Force determination in lateral magnetic tweezers combined with TIRF microscopy.

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.

Terahertz emission via ultrashort-pulse excitation of magnetic metal films.

We observe terahertz emission by optical rectification of an intense 1.5-eV, 50-fs pulse in single-crystal iron thin films grown by molecular beam epitaxy. The azimuthal dependence of the emission indicates the presence of a magnetic nonlinearity and a nonmagnetic surface nonlinearity.

Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production.

Interleukin-12 (IL-12) is a key regulator of cell-mediated immunity that has therapeutic potential in cancer and infectious disease. In a previous Phase 1 dose escalation study of a single test dose of recombinant human IL-12 (rhIL-12) followed 14 days later by cycles of five consecutive daily intravenous injections every 3 weeks, we showed that a dose level up to 500 ng/kg could be administered with acceptable levels of safety. Based on these results, a Phase 2 study was conducted. In the Phase 2 study, however, administration of rhIL-12 at this same dose level resulted in severe toxicities with some patients unable to tolerate more than two successive doses. Of the 17 patients receiving rhIL-12 in the Phase 2 study, 12 patients were hospitalized and two patients died. A thorough scientific investigation to determine the cause of this unexpected toxicity failed to identify any difference in the drug products used or the patient populations enrolled in the Phase 1 and Phase 2 studies that could have accounted for the profound difference in toxicity. The focus of the investigation therefore shifted to the schedule of rhIL-12 administration. We determined that a single injection of rhIL-12 2 weeks before consecutive dosing included in the Phase 1 study, but not in the schedule of administration in the Phase 2 study, has a profound abrogating effect on IL-12-induced interferon-gamma (IFN-gamma) production and toxicity. This observation of schedule-dependent toxicity of IL-12 has been verified in mice, as well as nonhuman primates. In this regard, a single injection of IL-12 before consecutive daily dosing protected mice and cynomolgus monkeys from acute toxicity including mortality and was associated with an attenuated IFN-gamma response. Because of this unique biologic response, careful attention to the schedule of administration is required to assure safe and effective clinical development of this highly promising cytokine.

The application of in vitro models of drug metabolism and toxicity in drug discovery and drug development.

In vitro models are being used increasingly during all phases of the drug development process in concert with the more traditional in vivo toxicological and pharmacokinetic evaluations. These in vitro models may be classified empirically as either validated in vitro screens, value-added screens or 'ad-hoc' mechanistic screens. The application of these screens is discussed with respect to their level of validation, standardization, uses of human tissue, level of iteration with in vivo studies, regulatory position and utility in the drug discovery and development process. The predictability and reproducibility of these screens is discussed, as well as future trends in regard to emerging technology and its application.

Cluster analysis of adult children of alcoholics.

To determine if adult children of alcoholics (ACOA) are heterogeneous with respect to personality characteristics, 97 ACOAs took the California Psychological Inventory (CPI), and the results were analyzed through cluster analysis techniques. The resulting three-cluster solution showed that, in one subgroup (44%), all CPI scales were at or above the mean; another subgroup (40%) achieved scores at or slightly below the mean; and the third subgroup's (16%) scores were considerably below the mean. Therefore, clinical claims regarding ACOAs may only be accurate for a small proportion of ACOAs. Research on resiliency in children may explain why many ACOAs have normal personality profiles.

Chemical dependency in students with and without learning disabilities.

To determine if students with learning disabilities (LD) demonstrate a higher frequency of chemical dependency than students without learning disabilities (NLD), a total of 191 adolescents with LD (101 males and 90 females) were given the Substance Abuse Subtle Screening Inventory (SASSI). The sample consisted of 88 students with LD and 103 NLD students between the ages of 12 and 18. The SASSI is an objectively scored self-report inventory that accurately classifies adolescents as chemically dependent (CD) or not chemically dependent (NCD). A significantly higher proportion of students with LD than NLD students were classified as CD. Of the 30 students who were classified as CD, 70% were students with LD. A discriminant analysis indicated that the presence or absence of a learning disability was a better predictor of classification as CD or NCD than gender, ethnicity, age, socioeconomic status, or family composition. The implications for evaluation and educational planning for students with learning disabilities are discussed.

