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OBJECTIVE: To examine the effect of unemployment on natural killer cell cytotoxicity (NKCC) and, in a subsample of persons who become re-employed, to determine if, after termination of the stressor, immune values recover to levels similar to matched controls. METHODS: One hundred unemployed and 100 matched employed healthy men and women, aged 29 to 45 years, were followed for 4 months with monthly blood samples taken to measure NKCC, the ability of NK cells to kill target cells. Twenty-five participants obtained employment before the end of the study, leaving 75 unemployed (and 75 employed) participants in the main sample. For unemployed participants who obtained employment before the end of the study, subsample analyses compared NKCC levels before and after obtaining a new job. RESULTS: The persistently unemployed sample had significantly lower NKCC levels for all three effector:target ratios (100:1, p = .0004; 50:1, p = .002; and 25:1, p = .02) when compared with the matched employed sample. There were no significant gender effects. In the subsample analyses, NKCC was significantly higher after the participants became employed, compared with their unemployed period, with substantial "recovery" of immune function (44%-72%) compared with values from the steadily employed group. CONCLUSIONS: Chronic stress is associated with persistent NKCC impairment. When the chronic stressor is terminated, however, the immune cell functional capacity quickly begins to recover. We believe this is the first study in humans to document immune function recovery after the definable end of a chronic stressor.

Natural killer (NK) cells are a class of lymphocytes important in immune resistance to viral and other serious diseases. The cytotoxic function, or ‘killing activity’ of NK cells has become important in studies of the effects of stress and other psychosocial factors on physical health. Unfortunately, research on NK cell function has been plagued by discrepancies in the methods of interpreting NK cytotoxicity data. We briefly review some of the variations in measuring NK cell activity and present a new model for interpreting these results, introducing maximal target cell lysis (A) and the slope of the cytolytic curve (k) as parameters that attempt to make full use of the information and the statistical power in NK cell cytotoxicity data. Examples of these interpretation methods are presented using NK cytotoxicity data from a group of metastatic breast cancer patients. This approach will be useful in applications of NK cell measurement in psychoneuroimmunology research. Copyright © 2006 John Wiley & Sons, Ltd.

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.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.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.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.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.

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.

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.

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.

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.

To evaluate the immunogenic potential of gadolinium-based magnetic resonance imaging (MRI) contrast agents, Sprague-Dawley rats were sensitized with gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) dimeglumine and with Gd-DTPA covalently linked to either human serum albumin, dextran, or polylysine. IgG antibodies directed against Gd-DTPA were detected in immune sera by an enzyme-linked immunosorbent assay (ELISA), and were confirmed by competitive inhibition of antibody binding using free Gd-DTPA dimeglumine. Antiserum induced by immunization with human serum albumin-(Gd-DTPA) was characterized by a monophasic competition curve with 50% inhibition (IC50) = 5.5 x 10(-4) M when Gd-DTPA dimeglumine was used as both the well-coating and the displacing agent in a competition ELISA. Antiserum induced by Gd-DTPA dimeglumine alone was characterized by a biphasic competition curve with IC50 = 6.5 x 10(-7) M and 7.9 x 10(-4) M. Antisera obtained after exposure to either dextran-(Gd-DTPA) or polylysine-(Gd-DTPA) were of insufficient titer for characterization. The detection of antibodies specific for Gd-DTPA suggests in vivo protein binding with formation of hapten-carrier conjugates. This hypothesis is supported by increased relaxivity values observed when Gd-DTPA dimeglumine is incubated in serum rather than in water. Gd-DTPA dimeglumine and albumin-(Gd-DTPA) are immunogenic in rats under idealized experimental conditions. Additional studies will be necessary to determine the potential for immunologic response in humans to gadolinium chelates under conditions of exposure inherent in clinical use.

Nitrogen dioxide (NO2) is a common oxidant air pollutant. Animal studies have suggested that NO2 exposure causes a decrease in the numbers of some splenic lymphocyte subtypes and impairs lymphocyte-dependent immune responses. To investigate whether ambient levels of NO2 alter circulating and bronchoalveolar lavage fluid (BALF) human lymphocytes, we studied five healthy nonsmoking adult volunteers. In each subject, blood and bronchoalveolar lavage fluid was obtained and then, more than 2 weeks later, volunteers were exposured to 0.60 ppm NO2 for 2 hr with intermittent light to moderate exercise on 4 separate days within a 6-day period. We measured standard tests of pulmonary function (airway resistance, thoracic gas volume, maximal expiratory flow) and had the subjects rate the severity of respiratory symptoms before and after each NO2 exposure. Circulating and BALF lymphocytes were labeled with fluorochrome-conjugated monoclonal antibodies to human lymphocyte antigens and a flow cytometer was used to count lymphocyte subtypes. Neither any single day's exposure nor all four exposures caused a change in symptoms or in the results of tests of pulmonary function. The total number of circulating lymphocytes obtained after NO2 exposure was slightly greater than at baseline (1792 +/- 544 vs 1598 +/- 549 cells/mm3 at baseline; P = not significant) but the proportions of lymphocyte subtypes did not differ. In the BALF obtained after NO2 exposure and in the baseline state, the total number of lymphocytes and the percentages of T cells (CD 3), B cells (CD 20), T cytotoxic-suppressor cells (CD 8), T helper-inducer cells (CD 4), and large granular lymphocytes (CD 57) also did not differ after NO2 exposure. A slightly but significantly greater proportion of natural killer cells (CD 16) was found in the BALF obtained after NO2 exposure (7.2 +/- 3.1 vs 4.2 +/- 2.4% of total lymphocytes). We conclude that repeated exposures of healthy nonsmoking adults to 0.60 ppm NO2 are not associated with clinically significant symptoms, changes in airway caliber, or alterations in circulating and BALF lymphocyte subtypes. We suggest that brief, daily exposures to NO2 at levels higher than those achieved in urban atmosphere are unlikely to provoke acute respiratory impairment in healthy, nonsmoking adults.

