A comparison of microLC/electrospray ionization-MS and GC/MS for the measurement of stable isotope enrichment from a [2H2]-glucose metabolic probe in T-cell genomic DNA.

Measurement of the proliferation of lymphocytes and other high-turnover cell populations in vivo can be accomplished through the incorporation of an isotopically labeled DNA precursor into actively dividing cells and the subsequent determination of the isotope enrichment in the isolated genomic DNA from selected cell populations. Two published gas chromatography/mass spectrometry (GC/MS) methods were successfully modified by our laboratory whereby a postinjection methylation reaction, rather than silylation or acetylation, was used to form a volatile derivative of deoxyadenosine (dA). We also developed a second robust microcapillary liquid chromatography-electrospray ionization (microLC-ESI)/MS method that is faster and more sensitive than the GC/MS method and does not require sample derivatization. Following administration of [6,6-(2)H(2)]-glucose to human immunodeficiency virus-infected patients, peripheral blood was drawn; cells were obtained by lymphapheresis and fractionated. DNA was isolated from the desired cell subtypes and enzymatically hydrolyzed to the free deoxyribonucleosides. The digest was analyzed using both capillary GC/MS and microLC/ESI-MS to measure the levels of the dA and [(2)H(2)]-dA or their reaction products. Sample enrichments were calculated by comparison to standard curves prepared from dA and [(2)H(2)]-dA. The microLC/ESI-MS method required fewer cells, less sample preparation, shorter analysis times, and a single calibration curve. Overall, the microLC/ESI-MS method is superior to the GC/MS method in terms of precision and accuracy, while providing a 4-fold increase in sensitivity (from 20 pmol at 0.2% [(2)H(2)]-dA enrichment to 5 pmol at 0.1% [(2)H(2)]-dA enrichment).

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

CD4 T cell expansions are associated with increased apoptosis rates of T lymphocytes during IL-2 cycles in HIV infected patients.

OBJECTIVE AND DESIGN: In an attempt to determine the mechanisms underlying the CD4 T cell expansions in patients receiving intermittent interleukin (IL)-2, a cohort of 10 HIV infected patients were studied during a 5-day cycle of IL-2 to measure rates of apoptosis, the expression of activation markers in CD4 and CD8 T cell subsets and the serum levels of proinflammatory cytokines. All patients were receiving highly active antiretroviral therapy. METHODS: Peripheral blood mononuclear cells were tested pre- and at the completion of IL-2 treatment with annexin V/7-AAD for the measurement of apoptosis. Phenotypic analyses of T lymphocytes were performed in parallel. Serum levels of interferon (IFN)gamma, granulocyte-macrophage colony stimulating factor, IL-6 and tumor necrosis factor (TNF)alpha were tested by enzyme-linked immunosorbent assay. RESULTS: IL-2 increased the spontaneous apoptosis rates of CD4 and CD8 T lymphocytes (P = 0.003). Expression of HLA-DR, CD38 and CD95 increased on both CD4 and CD8 T lymphocytes whereas CD25 induction was observed exclusively on CD4 T cells. Significant increases of serum IL-6 and TNFalpha levels were noted in all patients whereas viral loads remained unchanged. CONCLUSION: Administration of IL-2 for 5 days in HIV infected patients leads to enhanced apoptosis of both CD4 and CD8 T cells despite an eventual increase of the CD4 T cell count. A profound activation state with induction of activation markers on T cells and high levels of TNFalpha and IL-6 accompanies the increased apoptosis during the IL-2 cycle. These data suggest that the CD4 expansions seen in the context of intermittent IL-2 therapy are the net result of increases in both cell proliferation and cell death.

Correlation of human CD56+ cell cytotoxicity and IFN-gamma production.

