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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.

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.

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.

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.

The possible use of tetanus toxoid as a carrier which would heighten immune responses to spermatozoa was studied in mice. Animals previously immunized to tetanus toxoid showed the greatest humoral antibody response to spermatozoa covalently coupled with tetanus toxoid. This response was significantly greater than the response of mice preimmunized with tetanus toxoid and then injected with sperm mixed with tetanus toxoid or of nonpreimmunized mice injected with tetanus toxoid-coupled spermatozoa. These results suggest that immunization with tetanus toxoid-coupled sperm can evoke increased humoral responses in carrier primed mice.

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.

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.

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.

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.

T cells responsive to macrophages (M phi) in the autologous mixed lymphocyte reaction (AMLR) contain those cells that can be induced to proliferate by soluble antigens. Negative solution (5-bromo-2-deoxyuridine and light) of T cells activated by autologous M phi also removed those cells required for reactivity to Candida albicans and purified protein derivative. Positive selection of T cells responsive to autologous M phi yields a population that is simultaneously enriched in antigen reactivity. Some patients demonstrating cutaneous anergy and diminished in vitro blast transformation in response to soluble antigen also lack T cells responsive to the AMLR to M phi. When considered in conjunction with previously reported data, these findings indicate the AMLR occurring between T cells and M phi in the absence of soluble antigen represents self recognition occurring between antigen-reactive T cells and antigen-presenting M phi.

Five commercial assays were compared to a standardized complement fixation (CF) test for the detection of antibody to cytomegalovirus. Two hundred and thirty serum specimens were analyzed. In addition, nine pairs of acute- and convalescent-phase sera were tested by two of the commercial assays. The assays were compared as to sensitivity, specificity, and positive and negative predictive value, as well as incidence of false-positive and -negative results. Samples which did not agree in all the assays were retested and tested with an indirect fluorescent-antibody assay. Of 228 specimens, 103 (45.2%) were positive by CF. Of the 230 samples, 2 (0.9%) were inconclusive by CF and readable in the other assays. Of the 230 specimens, 97 (42.2%) were positive by an enzyme immunoassay (EIA; Litton Bionetics), 100 (43.5%) were positive by a second EIA (Abbott Laboratories), 104 (45.2%) were positive by a third EIA (M. A. Bioproducts). One hundred and eight (47.0%) were positive by indirect hemagglutination (IHA; Cetus Corporation), and 110 (47.8%) were positive by latex agglutination (LA; Hynson, Westcott and Dunning). Sensitivity and specificity were similar with all the assays (93 to 100%). The greater numbers of positive results by IHA and LA were confirmed by repeat CF testing at less than 1:8 dilution, and by indirect fluorescent-antibody assay. Acute- and convalescent-phase serum pairs showed a significant rise in antibody titer when tested by anticomplement immunofluorescence, IHA, and LA. There was good agreement among the assays, with LA having the highest sensitivity.

A fluorescence immunoassay for detection of immune complexes bound to solid-phase C1q was developed. The method was standardized by using human aggregated immunoglobulin G (IgG) to simulate immune complexes. A linear relationship existed between the concentrations of the aggregated IgG standards and the resulting fluorescent intensity. The method was found to be reproducible and capable of detecting as little as 10 micrograms of aggregated IgG per ml of heat-inactivated human serum. Antigen-antibody complexes prepared in vitro were detectable from equivalence to moderate antigen excess. Endogenous serum C1q inhibited the binding of aggregated IgG to solid-phase C1q. Pretreatment of test sera with EDTA was ineffective in eliminating this competitive effect. Heating the sera at 56 degrees C alleviated, but did not abolish, interference of endogenous C1q. Elevated levels of immune complexes were detectable in sera fro seven of nine patients wit systemic lupus erythematosus, provided the samples were heat inactivated before testing. Heparin and DNA were also found to interfere with the detection of aggregated IgG added to human serum. Assay values were falsely decreased due to competitive inhibition by these anions. Lipopolysaccharides from a variety of bacterial preparations produced no detectable interference. A comparative study was conducted on samples that had previously been tested by fluid-phase C1q-binding radioimmunoassay. The two methods were concordant in assigning normal or elevated levels of immune complexes in 70% of the samples tested. This solid-phase fluorescence immunoassay is proposed as a possible alternative to radioimmunoassay for the detection of circulating immune complexes.