To date microsphere-based assays in flow cytometry have focused on the detection of antibody or antigen. Most studies have been research based to evaluate the performance of the technique relative to conventional techniques. However, there have not been any carefully controlled studies of the sensitivity and specificity, as well as analytic sensitivity of the FMIA technique. As such, it is difficult to document advantages of this tecnique clearly. The data suggest that FMIA is considerably more sensitive than conventional techniques, and the ability to analyze for multiple analytes in one sample dilution is attractive. This ability to simultaneously analyze for multiple samples is primarily dependent upon the size difference as sensed by FALS of the microspheres. However, it is also possible to use microspheres of the same size but that differ in either fluorescence or RALS signal. If microspheres of the same size are used but one fluoresces red and the signal in the assay uses a green fluorochrome, then the two microspheres can be separated by their red fluorescence. Using this technique, one can increase the number of microspheres that can be used in an assay. It is also possible to use microspheres of the same size but with different abilities to scatter the incident light at right angles. The use of these microspheres is then similar to the nonfluorescent versus red microspheres. By the judicious combination of microsphere size, it is possible to easily differentiate eight different microspheres. With the addition of a fluorescebt dye and/or differences in right-angle light-scatter capabilities, the number of different microspheres that can be used simultaneously becomes quite large. In practice, the number of microspheres that can be differentiated is no doubt greater than the number of analytes that need to be assayed in one assay. Although the apparent increase in sensitivity and the ability to simultaneously detect and quantitate numerous analytes are important attributes of FMIA, there are drawbacks to this method. Although the FMIA lends itself well to one-step no-wash procedures, when wash steps are necessary they are time-consuming and ineffecient. Most wash steps in FMIA use centrifugation of the microspheres to remove them from the reagent. There is a significant loss of microspheres in these wash steps, which are time-consuming. There are studies ussing vacuum filtration of the suspension to separate the microspheres from the reagents. A number of different groups are pursuing an automated or semiautomated method for the efficient washing and reagent delivery system for FMIA. Commercial systems are being developed that may allow for the easier handling of these reagents. Numerous groups are investigating the use of microspheres and flow cytometry primarily in immunoassay development. The procedure has the advantages of the simultaneous yet discrete analysis of multiple analytes and the inherent increase in sensitivity using fluorescence over other signals. There will no doubt be wider applications

Sera from human immunodeficiency virus (HIV)-infected study subjects and controls were analyzed by enzyme-linked immunosorbent assay using 10 synthetic peptides to identify epitopes of HIV envelope glycoproteins (ENVgp) that were recognized by antibodies. Two epitopes of HIV ENVgp, ENVP466 (amino acids 466-481) and ENVP497 (amino acids 497-509), were recognized by antibodies in the sera of most HIV-infected individuals. The frequency of individuals with detectable serum antibodies to these two epitopes was not associated with the stage of HIV disease. Purified antibodies to ENV497 had only very weak neutralizing activity against infectious HIV. These data suggest that a particular dominant type of antibody response to HIV's ENVgp has minimal protective effects. These and other studies to identify and stimulate immune responses to selected epitopes of HIV antigens may be useful in the design of vaccines to prevent or treat HIV infections.

Peripheral blood monocytes (PBM) can selectively lyse malignant or virus-infected cells. We investigated the effects of target cell infection with HIV-1 on PBM cytolytic function. Cytokine-activated PBM lysed uninfected, HSV-1-infected or vaccinia virus-infected tumor cells, but did not lyse the same cell lines when infected with the human immunodeficiency virus type 1 (HIV-1). HIV did not impair PBM viability, and actinomycin D (Act D) pretreatment of HIV-infected target cells restored their susceptibility to PBM-mediated lysis. Either antibody to CD4 (Leu3a) or a recombinant vaccinia virus that induces expression of the HIV envelope protein, also inhibited target cell lysis by PBM. These studies indicate that CD4 can function as a mediator of PBM cytolytic function, and that target cell expression of the HIV-1 envelope protein may inhibit monocyte-mediated antitumor responses.

The Transfusion Safety Study (TSS) is a multicenter, cooperative investigation of factors that may determine the occurrence and modify the expression of transusion-transmitted infections. A flow cytometry laboratory was established in each of the six participating centers in order to avoid alterations in cell phenotypes which may be caused by shipping delays, temperature changes, and handling. As a consequence, in order to assure compatibility of results, stringent standardization, quality control, and proficiency testing procedures were developed. This paper documents (i) the effect of time from phlebotomy to specimen staining and then to analysis for the antibodies used in the study; (ii) the effects of variations in light scatter cursor location for certain antibodies; (iii) a quality control program and data management and analysis system, each specifically designed for the study; and (iv) presents extensive data on age- and sex-related reference (normal) ranges for the several individual and paired monoclonal antibodies used in the study. Problems encountered, including obtaining reliable absolute lymphocyte counts, interference by nucleated erythrocytes, and sources of variability in results, are discussed. This study is meant to serve as a reference for future TSS publications.

