Immunomodulatory effects of anti-CD4 antibody in host resistance against infections and tumors in human CD4 transgenic mice.

Anti-CD4 antibodies, which cause CD4(+) T-cell depletion, have been shown to increase susceptibility to infections in mice. Thus, development of anti-CD4 antibodies for clinical use raises potential concerns about suppression of host defense mechanisms against pathogens and tumors. The anti-human CD4 antibody keliximab, which binds only human and chimpanzee CD4, has been evaluated in host defense models using murine CD4 knockout-human CD4 transgenic (HuCD4/Tg) mice. In these mice, depletion of CD4(+) T cells by keliximab was associated with inhibition of anti-Pneumocystis carinii and anti-Candida albicans antibody responses and rendered HuCD4/Tg mice susceptible to P. carinii, a CD4-dependent pathogen, but did not compromise host defense against C. albicans infection. Treatment of HuCD4/Tg mice with corticosteroids impaired host immune responses and decreased survival for both infections. Resistance to experimental B16 melanoma metastases was not affected by treatment with keliximab, in contrast to an increase in tumor colonization caused by anti-T cell Thy1.2 and anti-asialo GM-1 antibodies. These data suggest an immunomodulatory rather than an overt immunosuppressive activity of keliximab. This was further demonstrated by the differential effect of keliximab on type 1 and type 2 cytokine expression in splenocytes stimulated ex vivo. Keliximab caused an initial up-regulation of interleukin-2 (IL-2) and gamma interferon, followed by transient down-regulation of IL-4 and IL-10. Taken together, the effects of keliximab in HuCD4/Tg mice suggest that in addition to depleting circulating CD4(+) T lymphocytes, keliximab has the capability of modulating the function of the remaining cells without causing general immunosuppression. Therefore, keliximab therapy may be beneficial in controlling certain autoimmune diseases.

Metal nanoparticle-based electrochemical stripping potentiometric detection of DNA hybridization.

A new nanoparticle-based electrical detection of DNA hybridization, based on electrochemical stripping detection of the colloidal gold tag, is described. In this protocol, the hybridization of a target oligonucleotide to magnetic bead-linked oligonucleotide probes is followed by binding of the streptavidin-coated metal nanoparticles to the captured DNA, dissolution of the nanometer-sized gold tag, and potentiometric stripping measurements of the dissolved metal tag at single-use thick-film carbon electrodes. An advanced magnetic processing technique is used to isolate the DNA duplex and to provide low-volume mixing. The influence of relevant experimental variables, including the amounts of the gold nanoparticles and the magnetic beads, the duration of the hybridization and gold dissolution steps, and the parameters of the potentiometric stripping operation upon the hybridization signal, is examined and optimized. Transmission electron microscopy micrographs indicate that the hybridization event leads to the bridging of the gold nanoparticles to the magnetic beads. Further signal amplification, and lowering of the detection limits to the nanomolar and picomolar domains, are achieved by precipitating gold or silver, respectively, onto the colloidal gold label. The new electrochemical stripping metallogenomagnetic protocol couples the inherent signal amplification of stripping metal analysis with discrimination against nonhybridized DNA, the use of microliter sample volumes, and disposable transducers and, hence, offers great promise for decentralized genetic testing.

Gold nanoparticle-enhanced microchip capillary electrophoresis.

We describe here the use of gold nanoparticles in conjunction with chip-based capillary electrophoresis to improve the selectivities between solutes and to increase the efficiency of the separation. We coated the microchannel wall of a microfluidic device with a layer of poly(diallyldimethylammonium chloride) (PDADMAC) and then collected on it citrate-stabilized gold nanoparticles. The resolutions and the plate numbers of the solutes were doubled in the presence of the gold nanoparticles. Such selectivity improvements reflect changes in the observed mobility accrued from interactions of solutes with the particle surface. The electrochemical detection and the quantitation of the solutes were not effected by the PDADMAC and the gold nanoparticles.

