Single-step immunosorbent preparation of F-protein from mouse liver with conservation of the allo-antigenic site, and determination of concentration in liver and serum.

A single-step immunopurification procedure is described for murine protein F, in which the T-cell-defined allo-antigenic site on the protein is fully conserved. The procedure is based on the use of a newly developed monoclonal antibody. The protein is isolated as a 42,500 mol. wt (F.1) and a 43,000 mol. wt (F.2) monomer. The content in liver, as estimated by radioimmune inhibition assay, is 0.083% and the yield is approximately one third. An assay of immunogenic activity in adoptive transfer, which detects the T-cell-defined site, provides a similar estimate of content in liver. The adoptive transfer assay yields concns of F-protein in serum of young mice of 0.5-1.2 X 10(-9)M, the lowest concn of protein known to induce complete immunological tolerance.

Flow cytometrically-sorted residual HLA-DR+T6+ Langerhans cells in topical steroid-treated human skin express normal amounts of HLA-DR and CD1a/T6 antigens and exhibit normal alloantigen-presenting capacity.

Topical corticosteroids decrease the number of HLA-DR+T6+ Langerhans cells (LCs) and the antigen-presenting capacity of epidermal cells (ECs). We have investigated the properties of residual HLA-DR+T6+ LCs in steroid-treated human skin. Flow cytometric analysis revealed that clobetasol propionate 0.05% applied twice daily for 7 d reduced the percentage of HLA-DR+T6+ LCs in EC suspensions to 46% of control (from a mean percentage +/- sem of 2.49 +/- 0.30 in control skin to 1.15 +/- 0.22 in steroid-treated skin), but did not significantly alter the relative amounts of HLA-DR and CD1a/T6 antigens per individual HLA-DR+T6+ cell. HLA-DR+T6- and HLA-DR-T6+ cells were not detected in either group. Steroid therapy significantly decreased the allostimulatory capacity of unsorted ECs. By contrast, in parallel experiments in which the same EC suspensions were greatly enriched (85% to 90%) for HLA-DR+T6+ LCs by flow cytometric sorting, the allostimulatory capacity of purified LCs from steroid-treated skin was not significantly different from control. Residual HLA-DR+T6+ LCs, which preserve their antigenic markers and alloantigen-presenting function, may be relatively unaffected because they have only recently immigrated into the epidermis, or they may represent a subgroup of steroid-resistant LCs. Alternatively, given the dose response relationship between topical steroid potency and decrease in HLA-DR+T6+ LC numbers, the apparent steroid resistance of residual HLA-DR+T6+ LCs may reflect heterogenity in the density of expression of LC steroid receptors.

Countercurrent immunoelectrophoresis on cellulose acetate.

A technique for the detection and demonstration of antigens or antibodies by means of countercurrent immunoelectrophoresis on cellulose acetate (CAM) is described. The method is simple, sensitive and rapid. There is no need for preliminary preparation. The samples are contained in wells punched in the CAM. The equipment required is commonly available in routine laboratories, and the whole procedure can be performed and the results obtained within 90 min.

Evidence that rapamycin has differential effects of IL-4 function. Multiple IL-4 signaling pathways and implications for in vivo use.

The immunosuppressive drug rapamycin, which inhibits the response of T cells to growth-promoting lymphokines, has been considered to act as a general inhibitor of cytokine action. Our investigations into the effect of rapamycin on human IL-4, a cytokine controlling B and T cell function, show this not to be the case. Unexpectedly, rapamycin actually synergized with IL-4 in both the upregulation of CD23 expression and the down-regulation of the type II (p75) TNF receptor, while in the same B cell line, rapamycin simultaneously inhibited the IL-4-dependent production of TNF alpha and beta. These results raise the possibility that multiple IL-4 signaling pathways may be responsible for the pleiotropic effects of IL-4, and have important implications for both the experimental and possible clinical in vivo use of rapamycin as a selective immunosuppressant.

Regulatory interactions among autologous T cell clones. Human bifunctional T cell clones regulate the activity of an autologous T cell clone.

In contrast to the ease of cloning and characterizing, at the molecular level, helper and cytotoxic T cells, suppressor T cells remain an enigma, and their existence as discrete entities is being increasingly challenged. Here we review evidence that CD4+ regulatory clones, capable of expressing both helper and suppressor functions, may account for much of the suppressor function. It is suggested that a single T cell clone, depending on the signals it receives from its environment, may release either helper or suppressor cytokines. Studying such clones under defined conditions (providing suppressor signals), may preclude detection of their helper capacity. Since some therapeutic approaches in various human diseases are based on the manipulation of helper and suppressor functions, the question whether committed suppressor cells exist has important practical implications in medicine.