Respiratory and immunological responses of guinea pigs to enzyme-containing detergents: a comparison of intratracheal and inhalation modes of exposure.

Guinea pigs were exposed once a week for 10 weeks by intratracheal exposure to solutions of 3, 1, 0.3, or 0.1 micrograms of the enzyme protein, Subtilisn Carlsberg (Alcalase), in 250 micrograms of a detergent base. Other groups of guinea pigs were exposed by inhalation (6 hr per day, 4 days a week) to 1 mg/m3 of the aerosolized detergent base containing either 3.5, 1.1, 0.3, or 0.1% Alcalase protein. Evaluations of gross respiratory responses immediately following each intratracheal exposure revealed a significant dose response in respiratory symptoms measurable after the fourth exposure and continuing throughout the study. In the inhalation experiment, during Weeks 4 through 10, animals were observed to have respiratory symptoms which were dependent upon both the dose of enzyme and on total exposure to the enzyme/detergent atmosphere. For both intratracheal and inhalation routes of exposure, the initial appearance of respiratory symptoms coincided with the first appearance of measurable serum allergic antibodies specific to Alcalase. The allergic antibody levels increased with time and dosage by both routes of exposure, and the antibody titers generated by the intratracheal administration of antigen were comparable to those generated by the inhalation route of exposure. These results indicate that the intratracheal technique is appropriate for the evaluation of the respiratory allergic response to a protein.

Personality characteristics of adult children of alcoholics, other adults from dysfunctional families, and adults from nondysfunctional families.

This study used the California Psychological Inventory (CPI) to examine the personality characteristics of adult children of alcoholics. The sample was noncollege age adults (N = 174). Subjects responding to advertisements were grouped into Adult Children of Alcoholics (ACOA) (56%), Adults with Dysfunctional Family Histories (ADFH) (21%), or Adults without Identified Dysfunctional Family Histories (AIDFH) (23%). Results indicated that the ACOA group was different from the AIDFH group on nine of the 28 CPI scales, but there were no significant differences between the ACOA and ADFH groups. The validity of clinical impressions of ACOAs and the utility of considering ACOAs as a distinct field of study are discussed.

Characteristics of adult children of alcoholics.

Ninety-seven adult children of alcoholics (ACOAs), 36 adults with dysfunctional family histories, and 41 adults without identified dysfunctional family histories were compared on self-reports of 20 adult characteristics. Significant differences among the groups were found on 4 of the characteristics, with the ACOA group showing the highest frequency of occurrence on 17 of them. No differences due to gender of the alcoholic parent were found, and there was no group by gender interaction. The results suggested that the clinical characteristics attributed to ACOAs may have some empirical validity but the ACOAs seem fairly similar to adults with other types of dysfunctional family histories.

Immunosuppression and cancer: the ciclosporin case.

Experimental and clinical data relevant for the evaluation of the carcinogenic potential of the immunosuppressant ciclosporin are reviewed. Ciclosporin binds reversibly to the cytosolic receptor protein ciclophilin. Ciclophilin is likely involved in the blockade of lymphocyte activation-induced gene transcription of various growth factors, especially interleukin-2. The drug has no effect on the transcription of housekeeping genes nor does it activate any gene. Ciclosporin may inhibit tumor cell growth, notably those which are growth factor dependent. At high concentration virus-transformed cells, especially Epstein-Barr-infected B-lymphocytes, may escape the control of specific cytotoxic T-lymphocytes. Ciclosporin has no genotoxic activity, and has no DNA-binding property. In experimental studies ciclosporin did not cause cancer in the absence of an initiating event (e.g. chemical mutagen). However, by its immunosuppressive property, the drug may allow the growth of initiated tumor cells in vivo, an effect which is dose-dependent. In clinical use ciclosporin immunosuppression is associated with an increased incidence of lymphoproliferative disorders and other malignancies particularly of the skin when compared with a normal, not immunosuppressed population. Conventional immunosuppression (azathioprine, antilymphocyte globulin, prednisone) also demonstrates comparable risks to develop tumors. Lymphoproliferative lesions regress after dose reduction or cessation of treatment. Furthermore, combinations of various immunosuppressants with associated 'over-immunosuppression' may result in a higher incidence of viral infection and malignancy. In summary, chemical immunosuppression carries the intrinsic risk of tumor growth. In the case of ciclosporin this effect is dose dependent. Thus, the risk may be reduced by low dosage and by avoiding combination therapies with additional immunosuppressants.