While prognostic factors in early stage breast cancer are well documented, few studies have examined predictors of the rate of metastatic progression. The purpose of this study was to examine cytotoxic T-cell lymphocyte (CTL) count as a marker of disease status in women with metastatic breast cancer. This study examined CTL subset counts as predictors of subsequent survival in 113 women with metastatic or recurrent breast cancer. Samples were measured by flow cytometry using monoclonal antibodies for cell surface antigens for percentages and absolute numbers of CTLs (CD3/CD8), total lymphocytes, T cells (CD3), helper T cells (CD3/CD4), and total white blood cell (TWC) count. Higher CTL counts emerged as a significant predictor of longer survival up to 7 years later (Wald = 7.40, p = 0.007; Cox regression model). The relationship of higher CTL count with enhanced survival was independent of the effects of medical treatment. CTLs were significantly associated with TWC count ( r = 0.42, p < 0.001). However, TWC count was not associated with subsequent survival time. Higher CTL count was associated with Karnofsky performance status ( r = 0.27, p = 0.004). However, after adjustment for the Karnofsky score, the CTL count/survival relationship remained significant (Wald = 4.33, p = 0.038). In conclusion, there is a robust relationship between CTL count and survival that is independent of the effects of medical treatments, TWC count, and Karnofsky performance status. Moreover, a reduced CTL count may be a mediator or marker of more rapid disease progression in metastatic breast cancer. �

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.

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.

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.

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.

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.

HGP-30-KLH vaccine in alum at doses of 10, 25, 50, and 100 micrograms/kg administered intramuscularly at weeks 0, 4, and 10 appear well-tolerated clinically. Local pain at the injection site, appears to be the main clinical toxicity. Laboratory parameters are not affected by administration of the vaccine candidate except for perhaps mild urinalysis abnormalities at the highest dose. This vaccine candidate has no apparent immunotoxicity and does not appear to affect lymphocyte populations or T-cell functional studies. Low levels and transient antibodies develop in a minority of subjects early after immunization with the vaccine candidate. These responses were observed in the lowest dose range. Higher doses, and longer follow-up will be needed to confirm this observation. T-cell proliferative responses to KLH and KLH-HGP-30 are consistent and may not be dose dependent, but the proliferative responses are variable and more data need to be accumulated. Preliminary, there appears to be an HGP-30-induced CTL response of HGP-30-coated EBV-transformed autologous B cell lines. This study was approved under an IND for the California Department of Health Services' Food and Drug Branch. They have provided excellent support and regulatory guidelines for this project. Future work will extend and confirm these initial observations.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.

To assess the incidence of human immunodeficiency virus type 1(HIV-1) transmission by antibody (anti-HIV-1)-positive blood components, and to determine the immunologic and clinical course in HIV-1-infected recipients.We retrospectively tested approximately 200,000 donor blood component specimens stored in late 1984 and 1985 for anti-HIV-1, and we contacted recipients of positive specimens to determine their serologic status. They were compared with both recipients of HIV-1-negative transfusions and healthy (untransfused) controls. Subjects were seen at 3- to 6-month intervals for up to 4 years for clinical and immunologic evaluations.Of 133 recipients, 9 had other possible exposures. Excluding these cases, 111 of 124 (89.5%) were anti-HIV-1-positive (95% CI, 84.1% to 94.5%). The recipient's sex, age, underlying condition, and type of component did not influence infection rates. The cumulative risk for developing the acquired immunodeficiency syndrome (AIDS) within 38 months after transfusion was 13% (CI, 7.5% to 21.6%). At 36 +/- 3 months after the index transfusion, seropositive recipients had lower counts of CD2+CDw26+, CD4+, CD4+CD29+, and CD4+CD45RA+subsets and more CD8+I2+ lymphocytes than did recipients of anti-HIV-1-negative transfusions. The CD4+ and CD2+CDw26+subsets changed the most rapidly. The absolute CD8+ count remained normal.Transfusion of anti-HIV-1-positive blood infected 90% of recipients. The rate of progression to AIDS within the first 38 months after infection was similar to that reported for homosexual men and hemophiliacs. Although most lymphocyte subset counts changed over time, CD8+ counts were constant.

Bacillary angiomatosis and bacillary peliosis have been described in patients with human immunodeficiency virus (HIV) infection and drug-induced immunosuppression. Patients with these vascular lesi...