Time-frequency visualization of alcohol withdrawal tremors.

In this paper, we propose a signal processing method of assessing the severity tremors caused by alcohol withdrawal (AW) syndrome. We have developed an iOS application to calculate the Clinical Institute Withdrawal Assessment (CIWA) score which captures iPod movements using the built-in accelerometer in order to reliably estimate the tremor severity component of the score. We report on the characteristics of AW tremor, the accuracy of electronic assessment of tremor compared to expert clinician assessment, and the potential for using signal processing assessment to differentiate factitious from real tremor in patients seen in the emergency department, as well as in nurses mimicking a tremor. Our preliminary results are based on 84 recordings from 61 subjects (49 patients, 12 nurses). In general we found a linear relationship between energy measured by the accelerometer (in the 4.4-10 Hz range) and the expert rating of tremor severity. Additionally, we demonstrate that 75% of the recordings from patients with actual AW syndrome had a mean peak frequency higher than 7 Hz whereas only 17% of the nurses' factitious tremors were above 7 Hz, suggesting that tremor above 7 Hz could be a potential discriminator of real versus factitious tremors.

Physicians' knowledge of alcohol, tobacco and folic acid in pregnancy.

OBJECTIVE: To assess: (1) physicians' knowledge and clinical confidence regarding problematic substance use in pregnancy compared to folic acid, and (2) physicians' desire for education in this area and their preferred learning modalitiestools. DESIGN: Self-administered survey. SETTING: Family Medicine Forum 2004 in Toronto, Canada. PARTICIPANTS: Physicians attending Family Medicine Forum 2004 in Toronto who provide antenatal care. MAIN OUTCOME MEASURES: Knowledge of folic acid, smoking and alcohol in pregnancy. Clinical confidence and interest in resources regarding problematic substance use in pregnancy. RESULTS: Sixty-six surveys completed. Physicians answered 92.3% of folic acid questions correctly, compared to 82.0% for nicotine and 57.1% for alcohol. Scores were higher on questions about effects of nicotine and alcohol use in pregnancy than on questions about treatment options. A perceived inability to influence clinical outcomes and a lack of professional resources regarding substance use in pregnancy were also identified. Physicians were interested in learning more about problematic substance use in pregnancy, particularly from continuing medical education events, websites and pocket cards. CONCLUSION: Participants' level of knowledge regarding substance use in pregnancy was significantly lower than their knowledge of folic acid, as was their clinical confidence. This lack of knowledge was not attributable to disinterest and clearly more educational resources are needed to address this topic.

Identifying and managing problem drinkers.

Problem drinking is far more common than severe alcohol dependence and is associated with considerable morbidity and health care costs. Whereas patients with alcohol dependence respond best to intensive treatment, one or more brief sessions of physician advice and counseling reduces alcohol consumption among problem drinkers. The two most useful tools to identify problem drinkers are the CAGE and the drinking problem question.

Opiate-dependent patients receiving methadone. How physicians should manage therapy.

Methadone treatment can reduce illicit drug use, needle sharing, and the social costs and health risks of heroin addiction. It is superior to no treatment, detoxification, or treatment programs lasting less than 3 months. For most patients, the optimal methadone dose is 50 to 120 mg daily. Supervised, random urine drug specimens should be collected at least twice weekly. Long-term counseling is essential and should include information on the risks of needle sharing and on screening for HIV and hepatitis B and C.

Studies on the immune response to fixed antigens. II. Optimal conditions for inducing and eliciting helper function by fixed antigens and the mechanism responsible for this effect.

Heavily trinitrophenylated sheep red blood cells (RBC) (TNP128SRBC) and glutaraldehyde-treated SRBC (G-SRBC) induced helper function. This helper function could accelerate anti-SRBC and anti-TNP secondary responses to subsequent challenge with TNP0.14SRBC in different strains of mouse. Preferential induction of helper function over primary antibody response was obtained with fixed antigens only. High doses of fixed RBC (4 X 10(7) or 4 x 10(8)/mouse) could induce optimal helper function and high doses of the challenger TNP0.14SRBC (4 X 10(7) or 4 x 10(8)/mouse) could optimally recall this function and stimulate secondary responses. G-SRBC was a better inducer of helper function than low doses of nonmodified SRBC. TNP128SRBC-primed mice produced a significant anti-SRBC response after subsequent challenge with TNP0.14 mouse RBC. Reverse help was suggested as one of the possible mechanisms of this response. Early helper function which developed four days after priming with TNP128SRBC or G-SRBC could be transferred to irradiated recipients with T, but not B, cells. Late immunologic memory which appeared 18 days after priming with TNP128SRBC could be transferred to irradiated recipients only with a mixture of T and B cells.

