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Nonpharmacological treatments for insomnia in adolescents with depression are lacking. This qualitative study was a thematic analysis of the unique characteristics of and preferences for an insomnia treatment in a group of depressed adolescents. Fourteen adolescents with insomnia (age range = 14-19, mean = 17, SD ± 1.7; 71% female) and depression completed a 90-min focus group. Information was elicited about sleep disruptions, insomnia's impact on mood, and preferences for insomnia treatments. Themes included poor daytime functioning affecting sleep, lack of benefit from sleep medication, and bedtime rumination. Most identified sleep diaries as a barrier to treatment regardless of mode of delivery. Participants also preferred an in-person therapy. Insomnia therapy in adolescents should consider the unique characteristics of depression. Larger studies are warranted.

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Our cross-sectional study suggested that cytotoxic T lymphocyte (CTL) responses have a protective effect in squamous intraepithelial lesion (SIL) development. More CTL responses in women with human papillomavirus type 16 (HPV 16) infection without SILs than with SILs were detected. In the current longitudinal study, the role of CTL in clearing HPV 16 infection in women without SILs was investigated. Women with HPV 16 infection (n=51) were enrolled, along with HPV 16-negative control women (n=3). Twenty-two (55%) of 40 women who cleared HPV 16 infection had an E6 CTL response at least once, compared with none of 9 women who had HPV 16 persistence (P=.003). Such a difference was not demonstrated for E7; 25 (63%) of 40 women who cleared HPV 16 infection responded, versus 5 (56%) of 9 women with persistence (P=.720). It appears that lack of response to E6 is important in the persistence of HPV 16 infection.

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OBJECTIVE: Oral hairy leukoplakia (OHL) is a white lesion of the tongue that is caused by Epstein-Barr virus (EBV) and occurs mainly in people infected with human immunodeficiency virus (HIV). The aim of this study was to determine whether the presence of OHL reflects the absence of EBV-specific cytotoxic T lymphocyte (CTL) activity. SUBJECTS AND METHODS: EBV-specific CTL responses were measured in HIV-positive homosexual men with OHL, HIV-positive homosexual men without OHL, and HIV-negative homosexual men. Also, the phenotypes of cells responsible for EBV-specific responses were studied. RESULTS: Eighty percent (8/10) of HIV-positive subjects with OHL, 52% (12/23) of HIV-positive subjects without OHL, and 83% (15/18) HIV-negative subjects had a positive anti-EBV CTL response (P = 0.004, Kruskal-Wallis test). Two HIV-positive subjects showed a greater anti-EBV CTL response after developing OHL than before the appearance of OHL Additional experiments showed that CD8-positive T cells and CD4-positive T cells were responsible for the EBV-specific CTL responses. CONCLUSION: Our data show more EBV-specific CTL activities in HIV-positive individuals with OHL than in HIV-positive individuals without OHL. Whether the presence of EBV-specific CTL contributes to resolution of OHL remains to be clarified.

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OBJECTIVES: The authors discuss the usability of an automated tool that supports entry, by clinical experts, of the knowledge necessary for forming high-level concepts and patterns from raw time-oriented clinical data. DESIGN: Based on their previous work on the RESUME system for forming high-level concepts from raw time-oriented clinical data, the authors designed a graphical knowledge acquisition (KA) tool that acquires the knowledge required by RESUME. This tool was designed using Protégé, a general framework and set of tools for the construction of knowledge-based systems. The usability of the KA tool was evaluated by three expert physicians and three knowledge engineers in three domains-the monitoring of children's growth, the care of patients with diabetes, and protocol-based care in oncology and in experimental therapy for AIDS. The study evaluated the usability of the KA tool for the entry of previously elicited knowledge. MEASUREMENTS: The authors recorded the time required to understand the methodology and the KA tool and to enter the knowledge; they examined the subjects' qualitative comments; and they compared the output abstractions with benchmark abstractions computed from the same data and a version of the same knowledge entered manually by RESUME experts. RESULTS: Understanding RESUME required 6 to 20 hours (median, 15 to 20 hours); learning to use the KA tool required 2 to 6 hours (median, 3 to 4 hours). Entry times for physicians varied by domain-2 to 20 hours for growth monitoring (median, 3 hours), 6 and 12 hours for diabetes care, and 5 to 60 hours for protocol-based care (median, 10 hours). An increase in speed of up to 25 times (median, 3 times) was demonstrated for all participants when the KA process was repeated. On their first attempt at using the tool to enter the knowledge, the knowledge engineers recorded entry times similar to those of the expert physicians' second attempt at entering the same knowledge. In all cases RESUME, using knowledge entered by means of the KA tool, generated abstractions that were almost identical to those generated using the same knowledge entered manually. CONCLUSION: The authors demonstrate that the KA tool is usable and effective for expert physicians and knowledge engineers to enter clinical temporal-abstraction knowledge and that the resulting knowledge bases are as valid as those produced by manual entry.