Detailed immunologic studies were performed on nine patients with the Wiskott-Aldrich syndrome and selected family members. Striking clinical features included occurrence of bloody diarrhea, eczema, and thrombocytopenia during the first year of life in all patients. Recurrent infections were common, but there was notable lack of infections of the urinary system. Results of immunologic studies confirm previous reports of normal IgG levels, high IgA levels, and reduced IgM in most patients. Response to carbohydrate antigens measured by isohemagglutinin levels was diminished in some patients and normal in others. Reduction in delayed skin reactivity to a panel of skin test antigens was confirmed in all patients, and studies in vitro indicated diminished radioactive thymidine incorporation and lack of migration inhibitory factor production to these antigens as well. Response to optimal doses of phytohemagglutinin was normal in some patients and diminished in others, and in one patient was higher after 6 days than after 3 days of culture. Complement levels were normal or elevated. In some patients, there was evidence for a defect in function of polymorphonuclear leukocytes as indicated by failure of these cells to adhere to nylon fiber columns, although results of nitroblue tetrazolium and bacterial killing tests were normal. Diminished monocyte IgG receptors were found in four of eight patients studied. Abnormalities of immunoglobulin levels were found in five of eight family members of patients. Other parameters, including hematologic studies, platelet counts, evaluation of cellular immunity, and monocyte IgG receptors, were normal in the family members.

Production of the human immunodeficiency virus (HIV) by cultured peripheral blood mononuclear cells (PMC) from many seropositive individuals is inhibited by the presence of CD8+ T lymphocytes. In a study of 10 subjects, high levels of virus replication could be detected in cultures of purified CD4+ cells, but not in unseparated PMC. Addition of highly purified, autologous CD8+ cells to the enriched CD4+ cells resulted in a dose-dependent inhibition of HIV growth and revealed that for some individuals, even low numbers of CD8+ cells can prevent replication of the virus. The data also indicated that culturing enriched CD4+ cells could greatly enhance detection of infectious virus in blood specimens and demonstrated that the CD4+ molecule is expressed on infected T cells isolated directly from the peripheral blood.

Lymphocyte reactivity was measured in human mixed lymphocyte (MLC) and mitogen-stimulated cultures in the presence of various steroids. When present during the entire culture period at concentrations between 1 and 20 µg/ml progesterone, estradiol, and testosterone blocked 3H-thymidine (3H-TdR) incorporation. Estriol and 16 α-hydroxy progesterone were without suppressive effects. The kinetics of steroid mediated suppression of thymidine incorporation and DNA synthesis was investigated at various times during lymphocyte activation. When progesterone was present only at the beginning of culture, subsequent DNA content, but not thymidine incorporation was suppressed. However, if added late during the activation process, progesterone reduced thymidine incorporation independent of cellular DNA content. If present during the entire culture period both were suppressed. Cortisol, but not progesterone irreversibly inhibited lymphocyte thymidine incorporation if added during the first 12 hr of culture. A reversible blockade of lymphocyte thymidine uptake could be observed with progesterone even during the final hours of mitogen or MLC cultures. Thus, effects of steroid hormones on cellular DNA synthesis and thymidine incorporation appear to be independent processes. Cell fractionation experiments demonstrated that progesterone blocked the uptake of 3H-TdR into the lymphocyte nucleoside pool thereby reducing its availability for incorporation into DNA. This study is consistent with the hypothesis that lymphocyte proliferation at the maternal fetal-interface during pregnancy may be under specific regulation by progesterone. The relatively high local concentrations of the hormone produced by trophoblast are immunosuppressive whereas the substantially lower peripheral steroid levels are entirely inadequate to suppress systemic immunity. We suggest that at least one important role of progesterone in maintaining pregnancy is its regulation of cellular immunity in the maternal-fetal bed.

By using the cell-mediated lympholysis (CML) test, it was found that lymphoid cells from pregnant BALB/c females (gravid by C57BL/6 males) were not directly cytotoxic for 51Cr-labeled target cells posessing the same haplotype as the paternal parent. During various stages of gestation and degrees of parity, splenic lymphocytes from interstrain (BALB/c × C57BL/6) pregnant mice were equivalent to those from virgin BALB/c females in their in vitro proliferative responses to paternal strain cells (C57BL/6) in the one-way mixed lymphocyte reaction (MLR) and also in their ability to develop in vitro into cytotoxic effector cells toward paternal strain alloantigens. Suppressor activity on the part of splenocytes from pregnant mice could not be detected in the MLR and CML assay. Heat-inactivated serum from allogeneically pregnant BALB/c mice, when compared to serum from virgin or syngeneically pregnant females, regularly depressed both proliferative and effector phases of the MLR between related parental strain cells. The degree of inhibition was related to the final concentration of pregnant serum in culture with 1 to 3% serum producing greater than 75% inhibition. The inhibitory serum was not cytotoxic by using a sensitive 51Cr release assay, and the suppressive effect was associated with histocompatibility differences between the BALB/c and C57BL/6 mating combination. Thus although pregnancy across strong H-2 differences acts as a poor in vivo inducer of the effector component of cellular immunity, the in vitro proliferative functions of spleen cells from pregnant mice are intrinsically normal, but may be modulated by factors present in pregnancy serum.

