as murine DETC in particular, it is clear that, compared with our understanding of alpha beta cells, major gaps still exist in our understanding of these cells. Persisting questions about DETC include: precise identification of the ligands for their homogenous T-cell receptors, the cellular and molecular requirements for their activation, their full range of functional activities, the reason(s) for the absence in normal human skin of a precise morphologic and phenotypic homologue, and, perhaps most important, their biologically relevant role(s) in cutaneous physiology, immunity, and/or pathology.

TCR gamma/delta+ dendritic epidermal T cells as constituents of skin-associated lymphoid tissue.

The epidermis of all strains of normal mice is populated by two distinct dendritic, bone marrow-derived cells: Langerhans cells and CD4-CD8- Thy-1+ dendritic epidermal T cells (DETC). The overwhelming majority of DETC are an unusually homogeneous population of thymic-dependent cells which express CD3-associated T-cell antigen receptors (TCRs) of the gamma/delta type, thereby distinguishing them from conventional CD4+CD8- or CD4-CD8+ T cells expressing CD3-associated alpha/beta TCR. Most DETC are ontogenetically primitive, derived from early fetal thymocytes with a preferential, but poorly understood tropism for the epidermis. Like the TCR on other populations of gamma/delta cells, which preferentially populate other epithelia such as in the gut and lung, the TCR on most DETC selectively utilize particular variable (V) gene segments (i.e., V gamma 3 and V delta 1 for DETC vs V gamma 5 and V delta 4 or V delta 6 for intestinal intraepithelial lymphocytes). However, unlike other gamma/delta populations whose TCR junctional regions exhibit marked heterogeneity, DETC junctional diversity is extremely limited. This lack of TCR heterogeneity among DETC suggests they recognize a narrow range of physiologic ligands (antigens) and that this recognition is restricted not by conventional polymorphic class-I or class-II MHC molecules, but rather by relatively nonpolymorphic self MHC-like molecules of the class Ib MHC type [e.g., Qa, TL, and CD1 (T6)]. Additional studies are required to clarify precisely what DETC recognize, their relevant biological functions, as well as their relationship(s) to the gamma/delta cells recently identified in human skin.

Superantigenic staphylococcal exotoxins induce T-cell proliferation in the presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.

Several staphylococcal toxins are among a growing number of immunostimulatory molecules called "superantigens" because of their ability, when presented by appropriate major histocompatibility complex class II+ accessory cells, to activate essentially all T cells bearing particular T-cell receptor V beta gene segments. We have examined the ability of murine epidermal Langerhans cells and/or keratinocytes to act as accessory cells in the T-cell response to the superantigens staphylococcal enterotoxin B and exfoliative toxin, also known as epidermolysin. Purified murine splenic T cells were stimulated with staphylococcal enterotoxin B or exfoliative toxin in the presence of Langerhans cells--enriched epidermal cells from normal mice or epidermal cells isolated from mice pretreated with recombinant interferon-gamma, a procedure that induces the expression of major histocompatibility complex class II molecules on keratinocytes. The data show that both Langerhans cells and class II-bearing keratinocytes can act as accessory cells in the T-cell response to staphylococcal enterotoxin B and exfoliative toxin. We also observed that both human and murine keratinocytes cultured in the presence of staphylococcal enterotoxin B or exfoliative toxin produce increased amounts of cytokine(s) capable of stimulating thymocytes and D10 cells, and that this toxin activity is independent of the level of expression of class II on keratinocytes. Studies by enzyme-linked immunosorbent assay showed that staphylococcal enterotoxin B stimulates keratinocytes to produce tumor necrosis factor-alpha but not interleukin-1, suggesting tumor necrosis factor-alpha and perhaps other cytokines are responsible for the T-cell proliferative activity. These results demonstrate that two distinct epidermal constituents (i.e. Langerhans cells and keratinocytes) can serve as accessory cells in the responses of T cells to superantigenic bacterial toxins. It is possible that such toxins contribute to the pathogenesis of a variety of skin diseases by either locally activating T cells bearing particular V beta genes and/or enhancing keratinocyte production of immunomodulatory cytokines.

Repairing the bond in important relationships: a dynamic for personality maturation.

OBJECTIVE: The author sought to establish a common set of interactional dynamics involved in growth-facilitating interpersonal relationships. METHOD: Empirical studies of the three most common dyadic relationships associated with personality growth were reviewed: infant-mother attachment behaviors, the psychotherapeutic alliance, and the marital relationship. RESULTS: Empirical and clinical studies support the conclusion that growth is facilitated when a strong affective bond is established with an important other and the inevitable disruptions of this bond are repaired. CONCLUSIONS: Personality maturation across the lifespan has been attributed to the internalization of admirable qualities of important others. The data surveyed suggest that the interactional dynamics associated with internalization involve establishing a strong affective bond with an important other and repairing this bond's inevitable ruptures. It is both the rupture and the repair that are crucial.

Dying with friends: implications for the psychotherapist.

The processes that augment the continued development of psychotherapeutic competence are little understood. One readily available source of learning is the life experiences of the therapist, particularly those situations which invoke intense affects and may increase empathic capacity. Some life experiences, however, lead to defenses against affect. In particular, situations in which the therapist feels helpless and those which confront the therapist with his or her own death may precipitate intense defenses. Several periods of such defensiveness resulting from discussions with four dying friends are explored and their implications for psychotherapeutic competence are analyzed.

For better or worse: interpersonal relationships and individual outcome.

At a time of strong biological emphasis in psychiatry, it is important to emphasize that relationships with important others may play a crucial role in individual outcome. Psychoanalytic theories in the form of object relations, self psychology, and relational psychoanalysis reflect this emphasis, and at a broader level, the interpersonal school of psychiatry focuses selectively on the role of relationships in health and illness. Ten central premises of the interpersonal school are presented, followed by brief, selective reviews of three bodies of empirical data: studies of well-functioning marriages and families, the role of adult relationships in undoing the adult consequences of destructive childhood experiences, and the relationship of marital variables to the onset and course of depressive disorders. Clinical experience and research findings suggest that clinicians treating couples and families may be helpful by using techniques designed to both increase the intensity of affective bonds and repair the inevitable disruptions of those bonds. It is also noted that recent psychophysiologic studies suggest that derivatives of intense affective bonds and their disruptions, in the form of confiding and conflict, may influence both vascular reactivity and cellular immune competence. These studies suggest that "for better or worse" may have physiologic as well as psychological implications.