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The host's immune response to cervical human papillomavirus (HPV) infection is poorly understood. In a longitudinal cohort of women with cervical HPV infections, defined by PCR-based HPV DNA testing, we used exfoliated cervical cells and reverse transcription-PCR to examine the cervical mucosal mRNA expression of cytokines involved in regulating cell-mediated immunity. We identified seven HPV-positive subjects who were found to have cleared their HPV infections 4 months later. In all seven, a T-helper type 1 (Th1) cytokine pattern (expression of gamma interferon and absence of interleukin-4) preceded clearance. The more variable cytokine patterns seen in HPV-negative subjects suggest that the Th1 pattern in the women with subsequent clearance was a response to the HPV infection. This contention is supported by additional cross-sectional data showing a Th1 pattern in a majority of HPV-positive women. This study establishes a feasible means for assessing local cytokine expression in the cervical milieu and demonstrates that a Th1 cytokine response is associated with subsequent clearance of cervical HPV infection.

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Cytotoxic T-lymphocyte (CTL) responses to E6 and E7 were previously shown to be more commonly detectable in human papillomavirus type 16 (HPV-16)-positive women without squamous intraepithelial neoplasia (SIL) than in HPV-16-positive women with SIL (M. Nakagawa, D. P. Stites, S. Farhat, J. R. Sisler, B. Moss, F. Kong, A. B. Moscicki, and J. M. Palefsky, J. Infect. Dis. 175:927-931, 1997). The objective of this study was to characterize the phenotype(s) of the effector cell population responsible for HPV-16 E6- and E7-specific cytotoxic responses. Peripheral blood mononuclear cells were stimulated with HPV-16 E6 or E7 fusion protein. Cells from an autologous B-lymphoblastoid cell line, infected with vaccinia virus expressing E6 or E7, served as target cells. The effector cells were characterized by using natural-killer-cell removal, antibody blocking, and T-cell subset separation. Our results suggest that both CD4 and CD8 T lymphocytes contribute to HPV-16 E6- and E7-specific CTL responses although their relative contributions vary from individual to individual. On the other hand, natural killer cells in the effector cell population contribute to background activities but not to HPV-specific responses in this assay system.

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This study investigated whether acute and persistent stressors and life change events were followed by changes in immune status, and whether dispositional optimism moderated these relationships. Thirty-nine healthy women ages 18-45 were followed prospectively for 3 months, with weekly assessment of acute and persistent stressors and monthly assessment of life events and immune parameters (NK cell cytotoxicity, and CD4 and CD8 T cell subsets). The study used an autoregressive linear model to examine how weekly appraised acute and persistent stress levels were associated with immune parameters in the subsequent week. Analyses revealed that the immune outcomes were differentially affected by acute and persistent stressors. Further, the association between acute stress and subsequent immune parameters was buffered by an optimistic perspective. However, when stress persisted at high levels, optimists showed more subsequent immune decrements than pessimists.

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Research has provided growing evidence of links between the social environment and cancer progression. Indeed, social support in the form of marriage, frequent daily contact with others, and the presence of a confidant may all have protective value against cancer progression. Furthermore, retrospective data suggest that major stressful life events are more prevalent in patients with relapse or malignancy, and thus may contribute to cancer morbidity. Initial studies of the effects of psychosocial intervention with cancer patients have provided some promising results. In three randomized prospective trials, protective effects of psychosocial interventions on cancer progression have been confirmed, while one matching and one randomized study showed no survival effect after psychosocial treatment. Though more research is clearly needed in this area, this body of evidence suggests that psychosocial factors have potentially powerful modulating effects on the course of disease. Here we review evidence of one possible mechanism whereby psychosocial factors may influence disease-resistance capabilities: the neuroimmune connection. Suppressive effects of stress on immune function are well documented, and these effects have been shown to be modulated by social support. Thus, it is reasonable to hypothesize that supportive social relationships may buffer the effects of cancer-related stress on immunity, and thereby facilitate the recovery of immune mechanisms that may be important for cancer resistance. Data addressing this hypothesis are reviewed.