Simultaneous dual immunofluorescence and flow cytometry was used to study sixteen lymphocyte phenotypes in 209 men including: healthy homosexuals, lymphadenopathy patients (LAN), and AIDS patients. Significant differences between the distribution of lymphocytes in healthy homosexuals and healthy heterosexuals were decreased percentages of helper/inducer T cells (Leu 3), increased cytotoxic/suppressor T cells (Leu 2), and consequently a decreased Leu 3/Leu 2 ratio. The increased Leu 2 cells were identified as functionally cytotoxic subset Leu 2+ 15- phenotype rather than suppressor cells which are Leu 15+. Leu 2 and Leu 3 bearing cells exhibited an excess of membrane-bound immunoglobulins which were easily elutable at 37 degrees C. An increased percentage of an HLA-DR framework determinant bearing T cells were also detected. Within the NK cell family, Leu 7 cells were moderately increased and the functionally unidentified Leu 2+ 7+ population was strikingly elevated. LAN or AIDS patients were compared to healthy homosexual controls. Lower percentages of Leu 3 cells and higher percentages of Leu 2 cells were evident in LAN patients. These subsets were similar in LAN and AIDS patients. The increase in Leu 2+ cells was due to the Leu 2+ 15- cytotoxic subset. Fewer T cells had immunoglobulin in LAN and AIDS. A definite increase in Leu 2+ DR+ cells but not Leu 3+ DR+ cells occurred in AIDS compared to LAN or healthy controls. NK cell changes already present in healthy homosexuals persisted in LAN and AIDS patients. No differences in the distribution of B cells was detected in any intergroup comparisons. Changes in monocytes or pan-T cells were relatively insensitive measures of immunologic alterations among any of the groups. These results indicate many of the changes in lymphocyte subsets seen in AIDS and LAN subjects are already present in a carefully screened population of healthy homosexuals in San Francisco. Many of the changes in Leu 2 and NK family of cells suggest a possible adaptive response to viral or neoplastic challenge. Whether these interesting phenotypic alterations relate to functional changes in response to such challenge of the identified subsets waits further investigation.

Progesterone and glucocorticoids share important anti-inflammatory and immunosuppressive properties. Both hormones have potent anti-proliferative effects in MLR, mitogen activation and cytotoxic T-cell generation. We investigated the cellular target of this in vitro anti-proliferative activity by comparing the effects of progesterone and cortisol on lymphocyte-monocyte interaction in concanavalin (Con A) induced human T-cell activation. Three different in vitro systems for assessing monocyte dependent T-cell activation by Con A were used: (1) limiting concentration of monocyte, (2) preincubation of isolated populations of monocytes and T cells with steroids and (3) role of steroid on action of Interleukin-1 (IL-1) activity. Monocytes separated from human peripheral blood leukocytes by flotation gradients and adherence to plastic were cultured at concentrations of 0.5-10% with constant numbers of isolated autologous T cells. Inhibition of Con A activation in cortisol (0.1-10 micrograms/ml) treated cultures was inversely proportional to percent monocytes, whereas in progesterone (2.0-20 micrograms/ml) treated cultures, inhibition was independent of monocyte concentration. Separated monocytes preincubated with progesterone and cultured with fresh T cells supported normal (108 +/- 7% control) levels of activation, but progesterone treated T cells and fresh monocytes responded at about 60% control levels. Similar experiments with cortisol (1 or 10 micrograms/ml) revealed significantly reduced responses when either cell population was preincubated with steroid. IL-1 induced by LPS stimulation of monocytes was blocked in its ability to stimulate Con A induced T cell proliferation with either steroid present during the assay of IL-1. These data provide additional support for local immunosuppression by steroids in the placenta during pregnancy. They suggest that progesterone selectively blocks T cell activation by a direct effect on T cells, whereas cortisol interferes with both monocytes and T cells.

A rapid, sensitive indirect immunofluorescence assay has been developed for detection of antibodies to the acquired immune deficiency syndrome (AIDS)-associated retrovirus (ARV). The human T-cell line HUT-78 was chronically infected with ARV-2 and used to detect antibodies to virus-specific cytoplasmic antigens. Because the helper T-cell marker Leu-3 is substantially reduced in this cell line after ARV infection, it appears to be an important receptor for virus infection. Nearly all patients with AIDS and most cases with related conditions showed antibodies against ARV. Some healthy individuals in risk groups for developing AIDS also had antibodies to the agent. In contrast, no antibodies to the virus were found in any individuals outside the risk groups for developing AIDS or with diseases other than those associated with AIDS. The titers of antibodies to ARV and to Epstein-Barr virus varied independently from each other. The level of anti-ARV antibodies in a patient's serum was found to reflect the severity of the disease; it was lower in individuals with more severe manifestations. Taken together, these data support the role of ARV in AIDS and its related disorders.

A reproducible method was developed to determine the ability of chlamydial antigens to stimulate lymphocytes from volunteers. In tests repeated 4 to 14 times, the cells from a given volunteer gave a relatively narrow range of responses, but there were great differences in the mean response of different volunteers. In the entire group of 52 volunteers, lymphocyte stimulation was significantly associated with the presence of antibody, but in a given individual results of one test did not aid in predicting the results of the other. A majority of persons with either antichlamydial antibody or elevated lymphocyte stimulation, or both, did not have a history of signs or symptoms within a spectrum of chlamydial diseases. This may reflect the great frequency of asymptomatic infection with these organisms. The lymphocytes of some individuals were stimulated to a significantly greater degree by antigens of one chlamydial species (Chlamydia trachomatis or C. psittaci) than by the other. These and other cell-mediated reactions in human chlamydial infections, and their possible medical significance, are under continued study.