The use of an IFN-gamma ELISPOT assay to evaluate cellular immune responses has gained increasing popularity, especially as a surrogate measure for cytotoxic T lymphocyte (CTL) responses. We have compared the IFN-gamma ELISPOT assay and the traditional(51)Cr release assay for detection of human natural killer (NK) cell activity. The cell populations used for evaluation of these assays included freshly isolated and IL-2-activated peripheral blood mononuclear cells (PBMC). CD56-positive cells were demonstrated to be the primary source of the IFN-gamma signal when PBMC were evaluated with NK-sensitive targets in the IFN-gamma ELISPOT assay. IFN-gamma ELISPOT and(51)Cr release assays showed excellent correlation suggesting that NK activity can be reliably evaluated with methods other than the traditional(51)Cr release assays.

Impact of HIV-1 infection and highly active antiretroviral therapy on the kinetics of CD4+ and CD8+ T cell turnover in HIV-infected patients.

To evaluate the effects of HIV infection on T cell turnover, we examined levels of DNA synthesis in lymph node and peripheral blood mononuclear cell subsets by using ex vivo labeling with BrdUrd. Compared with healthy controls (n = 67), HIV-infected patients (n = 57) had significant increases in the number and fraction of dividing CD4(+) and CD8(+) T cells. Higher percentages of dividing CD4(+) and CD8(+) T cells were noted in patients with the higher viral burdens. No direct correlation was noted between rates of T cell turnover and CD4(+) T cell counts. Marked reductions in CD4(+) and CD8(+) T cell proliferation were seen in 11/11 patients 1-12 weeks after initiation of highly active antiretroviral therapy (HAART). These reductions persisted for the length of the study (16-72 weeks). Decreases in naive T cell proliferation correlated with increases in the levels of T cell receptor rearrangement excision circles. Division of CD4(+) and CD8(+) T cells increased dramatically in association with rapid increases in HIV-1 viral loads in 9/9 patients 5 weeks after termination of HAART and declined to pre-HAART-termination levels 8 weeks after reinitiation of therapy. These data are consistent with the hypothesis that HIV-1 infection induces a viral burden-related, global activation of the immune system, leading to increases in lymphocyte proliferation.

Effects of the Th1 and Th2 stimulatory cytokines interleukin-12 and interleukin-4 on human immunodeficiency virus replication.

The cytokines interleukin-12 (IL-12) and IL-4 play important roles in the development of Th1-like (type-1) and Th2-like (type-2) T-cell responses, respectively, and there is evidence that type-1/type-2 T helper imbalances are important in the pathogenesis of human immunodeficiency virus (HIV) disease. With this background, we examined the effects of these cytokines on HIV replication. Neither stimulated HIV replication in fresh peripheral blood mononuclear cells (PBMC). However, in prestimulated PBMC, IL-12, and to a greater extent, IL-4 as well as IL-2, induced production of HIV p24 antigen over 7 days of culture (no cytokine 3,900 x/divided by 1.31 [GM x/divided by SEM] pg/mL; IL-12, 34,300 x/divided by 1.39 pg/mL; IL-4, 283,000 x/divided by 1.14 pg/mL; and IL-2, 328,000 x/divided by 1.31 pg/mL). Neither IL-12- nor IL-4-induced HIV replication was attributable to induction of IL-1, IL-2, tumor necrosis factor (TNF)-alpha, or TNF-beta. Both IL-12- and IL-4-induced HIV replication was associated with selective loss of the CD4+ subset in stimulated cultures. IL-4 stimulated HIV replication in monocyte/macrophages, while IL-12 had little or no effect in these cells. Finally, HIV replication stimulated by IL-12 or IL-4 was inhibited by dideoxynucleosides. Thus, IL-12 and IL-4 enhance HIV replication and HIV-induced cell death in prestimulated PBMC. Through killing of the CD4+ T cells stimulated by these cytokines, this may result in inappropriate type-1/type-2 responses in HIV-infected patients and contribute to their Th1 immunodeficiency.

Characterization of MHC class I restricted cytotoxic T cell responses to tax in HTLV-1 infected patients with neurologic disease.