Voltammetry on microfluidic chip platforms

Microfluidic chip devices are shown to be attractive platforms for performing microscale voltammetric analysis and for integrating voltammetric procedures with on-chip chemical reactions and fluid manipulations. Linear-sweep, square-wave, and adsorptive-stripping voltammograms are recorded while electrokinetically "pumping" the sample through the microchannels. The adaptation of voltammetric techniques to microfluidic chip operation requires an assessment of the effect of relevant experimental variables, particularly the high voltage used for driving the electroosmotic flow, upon the background current, potential window, and size or potential of the voltammetric signal. The exact potential window of the chip detector is dependent upon the driving voltage. Manipulation of the electroosmotic flow opens the door to hydrodynamic modulation (stopped-flow) and reversed-flow operations. The modulated analyte velocity permits compensation of the microchip voltammetric background. Reversal of the driving voltage polarity offers extended residence times in the detector compartment. Rapid square-wave voltammetry/flow injection operation allows a detection limit of 2 x 10(-12) mol (i.e., 2 pmol) of 2,4,6-trinitrotoluene (TNT) in connection with 47 nL of injected sample. The ability of integrating chemical reactions with voltammetric detection is demonstrated for adsorptive stripping measurements of trace nickel using the nickel-dimethylglyoxime model system. The voltammetric response is characterized using catechol, hydrazine, TNT, and nickel as test species. The ability to perform on-chip voltammertic protocols in advantageous over nanovial voltammetric operations that lack a liquid-handling capability. Coupling the versatility of microfluidic chips with the rich information content of voltammetry thus opens an array of future opportunities.

Microfabricated electrophoresis chips for simultaneous bioassays of glucose, uric acid, ascorbic acid, and acetaminophen.

A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.

Atiprimod (SK&F 106615), a novel macrophage targeting agent, enhances alveolar macrophage candidacidal activity and is not immunosuppressive in Candida-infected mice.

Azaspiranes are novel macrophage-targeting agents with activity in preclinical animal models of autoimmune disease and transplantation. The purpose of this work was to determine the effects of atiprimod (SK&F 106615), an azaspirane being developed for the treatment of rheumatoid arthritis, on rat pulmonary alveolar macrophage (AM) function and immunocompetance in Candida-infected mice. AM from rats treated with 20 mg/kg/day of atiprimod for 15 days demonstrated enhanced killing of Candida albicans ex vivo. Concentration-dependent increases in candidacidal activity were also observed as early as one hour after exposure in vitro in AM from untreated normal rats. Treatment of AM with atiprimod in vitro did not increase particulate-stimulated superoxide production or phagocytosis of Candida but decreased their ability to concentrate acridine orange, indicating an increase in lysosomal pH. Increased candidacidal activity was inhibited by superoxide dismutase and catalase, suggesting a role for reactive oxygen intermediates (ROI). Atiprimod also increased free radical-mediated killing of Candida in the presence of H2O2, iron and iodide in a cell-free system. These findings indicated that treatment with atiprimod increased the candidacidal activity of rat AM in a free radical-dependent manner. The data also suggested that atiprimod did not increase ROI production by AM, but rather increased the efficiency of radical-mediated killing. This increase may be caused by cyclization of atiprimod, facilitating electron transfer and peroxidation of lipid membranes. In vivo studies in Candida-infected CBA mice showed that atiprimod (10 mg/kg/day), did not compromise immune function in the infected mice and could be differentiated from prototypical immunosuppressive compounds used for treatment of autoimmune diseases.

Single-organism model of host defense against infection: a novel immunotoxicologic approach to evaluate immunomodulatory drugs.