The role of cyclic AMP in immune responses remains controversial. Although cyclic AMP and compounds which elevate cyclic AMP levels have been shown to enhance immune responses, several recent reports have shown that the addition of cyclic AMP will depress PHA-induced blastogenesis. The data reported here involve the use of much wider dose ranges of PHA and cyclic AMP than reported heretofore. When supraoptimal doses of PHA are used, the addition of cyclic AMP, or drugs which raise intracellular cyclic AMP levels, results in an enhancement of DNA and RNA synthesis; this effect we have termed cyclic AMP-mediated high dose recovery. Measurements of intracellular cyclic AMP levels following exposure to PHA-MR69 and/or cyclic AMP reveal that elevated cyclic AMP levels occur throughout the cell culture period. This demonstrates that elevated cyclic AMP levels may alter macromolecular synthetic patterns at points in time well after the initial activating event.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.

The three year actuarial progression rate to the acquired immune deficiency syndrome (AIDS) in a cohort of men in San Francisco who were seropositive for the human immunodeficiency virus (HIV) was 22%. An additional 26 (19%) developed AIDS related conditions. Beta 2 Microglobulin concentration, packed cell volume, HIV p24 antigenaemia, and the proportion and number of T4 lymphocytes each independently predicted progression to AIDS. Beta 2 Microglobulin was the most powerful predictor. The 111 subjects tested who were normal by all predictors (40%) had a three year progression of 7%, and the 68 subjects who were abnormal by two or more predictors (24%) had a progression rate of 57%. Two thirds of all men who progressed to AIDS were in the last group. The median T4 lymphocyte count in subjects who did not progress to AIDS fell from 626 x 10(6) to 327 x 10(6)/l. HIV p24 antigenaemia developed in 7% of the subjects per year. The proportion who were abnormal by two or more predictive variables rose to 41%. At three years an estimated two thirds of the seropositive subjects showed clinical AIDS, an AIDS related condition, or laboratory results that were highly predictive of AIDS. It is concluded from the observed rates and the distribution of predictive variables at three years that half of the men who were seropositive for HIV will develop AIDS by six years after the start of the study, and three quarters will develop AIDS or an AIDS related condition.The 3-year actuarial progression rate to acquired immunodeficiency syndrome (AIDS) in a cohort of 288 men in San Francisco, California, who were seropositive for the human immunodeficiency virus (HIV) was 22%. An additional 26 (19%) developed AIDS related conditions. Beta 2 Microglobulin concentration, packed cell volume, HIV p24 antigenaemia, and the proportion and number of T4 lymphocytes each independently predicated progression to AIDS. Beta 2 Microglobulin was the most powerful predictor. The 111 subjects tested who were normal by all predictors(40%) had a 3-year progression rate 7%, and the 68 subjects who were abnormal by 2 or more, predictors (24%) had a progression rate of 57%. 2/3 of all men who progressed to AIDS were in the last group. The median T4 lymphocyte count in subjects who did not progress to AIDS fell from 6.26 trillion to 3.27 trillion per liter. HIV p24 antigenaemia developed in 7% of the subjects per year. The proportion who were abnormal by 2 or more predictive variables rose to 41%. At 3 years an estimated 2/3 of the seropositive subjects showed clinical AIDS, an AIDS related condition, or laboratory results that were highly predictive of AIDS. It is concluded that 1/2 of the men who were seropositive for HIV will develop AIDS by 6 years after the start of the study, and 3/4 will develop AIDS or an AIDS related condition.

During embryogenesis differentiation of cells originally derived from the single-celled zygote results in the emergence of an organism with highly complex humoral and cellular immune systems. In some species, particularly the mouse and the chicken, detailed descriptive and experimental analysis of the elements and interrelationships of the complex process of immunologic differentiation have been possible. Such studies have given rise to the original concept of a two-component immune system, one for humoral and the other for cellular immune reactions, each with its separate central organ, e.g., the bursa of Fabricius and the thymus in the chicken, respectively. The study of the ontogeny of immunity in man is still in its infancy and currently limited largely because of legal and moral restraints. However, through the use of tissue culture and other sensitive in vitro techniques, human immunologic embryology has recently emerged as a provocative and scientifically exciting endeavor. The following summarizes salient aspects of our current knowledge of the development of cellular and humoral immunity in the human fetus.

The effects of progesterone on murine suppressor cell function generated in allogeneic MLCs were investigated. BALB/c splenic lymphocytes stimulated in vitro with C3H/He cells significantly suppressed the proliferative response of BALB/c lymphocytes in a secondary MLC. This suppression was highly specific for the sensitizing alloantigens since the suppressor cells had no effect on the proliferative response of BALB/c lymphocytes to third-party alloantigens. In addition, BALB/c lymphocytes stimulated with syngeneic cells were observed to nonspecifically suppress the MLC response to a lesser extent. One to 10 micrograms/ml progesterone added at initiation to suppressor cell generating cultures diminished the ability of both alloantigen specific and nonspecific suppressor cell populations to suppress the proliferative response of homologous lymphocytes to alloantigens. Experiments with pyrilamine, an antihistamine, which blocks cytotoxic T lymphocyte (CTL) generation, suggests that progesterone has a direct inhibitory effect on suppressor cell function independent of its ability to block CTL induction. The effects of progesterone on suppressor cells were not due to shifts in peak response time in MLC or induction of radiosensitive cells in progesterone-treated cultures. Estradiol at doses between 5 and 10 micrograms/ml, and cortisol at dose of 1 microgram/ml, also significantly inhibited suppressor cell function. These results suggest that the steroid hormone milieu within the placenta may effect the activity of allogeneic or nonspecific suppressor cell activity.