Arsenic distribution in rabbits after Lewisite administration and treatment with British anti-Lewisite (BAL).

The standard treatment of Lewisite (dichloro(2-chlorovinyl)arsine) poisoning is by chelation with BAL (British anti-Lewisite, dimercaptopropanol). The present study investigated the effect of BAL treatment on the distribution of arsenic after Lewisite administration. Lewisite was administered subcutaneously at the LD10 and LD40 of the compound. Without BAL treatment arsenic was eliminated with a half-life in blood of between 55 and 75 hr and a blood clearance of 120 ml/hr/kg. Arsenic had a large volume of distribution of several liters per kilogram, indicating extensive distribution in tissues. The highest tissue concentrations, more than seven times blood concentrations, were found in the liver, lung, and kidneys. These organs maintained an approximately constant concentration ratio with blood during the sampling period. Concentrations in tissues with a blood-to-tissue barrier, such as the brain and the spinal cord, rose between 4 and 96 hr while blood concentrations declined more than fourfold over the same time period. BAL treatment by four equal, maximally tolerated doses over 12 hr substantially reduced arsenic concentrations in blood and tissues. For example, at 24 hr the concentrations in brain and liver (target organs for arsenic toxicity) were reduced by 65 to 89% over the range of Lewisite doses administered. The total exposure of brain and spinal cord was reduced by more than two-thirds by BAL treatment. Further, the blood clearance of arsenic was increased. BAL treatment enhanced the elimination of arsenic in two ways: by decreasing the tissue-to-blood partitioning which mobilizes arsenic into the blood stream, and by increasing the clearance of arsenic.

Factor analysis of the substance abuse attitude survey with college undergraduates.

Few standardized instruments measure attitudes and beliefs towards substance abuse. The Substance Abuse Attitude Survey, developed for measuring drug attitudes in medical education, was administered to 598 college undergraduates, and a factor analysis was performed. Three coherent and stable factors were identified, e.g., Stereotypes and Moralism, Treatment, and Permissiveness. Internal consistency and 6-wk. test-retest measures indicated moderate to high reliability factor structure. Results are discussed in terms of sample differences between this effort and a previous validation.

Pulmonary adenomas in A/J mice treated with silica.

This study was designed to test the pulmonary tumor response to intratracheally instilled silica in Strain A mice. Urethane was used as a positive control. Silica treatment was utilized to evaluate the effect of a potent fibrinogen on pulmonary adenoma formation in this unique animal model. Urethane produced an increase in pulmonary tumor response in this study in agreement with previous investigations. Also, the background incidence of adenomas was comparable to other studies. Silica treatment did not affect tumor incidence either in terms of percent of mice with adenomas or average number of adenomas per mouse.

Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review.

This review evaluates the available information on the effects of mainstream and environmental tobacco smoke on the immune system in animals and humans. The primary emphasis is on mainstream smoke since little information is available on the effects of environmental smoke. The effects of mainstream tobacco smoke on the immune system in humans and animals are similar. Animals exposed to mainstream tobacco smoke for periods of a few weeks generally exhibit a slight immunostimulation. However, subchronic and chronic exposure studies indicate that immunosuppressive changes develop. Lymphocyte proliferation in response to the mitogens PHA and LPS is decreased, suggesting compromise of cell function. Antibody production can be suppressed. Smoke-exposed animals that are challenged with metastasizing tumors or viruses have been shown to exhibit a higher incidence of tumorigenic and infectious diseases, respectively. Localized immunological changes in the lung can include reduction of bronchus-associated lymphoid tissue and immunoglobulin levels. Smoking-related changes in the peripheral immune system of humans have included elevated WBC counts, increased cytotoxic/suppressor and decreased inducer/helper T-cell numbers, slightly suppressed T-lymphocyte activity, significantly decreased natural killer cell activity, lowered circulating immunoglobin titers, except for IgE which is elevated, and increased susceptibility to infection. The effects of environmental tobacco smoke on the immune system, in contrast to mainstream tobacco smoke, have just begun to be investigated and information available in the literature, to date, is limited. Immunoreactive substances are known to be present in environmental tobacco smoke, but to date, environmental tobacco smoke has been more closely associated with irritation than sensitization. A few studies have indicated a potential for environmental smoke-induced hypersensitivity and suppression of immunoregulatory substances. In contrast, other investigators have failed to detect immunological or other biological changes associated with environmental smoke. Clearly, more research is needed to resolve these differences.