The immunotoxicologic effects of drugs on host defense have been studied widely using various animal models of infection. Here we describe a new approach to testing host defense by using a single organism (Candida albicans) in CBA/J mice. The model is configured to test 3 effector systems via different routes of inoculation to stimulate different effector arms of the immune response. Nonspecific immunity was evaluated by C. albicans colony-forming unit (CFU) count from the spleen at 2 hr (uptake) and > or = 22 hr (clearance) following intravenous inoculation. Cell-mediated immunity was assessed by CFU count from an intramuscular injection site 6 days postinoculation. Humoral immunity was assessed by anti-Candida antibody titer, following multiple subcutaneous immunizations with C. albicans. Finally, overall immunity was evaluated following intravenous injection using survival as the endpoint. Histopathological, immunohistochemical, and electron microscopic evaluation of selected tissues revealed the involvement of the expected cell types in the different effector systems. Several immunomodulatory drugs--dexamethasone, cyclosporine, liposomal muramyltripeptide phosphatidylethanolamine, and SK&F 105685--were evaluated in the C. albicans model. Dexamethasone impaired host defense against C. albicans by suppressing all endpoints measured. Similarly, cyclosporine showed broad immunosuppressive activity, with the exception of yeast uptake from the spleen. In contrast, muramyl tripeptide-phosphatidylethanolamine enhanced all but cell-mediated immunity to C. albicans. SK&F 105685 displayed both stimulatory and inhibitory effects on immune responses to the infection. Our studies demonstrate that a single organism-based approach can be a useful method for evaluating the immunological hazards of drugs on host resistance to infection.

In vitro and in vivo effects of clinically important camptothecin analogues on multidrug-resistant cells.

The cytotoxic alkaloid camptothecin (CPT) and several of its analogues, including the clinically relevant topotecan (TPT), irinotecan (CPT-11), and 9-aminocamptothecin, were evaluated for differential cytotoxic effect and DNA damage induction in multidrug-sensitive (AuxB1) and multidrug-resistant (MDR) (CHRC5) Chinese hamster ovary cells. CPT, 10-hydroxycamptothecin, and 10,11-methylenedioxycamptothecin produced equivalent amounts of cell growth inhibition and/or DNA single-strand breakage in the two cell lines. TPT, SN-38 (the active metabolite of CPT-11), and 9-aminocamptothecin were 12-, 9-, and 10-fold, respectively, less toxic to the MDR than to the wild-type cells. These findings are consistent with differences in yields of DNA single-strand breaks produced in AuxB1 and CHRC5 cells by 2-hr incubations with the various compounds. In both assays, the resistance ratios of the topoisomerase I inhibitors were approximately one-tenth those of known MDR drugs such as vinblastine or amsacrine. Thus, cultured cells that overexpress P-glycoprotein have the potential to develop some level of cross-resistance to all three topoisomerase I inhibitors currently in the clinic. The chemical basis for cross-resistance of cultured MDR cell lines to certain CPT analogues is not yet understood, but is likely more complex than positive charge alone. TPT had a reasonable therapeutic effect on B6D2F1 female mice implanted with MDR sublines of P388 leukemia, compared with its effect on mice implanted with wild-type P388 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective and subjective evaluation of psychiatric ward redesign.

The authors describe the redesign and evaluation of a 30-bed psychiatric unit. The design approach was user oriented, employing a psychoenvironmental model that postulates an interaction between the physical environment and the psychotherapeutic milieu. Objective and subjective instruments demonstrated that environmental redesign based on this model correlated with behavioral changes in clinically desirable directions. These changes also correlated with reduced psychopathology and might have been facilitated by "social organizers," design solutions that encourage social interaction focused on ward activities.

Psychiatric disorders in the context of evoolutionary biology. An ethological model of behavioral changes associated with psychiatric disorders.

This paper presents a model designed to account for behavioral changes associated with acute psychiatric illnesses. The model is developed from concepts and data presented in previous work by the authors and others and is strongly influenced by modern evolutionary theory. The model places a strong emphasis on the structural and organizational features of behavior and on testability.

Social ethology of acute psychiatric patients. The influence of sex, hospital environment, and spatial proximity.