A semiautomated fluorescent immunoassay system (FIAX) for detecting anti-nuclear antibodies (ANAs) in human serum was compared with conventional indirect immunofluorescence using microscope slides (Meloy Laboratories) coated with mouse fibroblasts. The FIAX system uses quantitative indirect immunofluorescence to measure the specific binding of ANA to a sampler coated with human epithelial cells. A total of 101 serum samples were examined for the presence of ANAs by employing both methods. At an initial 1:10 screening dilution, 23 samples were negative for ANAs by the slide method, whereas 21 samples were negative with the FIAX system. Using 2+, 3+, and 4+ subjective brightness, 68 (100%) samples were positive by the slide method, whereas 67 (98.5%) were positive with the FIAX system. ANA-positive samples were diluted twofold from 1:10 to 1:640, and positive titers were determined by both methods. Sixty (77%) of 78 positive samples titrated by FIAX came within +/-1 dilution of the titers determined by the slide method, and 75 (96%) of the samples fell within +/-2 dilutions. Results indicate good correlation between the FIAX system and indirect immunofluorescence for the detection of ANAs in human serum. The FIAX system has the advantage of speed, reproducibility, and the elimination of subjective microscopic assessment of ANA titers.

A sensitive assay for the simultaneous detection of anti-cytomegalovirus and anti-herpes simplex virus antibodies was developed. Two different sizes of polystyrene microspheres were coated with purified viral antigens. Human antiviral antibodies were detected with a biotin-streptavidin amplification procedure with phycoerythrin as the fluorescent label. Microsphere-associated fluorescence was quantitated with a flow cytometer. Sixteen percent of samples initially scored as seronegative for cytomegalovirus and 35% of samples initially scored as seronegative for herpes simplex virus by conventional assays were clearly found positive by the microsphere technique. This flow cytometric assay can simultaneously detect several specific antibodies at levels which are below the sensitivity of standard assays. The dynamic range of this assay is at least sixfold greater than that of enzyme immunoassays. This technique is amenable to numerous serologic assays and could greatly expand the clinical laboratory applications of flow cytometry.

Southern blot analyses revealed that cells from nearly 30% of childhood B cell precursor acute lymphoblastic leukemias (ALLs) contained more than two rearranged, nongermline bands for Ig heavy chain genes. DNA corresponding to these bands was molecularly cloned from two cases which showed three and seven rearranged bands, respectively. Nucleotide sequence analysis of the cloned DNA demonstrated that each band represented different VDJ or DJ rearrangements. While the same DJ joints were shared by several rearrangements, different DJ joints were found in the majority of rearrangements, precluding V region substitution as an explanation for the multiplicity of heavy chain rearrangements in these leukemias. Most of the V region segments involved in these rearrangements were restricted to VH region families that have been shown previously to be preferentially rearranged in human fetal B lineage cells. Sequence analysis of multiple copies of the same VDJ rearrangements from different cells revealed no somatic mutation, a mechanism responsible for detection of extra rearranged Ig DNA bands in certain other B lineage tumors. The data suggest that in some cases of ALL Ig heavy chain genes begin and continue to rearrange de novo within the neoplastic B cell precursor populations derived from an original malignant cell transformed at a stem cell stage of differentiation.

Six isolates of the human immunodeficiency virus (HIV) showed differences in their ability to productively infect glioma-derived cell lines and early-passage human brain cell cultures. Susceptibility to HIV infection correlated well with the expression of the astrocyte marker glial fibrillary acidic protein. The CD4 molecule was expressed on some, but not all, of the brain-derived cells; however, no correlation was observed between CD4 protein expression and susceptibility to virus infection. The results show that HIV can productively infect human brain cells, particularly those of glial origin, and suggest that these cell types in the brain can harbor the virus.

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.