Tumorigenicity of nickel subsulfide in Strain A/J mice.

The pulmonary tumor response of Strain A mice has been reported to be a rapid and efficient predictor of carcinogenic potential for a variety of chemicals. The route of exposure has usually been by intraperitoneal injection (i.p.) of solubilized materials. We compared intratracheal (i.t.) instillation as a more representative route typical of human exposures, with i.p. injection of nickel subsulfide, a potent animal carcinogen. Animals were sacrificed either 20 weeks after the first dosing, or were held until 45 weeks after the first dosing. Urethane, a positive control, produced a significant increase in pulmonary tumor response after i.t. instillation as well as i.p. injection. For nickel subsulfide treated animals, there was no evidence of a dose-related increase in pulmonary tumor response in any i.p. or i.t. treatment group when compared with age-matched controls.

In vitro effects of fibrous and nonfibrous silicon nitride on bovine pulmonary macrophages.

Bovine pulmonary macrophages were exposed in vitro to 0.3, 0.1, 0.03, or 0.01 mg/ml of a fibrous silicon nitride, nonfibrous (milled) silicon nitride comminuted from the fibrous powder, alpha-quartz (an active control), or glass beads (an inert control). Functional evaluation of the exposed cells indicated that the fibrous silicon nitride was as cytotoxic as quartz, while the nonfibrous silicon nitride was relatively inert. To further evaluate the mechanisms of cytotoxicity, the cells were exposed to 1 mg/ml of the control and test materials and biochemical studies were performed. Quartz increased release of both the cytoplasmic enzyme, lactate dehydrogenase (LDH), and the lysosomal enzyme, acid phosphatase (AP), consistent with both cell membrane and lysosome lysis. In addition, total protein levels were significantly depressed, suggesting significant impairment of cellular synthetic processes. LDH, but not AP, values were increased with fibrous silicon nitride treatment, but not with the nonfibrous silicon nitride. In contrast to quartz, which increased LDH levels by 65%, the fibrous silicon nitride only increased LDH levels by 11%. Scanning electron micrographs further indicated that the fibrous silicon is cytotoxic and poorly tolerated by macrophages. These studies provide further evidence of morphology as a primary determinant of cytotoxicity since the milled powder test article was comminuted from the fibrous material.

Pre-service teachers use of and attitudes toward alcohol and other drugs.

Drug attitude and use assessment of 598 undergraduate students revealed attitudinal differences between anticipated occupation groups and drug use patterns that paralleled prior studies which used college student samples. Results are discussed as they pertain to the education of those planning to enter the teaching profession.

In vitro interactions between pulmonary macrophages and respirable particles.

Pulmonary alveolar macrophage cells (PAM) are an important component of the pulmonary response to particles deposited in the deep lung. To more fully characterize the interactions between macrophages and particulate materials, a correlative light and electron microscopic technique was developed that allowed light microscopic identification of individual cell viability after in vitro particle exposure, followed by scanning and transmission electron microscopic analyses of specific PAM, including surface morphology, X-ray microanalytic evaluation of particle content, and internal cellular structure. Individual cell viability, particle content, and morphologic alterations were evaluated for three particle types: Ni3S2, TiO2, and glass beads. Cell death and stages of cell disruption including bleb cluster formation, loss of surface features, formation of membrane tears and holes, and cell degranulation correlated with Ni3S2 and TiO2 content. Glass beads were not associated with cell disruption or viability reduction. Correlative microscopic techniques were essential in describing particle-dependent effects on an individual cell basis.