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Cytotoxic T lymphocyte (CTL) responses to the human papillomavirus (HPV) type 16 E6 and E7 proteins were measured in 20 women with known HPV and cervical disease status. CTL assays were performed after stimulation with E6 or E7 fusion proteins using autologous B lymphoblastoid cells infected with vaccinia viruses expressing E6 or E7. CTL responses to E6 and E7 were detected in 6 (75%) of 8 and 5 (56%) of 9 HPV-16-positive women without cervical intraepithelial neoplasia (CIN), respectively. Responses to E6 or E7 were each detected in only 2 (29%) of 7 HPV-16-positive women with CIN. Responses to both antigens were found in 63% of women without CIN and 14% of those with CIN. CTL responses to E6 or E7 are more commonly detectable in HPV-16-positive women without CIN than in HPV-16-positive women with CIN, suggesting that CTL response may play a role in disease protection.

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Performances of anti-nuclear antibody testing by immunofluorescence assay (ANA-IFA) and enzyme immunoassay (ANA-EIA) were compared in relation to patient diagnosis. A total of 467 patient serum samples were tested by ANA-IFA (Kallestad; Sanofi) and ANA-EIA (RADIAS; Bio-Rad), and their age, sex, diagnosis, disease status, and medications were obtained through chart review. Reference ranges were established by testing 98 healthy blood donor samples. Eighty-six samples came from patients with diffuse connective tissue diseases, including systemic lupus erythematosus, discoid lupus erythematosus, or drug-induced lupus (n = 71); systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia), or Raynaud's syndrome (n = 8); Sjögren's syndrome (n = 5); mixed connective tissue disease (n = 5); and polymyositis or dermatomyositis (n = 3). The sensitivity, specificity, positive predictive value, and negative predictive value for ANA-IFA were 87.2, 48.0, 29.1, and 93.9%, respectively, for the reference range of < 1:160. For ANA-EIA, they were 90.7, 60.2, 35.8, and 96.4%, respectively, for the reference range of < 0.9. ANA-EIA offers equivalent sensitivity and higher specificity compared to ANA-IFA.

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The incidence of human papillomavirus (HPV)-related cervical intraepithelial neoplasia (CIN) and cervical cancer is increased with immunodeficiency, but the role of immune response, including cell-mediated immunity, in disease prevention is not well understood. In this study, T-cell proliferative responses to six synthetic peptides with predicted immunogenic determinants from the HPV-16 E4, E6, E7, and L1 open reading frames were analyzed in 22 sexually active women with new-onset CIN and 65 sexually active women without cervical disease, characterized by cytology, colposcopy, and HPV testing. T-cell proliferative responses were demonstrated to all six HPV-16 peptides. Although not statistically significant, rates of reactivity to E6 (24-45) were higher among sexually active women without disease (26%) than among women with current CIN (7%), as was the overall number of peptides stimulating a response. Women with CIN may not respond to selected HPV antigens as well as women without disease do.

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Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a heterogeneous sample.

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Local immune function is most likely a key influence in the establishment of human papillomavirus infections and its subsequent disease. Unfortunately, little information is known about local cervical immunity, and even less is known about human papillomavirus immunoreactivity. In addition, studies of local immunoreactivity have been hampered by the technical difficulty in obtaining cervical lymphocytes. The objective of the present study was to develop a simple method for the propagation of cervical lymphocytes from biopsy-size specimens. Cervical tissue was obtained from women undergoing a hysterectomy. Cervical samples measuring approximately 3 by 5 by 2 mm were minced and divided into two portions. One portion was digested by standard digestion methods and density gradient lymphocyte separation. The sample was then immunocharacterized for CD4 and CD8 cells by flow cytometry. The other portion was minced into 1-mm3 sections, and each section was placed into a separate well with tissue culture medium and interleukin-2. Lymphocyte counts and immunophenotypic analysis were performed after 18 to 20 days in culture. After 18 to 20 days in culture, the analysis demonstrated that this method of direct lymphocyte culture from a biopsy specimen yielded approximately 1 x 10(6) to 5 x 10(6) lymphocytes. Immunophenotypic studies of the digested sample at day 0 revealed CD4-to-CD8 ratios of between 0.7:1 and 3.5:1, and at days 18 to 20 they revealed ratios of between 2.3:1 and 98:1. In summary, we developed a simple technique for propagating cervical lymphocytes from small tissue samples for the study of the local immune response. Studies are under way to optimize lymphocyte growth and to preserve CD8 populations.