To understand the nature of the cytotoxic T cell response generated in human T lymphotropic virus type 1 (HTLV-1)-infected patients with HTLV-1-associated myelopathy/tropical spastic paraparesis, we cloned CTL from the peripheral blood and cerebrospinal fluid from patients with neurologic diseases and demonstrated the presence of HLA-A2, A3, and B14 restricted responses to the HTLV-1 p40x (tax) protein. We identified the minimal amino acid residues within the epitopes required for binding and recognition by HLA-A2- and B14-restricted CTL, identified the critical residues within the peptide sequence defining the HLA-A2-restricted response, and demonstrated that CTL can lyse T cells infected with HTLV-1. This study shows that the CTL response to HTLV-1 tax in patients with neurologic diseases is heterogenous in nature and is not confined to patients of a single HLA haplotype or to a specific region of the tax protein.

Infection of monocytes by human immunodeficiency virus type 1 blocked by inhibitors of CD4-gp120 binding, even in the presence of enhancing antibodies.

Infection of monocyte/macrophages (M/M) by a variety of viruses (including HIV-1) has been shown to be enhanced in the presence of low concentrations of antiviral antibodies. This process has been hypothesized as occurring through binding of the virus-antibody complex to Fc or complement receptors followed by endocytosis. In the current study, we explored whether such a mechanism might provide a CD4-independent route of infection by HIV-1 for any of several populations of M/M. In the absence of anti-HIV antibodies, replication of HIV-1 in M/M was blocked by viral binding inhibitors such as soluble CD4 or OKT4A mAb. Furthermore, while infection of the M/M populations by a low multiplicity of infection of HIV-1 was found to be somewhat enhanced by the presence of very low concentrations of anti-HIV antibodies, this process was also consistently inhibited by recombinant soluble CD4 and by OKT4A antibody. These results suggest that under the variety of conditions studied, CD4 binding was an essential step in the infection of M/M by HIV. Moreover, they are consistent with the notion that "enhancing" antibodies may serve to concentrate HIV onto CD4 receptors or, alternately, may act at other steps in the process of viral entry and replication.

Zidovudine in patients with human immunodeficiency virus (HIV) infection and Kaposi sarcoma. A phase II randomized, placebo-controlled trial.

STUDY OBJECTIVE: To evaluate the toxicity, effects on immune function, antitumor effects, antiretroviral effects, and pharmacokinetics of zidovudine therapy in patients with early human immunodeficiency virus (HIV) infection and Kaposi sarcoma. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: National Institutes of Health, a referral-based research institution (single site). PATIENTS: Physician-referred volunteer patients with HIV infection, Kaposi sarcoma, CD4+ lymphocyte counts greater than 0.2 x 10(9)/L, and no systemic symptoms or history of opportunistic infection. Of 41 patients enrolled, 4 had not met all entry criteria and were therefore not evaluable. INTERVENTIONS: Patients were randomized to one of four treatment groups for an initial 12-week treatment period: oral placebo (9 patients); zidovudine, 250 mg orally every 4 hours (9 patients); zidovudine, 0.5 mg/kg body weight intravenously every 4 hours (9 patients); and zidovudine, 2.5 mg/kg intravenously every 4 hours (10 patients). After at least 12 weeks of therapy at their assigned dose, patients were treated with oral zidovudine, generally 250 mg every 4 hours, with a mean 42-week follow-up. MEASUREMENTS AND MAIN RESULTS: Anemia and granulocytopenia were the major toxicities. Significant increases in platelet counts and declines in serum HIV antigen and IgG and IgM levels occurred in treated patients. Treated patients were more likely than those on placebo to clear HIV from the cerebrospinal fluid. There were no differences in tumor progression or CD4+ or CD8+ lymphocyte counts among the groups. CONCLUSIONS: Zidovudine was well tolerated and had antiretroviral activity in patients with early HIV infection and Kaposi sarcoma but it had no significant effect on the extent of Kaposi sarcoma or on immune function.