Differential effects of interleukin-10 on the expression of HLA class II and CD1 molecules induced by granulocyte/macrophage colony-stimulating factor/interleukin-4.

Interleukin (IL)-10 down-regulates HLA class II molecules, whether constitutively expressed or up-regulated by interferon-gamma or IL-4 on monocytes but not on B lymphocytes. In this study we show that IL-10 does not inhibit HLA class II expression induced by the combination granulocyte/macrophage colony-stimulating factor and IL-4 on monocytes, although it simultaneously abrogates the expression of CD1 molecules induced by the same combination of cytokines. CD1 molecules can act as element of genetic restriction for CD4- CD8- T lymphocytes, and the suppression of CD1 expression by IL-10 abolished antigen presentation to CD1-restricted CD4- CD8- T cell receptor-positive T cells. Although HLA class II expression was not down-regulated by IL-10, the antigen specific proliferative response of CD4+ T cells was nevertheless decreased. This was not caused by down-regulation of known co-stimulatory molecules such as B7.1, B7.2 and ICAM-1. IL-10 decreased the antigen specific proliferative response further by directly influencing the T lymphocytes. Our results indicate that IL-10 exerts some of its immunoregulatory functions by differential modulation of antigen presenting molecules, induced by the same combination of cytokines.

Effectiveness of physician-based interventions with problem drinkers: a review.

OBJECTIVE: To review the results of randomized controlled trials on the effectiveness of brief physician interventions with problem drinkers. DATA SOURCES: The MEDLINE and EMBASE databases were searched for articles published from 1966 and 1972 respectively, with the terms "problem/controlled/responsible/moderate/risk/drink"; "advice/drink"; "physician, nurse, general practitioner"; and "random." Forty-three articles were identified in the EMBASE search and 112 articles in the MEDLINE search. STUDY SELECTION: All trials examining the effectiveness of interventions by physicians in reducing alcohol consumption among problem drinkers attending a health-care facility were reviewed. Trials involving subjects attending an alcohol treatment clinic and those involving interventions delivered solely by nonphysicians were excluded. Eleven trials met the final selection criteria. DATA EXTRACTION: For each article, two of the authors independently assigned a score from 0 to 2 on a number of criteria for validity and generalizability. DATA SYNTHESIS: The four trials with the highest validity scores showed that men in the intervention groups reduced their weekly alcohol consumption by five to seven standard drinks more than the men in the control groups. Results for women were inconsistent. No convincing evidence of declines in alcohol-related morbidity among men or women was found. CONCLUSIONS: The trials support the use of brief interventions by physicians for patients with drinking problems. Although further studies are needed to determine their effect on morbidity and mortality, the public health impact of such interventions is potentially enormous. Further research is needed to determine which patients are best suited for brief interventions, the optimal intensity of treatment and which components of brief interventions are most effective. Research is also needed to establish which strategies are effective in inducing physicians to use brief interventions.

IL-3 in combination with IL-4, induces the expression of functional CD1 molecules on monocytes.

CD1 molecules have recently been shown to present bacterial antigens to CD4-CD8-TCR alpha beta + T lymphocytes. The expression of CD1 on monocytes can be induced by GM-CSF and is further enhanced by IL-4. GM-CSF and IL-3 receptors share a common chain and often have similar activities; however, only IL-3, but not GM-CSF, can stimulate CD4-CD8-TCR alpha beta + T lymphocytes directly. In this study we show that IL-3, in combination with IL-4, can also induce the expression of CD1 on monocytes to a level comparable to that induced by GM-CSF/IL-4. This induction of CD1 by IL-3 can be suppressed by IL-10. Furthermore, CD1-induced antigen presentation was similar whether CD1 was induced by IL-3/IL-4 or GM-CSF/IL-4. The ability of IL-3 to induce expression of CD1 molecules and to directly stimulate CD4-CD8- alpha beta + TCR T lymphocytes raises interest in the role of this cytokine in the development, differentiation and function of this T cell subset.