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Eleven HIV-positive patients with chronic oral candidiasis were supplemented with 60 to 120 mg of beta-carotene daily for 3 to 7 months. Lymphocyte profiles were evaluated at intervals to help assess immune competence. Although there was a modest increase in some lymphocyte values at 2 months, there was a significant decrease in numbers of CD4 and CD8 cells and CD4 percentage of lymphocytes after 6 months of beta-carotene supplementation. Serum triglyceride and liver enzyme levels were not affected by the beta-carotene supplementation. No improvement was observed in the control of the oral candidiasis. Under the conditions of the study, there was no indication that daily beta-carotene supplements enhanced immune competence or was of benefit in managing oral candidiasis.

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Human immunodeficiency virus type 1 (HIV-1) gp-120 potentially plays an important role in inducing functional suppression and depletion of CD4 lymphocytes following infection with HIV. In order to further understand the mechanisms involved in HIV-induced immunosuppression, we have studied the effects of recombinant HIV-1 gp120/SF2 and anti-gp120/SF2 antibodies on T cell receptor (TCR)-mediated proliferation of peripheral blood mononuclear cells (PBMCs) and isolated lymphocyte subsets from HIV-seronegative donors. In a dose-dependent manner, gp120 significantly reduces the proliferative responses of unfractionated PBMCs and highly enriched CD4 T lymphocytes when they are polyclonally stimulated through the TCR using WT31 (anti-alpha beta Ti chains) and anti-Leu 4 (anti-CD3 epsilon) in the presence of autologous accessory cells. The addition of divalent anti-gp120/SF2 to lymphocytes previously incubated with gp120 further reduces the proliferation to the levels seen after pretreating cells with divalent anti-CD4 (anti-Leu 3a). CD8 T lymphocytes, on the other hand, show no change in TCR-mediated proliferation following preincubation with either anti-CD4 or gp120/anti-gp120. We find no evidence for significant cell death by apoptosis using methods of DNA analysis or flow cytometry and DNA-specific dyes to account for the loss of CD4 lymphocyte proliferation. Interleukin-2 restores the proliferation suppressed by gp120/anti-gp120 suggesting the induction of reversible functional anergy.

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Dehydroepiandrosterone (DHEA) is a naturally occurring adrenal steroid reported to have immunomodulatory and antiviral activity in cellular and animal models as well as modest in vitro antiretroviral activity against human immunodeficiency virus (HIV). A phase I dose-escalation study was performed to evaluate the safety and pharmacokinetics of DHEA in subjects with symptomatic HIV disease and an absolute CD4 lymphocyte count between 250 and 600 cells/microliters. Thirty-one subjects were evaluated and monitored for safety and tolerance. The oral drug was administered three times daily in doses ranging from 750 mg/day to 2,250 mg/day for 16 weeks. Some immunological and virological parameters were monitored as well. The drug was well tolerated and no dose-limiting side effects were noted. Dose proportionality was evidenced neither by the serum DHEA nor by DHEA-S time-concentration curves for the three dosing groups. However, the study cohort appeared to consist of two subpopulations with markedly different bioavailability for a given DHEA dose. No sustained improvements in CD4 counts nor decreases in serum p24 antigen or beta-2 microglobulin levels were observed. However, serum neopterin levels decreased transiently by 23-40% at week 8 compared with baseline in all dosing groups. DHEA was well tolerated by patients with mild symptomatic HIV disease; evaluation of this agent for efficacy in HIV disease would require randomized, controlled trials.

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RATIONALE AND OBJECTIVES: Gadolinium-ethoxybenzyl-DTPA (Gd-EOB-DTPA) is a recently introduced experimental magnetic resonance (MR) contrast agent for hepatic imaging. Although liver enhancement has been investigated in a number of animal models, tolerance evaluations of Gd-EOB-DTPA injection have been limited. METHODS: The authors investigated acute hepatotoxicity in an isolated perfused rat liver model, cardiovascular effects in the anesthetized rat, and potential immunogenicity of Gd-EOB-DTPA using detection of specific antibodies. RESULTS: Using perfused rat liver model, no significant deviation could be observed for functional parameters, liver enzymes, or potassium release, comparing Gd-EOB-DTPA to a control, but there was a significant choleresis (+250% bile flow). Hemodynamic effects of Gd-EOB-DTPA were observed after femoral bolus injection, but only with relatively high dosages (0.3-0.5 mmol/kg, 10-fold the likely clinical dose in humans). Experimental conditions, idealized for antibody induction, failed to cause an IgG immune response to Gd-EOB-DTPA in the intact rat. CONCLUSIONS: The results further support preliminary conclusions that Gd-EOB-DTPA is a well-tolerated MR contrast agent.

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