Cell-mediated immune response and humoral response to chlamydial antigens were investigated in guinea pigs infected with the agent of guinea pig inclusion conjunctivitis (GPIC). Pronounced cell-mediated immune response to the homologous antigen, as well as to two other chlamydial antigens, 6BC (Chlamydia psittaci) and LB-1 (C. trachomatis), occurred in all infected animals. Cell-mediated immune response to GPIC, and to a lesser extent to 6BC and LB-1 as well, was enhanced with time after infection even without the re-inoculation of the infectious agent. Extensive cross-reactions among the three chlamydial antigens during the cell-mediated immune response appeared to be due to shared species-specific and group-reactive antigens. Serum antibody response was pronounced and uniform to GPIC; it was less marked to 6BC and LB-1, with fewer cross-reactions than seen in tests for cell-mediated immunity.

Immune complexes are formed in vivo when antigens are bound by antibodies as a normal consequence of immune reactions. These complexes are usually cleared from the circulation by Fc receptor-bearing cells that are widely distributed in the lymphoid system. In many autoimmune, infectious, and neoplastic diseases, however, immune complexes accumulate and cause significant pathological changes, such as inflammation, immunosuppression, and thrombocytopenia. Infection with the human immunodeficiency virus (HIV) predisposes the patient to other infectious agents, autoimmune phenomena, and neoplastic disease [1]. Because of these pathological changes, elevated levels of circulating immune complexes might be expected during HIV infection. In fact, numerous studies of patients infected with HIV have documented increases in immune complexes [1-4] and defects in the clearance of these complexes [5]. A few studies have also demonstrated the presence of HIV antigen or intact virus in these immune complexes [6-8]. To obtain a complete profile of serological responses to HIV, we measured circulating HIV antigen, antibody to p24, and HIV-containing immune complexes in selected groups of HIV-infected individuals. To make these measurements, we have developed a fluorescent, solid-phase, Clq-binding assay to detect antigen-specific immune complexes present during HIV infection. This assay has very high analytic sensitivity and uses a microsphere technique that we have previously described [9]. We report here the sensitivity and specificity of this assay and an analysis of the distribution of antigen-specific immune complexes in relation to levels of free HIV antigen in serum and antibody to p24 core.

Current knowledge of human T cell ontogeny is reviewed in terms of appearance of T cells in central and peripheral lymphoid organs, maturation of T cell markers, and development of immune functions. Extrapolation of growth curves derived from cell counts from fetal thymus, spleen, and bone marrow indicates the appearance of lymphocytes at 3.5 weeks gestation. Erosette-forming cells are present in thyrnus at 11 weeks and in peripheral organs 15 to 16 weeks gestation. β-2-Microglobulin is associated with all lymphoid cells by 13 weeks gestation. Lymphocyte responses to the T cell mitogen phytohemagglutinin (PHA) are first detected in thymus at 10 weeks and in spleen and blood 3 to 4 weeks later. Allogeneic responses in mixed lymphocyte reactions develop at about 7.5 weeks in fetal liver and later in thymus and peripheral organs. Lymphocytotoxicity for xenogeneic cells is a property of bone marrow cells and not thymocytes. Several aspects of development of a suppressor T cell in human neonates is discussed and related to similar findings in the mouse. These studies indicate a relatively high degree of maturation of human T cells during fetal life.

Exposure of human peripheral blood lymphocytes to increased doses of PHA (0–2000 μg/ml PHA) leads to a decrease in thymidine incorporation. The addition of exogenous cyclic AMP (10 −3 –10 −4 M ) to lymphocyte cultures exposed to such high levels of PHA results in a recovery of the ability to incorporate both thymidine and uridine. Using this system as a model for the study of cyclic nucleotide metabolism, experiments have demonstrated a differential sensitivity to changes in cyclic nucleotide levels among lymphocyte subpopulations exposed to PHA, concanavalin A, or pokeweed mitogen. Further evidence for differential sensitivity is also obtained using lymphocyte populations separated on the basis of rosette formation. Measurement of intracellular cAMP and cGMP levels in lymphocyte cultures exposed to PHA, concanavalin A, or pokeweed mitogen suggests that while cGMP is the mitogenic signal, cAMP may control deactivation, thereby exerting an overide effect on cGMP mediated events. In addition, metabolic studies suggest that exogenous cAMP may influence PHA-induced transformation up to 67 hr after culture initiation and that the effects of Ca 2+ ions and cyclic nucleotides are interrelated in the activation of lymphocytes by PHA.