Naturalistic observations were used to monitor overt behavior in acutely ill psychiatric patients during hospitalization. Patients were observed on an individual basis in three different hospital locales: the inpatient wards, and the occupational and recreational therapy areas. The analysis was designed to determine which behaviors varied across locations as a function of the distance separating subjects and their nearest neighbors. Sex-related differences in behavior were also examined. Results showed: a) a modal distance of 1.0 to 1.5 meters separating individuals in all three locations; b) closer interpersonal distances between individuals in occupational therapy; c) significant differences in the frequency of selected behaviors in different functional areas; and d) closer interpersonal distances for females and a higher incidence of affiliative behavior by females toward males at close distances. Results are discussed in terms of environmental influences on behavioral responsiveness and sex differences in sociospatial behavior.

Ethological assessment of stable and labile social behaviors during acute psychiatric disorders: clinical applications.

Results are presented which illustrate the use of naturalistic observation techniques to study ongoing patterns of postural, gestural, and spacing behaviors among psychiatric patients hospitalized for acute psychiatric disorders. Behavioral frequencies are analyzed in terms of stable and labile characteristics by week of hospitalization, clinical diagnosis, and clinical outcome. Findings are interpreted from both clinical psychiatric and ethological perspectives. The discussion emphasizes the potential value of observational data gathering techniques for disorder assessment and theory development.

Social interaction and the use of space on a ward of long term psychiatric patients.

Naturalistic observations were made on 24 chronic schizophrenic patients in order to determine whether patterns of spatial behavior in a ward setting resembled those reported by Esser et al. for similar population. Patients were ranked in a social interaction hierarchy. Interactional categories included altruistic, verbal, and assertive behavior, and behavior surrounding the exchange of cigarettes. Patient space use was analyzed on a group and individual basis according to preferences for particular chairs or particular areas of the ward. Groups were formed by dividing the interactional hierarchy into thirds: top, middle, and bottom. Results showed that patients who interacted the most (i.e., top interactors) tended to sit in the areas closet to the front of the ward. Middle interactors preferred the areas furthest from the front, and bottom interactors exhibited no preference for any area. Preferences remained stable over a 1-year period. More patients in this study (100 per cent) than in Esser's study (50 per cent) met the criterion of territoriality, as defined by Esser. These findings are inconsistent with those of Esser et al. Possible sources of discrepancy are discussed.

An ethological perspective on social behavior in long stay hospitalized psychiatric patients.

Ethological methods were employed to gather normative data on social behavior in long stay male inpatients in the ward environment. Most were diagnosed schizophrenic. Social bheavior was categorized into four main types: altruistic, assertive, cigarette, and verbal. Patients (N = 24) were divided into three groups on the basis of the amount of social interaction (top, middle, and bottom thirds of the interactional scale). With the use of these divisions, results indicated: a) marked variability between thirds in the amount of social behavior exhibited; b) differences in the kinds of social behavior utlized by each third--the top was predominantly verbal whereas the middle and bottom were predominantly nonverbal; c) differences in the modes of interaction between thirds--verbal behavior characterized interactions between the top and top, and top and bottom, whereas assertive behavior characterized interactions between the top and middle, middle and middle, middle and bottom, and bottom and bottom; and d) correlative relationships between various social and nonsocial parameters, the most noteworthy including the positive relationship between head up, eyes open, and high rate of social interaction, and the positive relationships between altruistic, cigarette, and verbal behavior. Discussion focused on issues of variability between patients, stability of behaviors over time, behaviors indicative of bonding and social attentiveness, profiles of behavior characteristic of each third of the interactional scale, and the ethological constructs of dominance and attention structure.

An ethological analysis of long stay hospitalized psychiatric patients. Senders and receivers in social interaction.

Ethological methods were employed to monitor social interaction among 24 long stay male patients on a psychiatric ward. Most were diagnosed as schizophrenic. Analysis was made using four molar behavioral groupings: assertive, altruistic, cigarette, and verbal. Within each grouping social behavior was divided into two categories: sending and receiving. Patients were ranked in an interactional scale and divided into three groups on the basis of the amount of social interaction: top, middle, and bottom third. Results showed that individuals in the top third tended to be senders of social behavior, whereas individuals in the middle and bottom thirds tended to be receivers. Individuals in the middle received relatively large amounts of assertive behavior. In addition, correlative relationships indicated positive associations between send and receive verbal and send and receive cigarette. No associatiociation existed between send and receive verbal and send and receive assertive. Findings are discussed in terms of a) sending and receiving profiles characteristic of each third of the interactional scale; b) the value of dividing social behavior into components of sending and receiving; and c) the role assertive behavior plays in the social organization of hospitalized groups.

Behavioral changes in hospitalized acute schizophrenics. An ethological perspective.

Ethological techniques were employed to monitor the behavior of hospitalized patients suffering from acute schizophrenic disorders during the first 4 weeks of hospitalization. Two groups were studied: a) patients whose clinical condition markedly improved (N = 3); and b) patients whose clinical condition showed little or no improvement (N = 3). Method, observational procedures, and the taxonomy of behaviors recorded closely followed previous research of the authors. Data are analyzed in terms of behavioral diversity, the frequency of individual behaviors, and grouped behaviors. Compared to unimproved patients, improved patients showed less behavioral diversity for pathological behaviors and a reception of more social behavior. An analysis of individual behaviors reveals differences between the two groups. Issues relating to the utility of observational methodology and differences between chronic vs. acute schizophrenic populations are discussed.

An ethological analysis of manic-depressive disorder.

Hospitalized manic-depressive patients were studied using ethological techniques. Within the sample three subgroups were created: a) manic patients whose psychiatric condition improved during the course of hospitalization (N = 3); b) manic patients who showed little or no improvement (N = 3); c) depressed patients (N = 2). Time-sampling procedures were employed to monitor the occurrence of a wide variety of behaviors in various parts of the hospital. Data were analyzed in terms of behavioral diversity, frequency, constancy, and profiles of behavior. Findings included: a) distinctive patterns of behavior characteristics of manic and depressed patients: manics had higher frequencies for most behavioral categories: b) characteristic patterns of behavior for patients who improved as compared to patients who showed no improvement: manic-improved patients showed a marked decrease in diversity as hospitalization progressed. Theoretical and clinical implications are discussed in relation to the kinds of data typically generated from ethological investigations.

Preparation and characterization of two isozymes of choline acetyltransferase from squid head ganglia. II. Self-association, molecular weight determinations, and studies with inactivating antisera.

The two isozymes of choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) from head ganglia of Loligo pealei have been examined by polyacrylamide gel electrophoresis, gel chromatography, and equilibrium sedimentation in the ultracentrifuge. Inactivating antisera, prepared to both native and dithiothreitol-treated isozymes 1 and 2 of squid choline acetyltransferase, were used to demonstrate the immunologic identity of isozymes 1 and 2. Each isozyme appeared to contain two non-identical catalytically active subunits, with molecular weights of approx. 37 000 and 56 000. A staining method was developed to visualize choline acetyltransferase activity in acrylamide gels. The method is based on the formation of a precipitate of manganese ferrocyanide at sites where free coenzyme A is released. By this method, and by analysis of gel slices, it was found that each of the isozymes can form aggregates of several different sizes. The formation of immune precipitates with the aggregates showed the identity of the multiple bands of enzyme protein resolved on disc gel electrophoresis. Isozyme 1 was most active as a small aggregate, whereas isozyme 2 was most active as a large aggregate. Both chromatography on Sephadex G-200 and isoelectric focusing yielded a number of active species with molecular weights ranging from 35 000 to 300 000. In addition, we demonstrated the dissociation of enzyme protein in the presence of 1.0 - 10(-2) M dithiothreitol, the formation of multiple precipitin bands by aged enzyme, and the identity of the different isoelectric fractions of each of the isozymes.