Human fetal lymphoid cells from thymus, spleen, blood, liver, and bone marrow of 22 fetuses (5–19 weeks of fetal age) were studied for their ability to respond to phytohemagglutinin and to adult allogeneic lymphocytes by the mixed lymphocyte reaction. The earliest detectable response was that by hepatic cells in the mixed lymphocyte reaction at 7.5 weeks of fetal age. Phytohemagglutinin reactivity was initially seen in the thymus at 10 weeks, in blood at 14.5 weeks, and in spleen at 13 weeks. Mixed lymphocyte reaction reactivity was first detected in the thymus at 12.5 weeks; it appeared somewhat later in peripheral organs. Few significant responses to phytohemagglutinin were detected in bone marrow or hepatic cells, and virtually no response to allogeneic cells was found in bone marrow. All lymphoid cells studied showed stimulatory ability in the mixed lymphocyte reaction. However, spleen, blood, and marrow cells produced higher stimulation of allogeneic cells than did thymic or hepatic cells.

The possibility of using tetanus toxoid as a carrier to enhance immune responses to spermatozoa was investigated in mice. The specificity of the antiglobulin test used to detect tetanous toxoid bound to mouse sperm was established. The greatest humoral antibody response to spermatozoa covalently coupled with tetanus toxoid was observed in mice previously immunized to tetanus toxoid. The antibody titers achieved were significantly (p lesser than .05) higher than those observed in animals preimmunized with tetanus toxoid and injected with sperm mixed with tetanus toxoid and of those raised in nonpreimmunized mice injected with tetanus toxoid-coupled spermatozoa. It is concluded that immunization with tetanus toxoid-coupled spermatozoa can elicit increased humoral responses in mice primed with carrier.

A consistent modification in B lymphocyte activation has been observed 1 month after infection of C3H/HeJ mice with scrapie. The mitogenic response to lipopolysaccharide of splenocyte cultures from experimental mice was reduced 30 to 60% as compared to controls. This reduction in mitogen responsiveness was transient but coincided with the onset of detectable splenomegaly and with the reported recovery of maximum yield of infectious scrapie agent in the spleen. The DNA synthetic response to lipopolysaccharide stimulation of splenocytes from scrapie-infected C3H/HeJ mice was depressed relative to controls only between 20 and 40 days after intracerebral inoculation. At all other times, experimental and control responses were identical. Scrapieassociated decreases in mitogenesis were found whether the spleen cell cultures contained splenocytes from individual mice, splenocytes pooled from several mice, or gradient-purified mononuclear cells. The responses of C3H/HeJ splenocyte cultures to phytohemagglutinin or concanavalin A stimulation were unaffected by scrapie infection.

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2 k ) or C57BL/6 (H-2 b ) mice hyperimmunized with 3.5-day-old Balb/c (H-2 d ) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2 d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2 b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.

The Transfusion Safety Study (TSS) is a cooperative investigation of factors that determine the occurrence of and modify the expression of transfusion-transmitted infections. A major component of its data is derived from lymphocyte immunophenotyping using a large panel of monoclonal antibodies and two-color flow cytometric analysis. The multicenter longitudinal character of TSS necessitates a uniformity of instrumentation, reagents, and protocols, as well as an intensive quality control program. The baseline assessment of a cohort of males 10 years of age and over with congenital clotting disorders (CCD) exemplifies the approach and some of the flow cytometry results. A comparison of anti-HIV-1 positive and negative subjects shows that more of the loss of T4+ cells was attributable to a decrease in the T4+4B4+ subset than the T4+2H4+ subset. There was an overall increase in CD8 cells, with a significant increase in the I2+T8+ and Leu7+T8+ cells, but a fall in NKH.1+T8+ cells. Monocytes, MO2+I2+ cells, increased. In CCD patients under the age of 10, both anti-HIV-1 positive and negative, there were absolute elevations in immunocytes, including CD4. There was also a distinctly different distribution of CD4 subsets. The suppressor inducer subset, 2H4+T4+, was increased relative to the helper inducer subset, 4B4+T4+, in the younger subjects.

Female mice of proven fertility were immunized with pure lactate dehydrogenase X (LDH-X) and adjuvant or with adjuvant alone and their fertility determined. Assessments of humoral and cellular immunity were performed at the termination of the experiment. A significant decrease in litter size occurred with the highest serum concentrations of anti-LDH-X antibody.

A group of 10 leukocyte and lymphocyte subsets were measured by simultaneous dual immunofluorescence and flow cytometry in a group of homosexual men from the San Francisco General cohort. Absolute numbers and percentages of lymphocytes were determined in 30 individuals who progressed to AIDS and 29 who did not over a 44-month period at annual intervals. At entry into the study, all subjects were asymptomatic, HIV seropositive, and had multiple changes in lymphocyte subsets compared to HIV-negative controls. In progressors, large changes occurred from the first visit to the last visit before progression in both absolute numbers and percentages of CD4 cells. The percentage of HLA-DR-bearing CD8 cells also increased. We utilized a proportional hazards model to assign a predictive value for progression to AIDS to lymphocyte subsets in both univariate and multivariate tests. Increased DR-positive CD8 cells, decreased CD4 cells, and increased CD8-positive, Leu 7-positive cells independently predicted progression to AIDS at P less than 0.006 (relative hazard 5.8-4.0). In a multivariate model, the most useful tests were either increased numbers or percentages of DR-positive CD8 cells. These data suggest parsimonious approaches to following HIV-positive individuals and further support the possibility of autoreactive T cells in the pathogenesis of HIV-associated diseases.