Deoxycoformycin-induced immunosuppression in patients with hairy cell leukemia.

Immune function in patients with hairy cell leukemia (HCL) was examined serially during treatment with alternating monthly cycles of recombinant interferon alpha-2a and 2'-deoxycoformycin (dCF). At presentation, most patients had normal numbers of T lymphocytes and their cells had normal proliferative responses to mitogens [phytohemagglutinin (PHA) and concanavalin A (Con A)] and alloantigens. Patients had severe monocytopenia, decreased delayed-type hypersensitivity (DTH) reactions, and decreased peripheral blood natural killer (NK) activity. Treatment caused a profound decrease in all lymphocyte subpopulations. T cells were more affected than B cells or NK cells. Numbers of CD4+ and CD8+ lymphocytes decreased to levels less than 200 cells/microliters in all patients during treatment. This decrease in T cell number was associated with a marked decrease in proliferative responsiveness to PHA, Con A, and alloantigens. These abnormalities persisted throughout the 14 months of treatment and have continued for up to 6 months beyond discontinuation of treatment. NK cell activity increased during treatment, but cycled depending on the phase of treatment; highest activities were observed after interferon (IFN)-alpha and lower levels of activity were observed after dCF. DTH responses generally did not improve during therapy. Levels of IgM, IgG, IgA, and IgD did not change during treatment, but IgE levels rose in most patients. All immunosuppressive effects were attributable to dCF since patients receiving IFN-alpha 2a alone did not exhibit these same immunosuppressive effects, and patients receiving dCF alone after IFN failure exhibited similar abnormalities. Despite this severe immunosuppression from dCF, life-threatening opportunistic infections have not been observed in our patient population. Six patients developed localized Herpes zoster infection among 21 patients who had received dCF. Pending the results of long-term follow-up, we recommend that dCF be reserved for patients who have failed splenectomy and IFN therapy.

Circulating IgA immune complexes in head and neck cancer, nasopharyngeal carcinoma, lung cancer, and colon cancer.

A double antibody enzyme-linked immunosorbent assay (ELISA) was developed to quantitate circulating immune complexed IgA (IgA IC) in human serum. The serum panel for this study consisted of normal blood donors, benign surgery (BS), head and neck cancer (HN), nasopharyngeal carcinoma (NPC), lung cancer (LC), and colon cancer (CC) patients. Immune complexes (IC) were isolated from these sera by precipitation with 3.5% polyethylene glycol (PEG), washed and then redissolved in 0.1 M phosphate-buffered saline pH 7.2. The amount of IgA IC present were then quantified using the double antibody IgA ELISA. This assay was found to be both sensitive (26.0 ng/ml) and reproducible (intra-assay coefficient of variation 4.0%). The mean IgA IC for each cancer group tested (HN = 11.38 +/- 12.54 micrograms/ml; NPC = 13.36 +/- 17.56 micrograms/ml; LC = 17.39 +/- 13.04 micrograms/ml; CC = 26.50 +/- 4.60 micrograms/ml) were significantly elevated (P = 0.001) over both the normals (5.12 +/- 4.09 micrograms/ml) and the benign surgery controls (5.92 +/- 5.04 micrograms/ml). In addition to providing a new tumor marker the presence of high levels of IgA IC in cancer patients could provide a source of tumor-specific antibody as well as antigen and provide reagents to study immune regulation in cancer patients.

A new class of synthetic biological response modifiers: the methylfurylbutyrolactones (Nafocare B).

A new class of synthetic biological response modifiers (BRMs) has been produced by combining a highly electrophilic reactant, 2-methyl-2, 5-dihydrofuran (a cyclic acetal of cis-3-acetyl acrolein), with L-ascorbic acid. The parent class of compounds can be referred to as methylfurylbutyrolactones (MFBL), previously termed Nafocare B. This parent molecule is amorphous, has a molecular weight of 252.7, and the chemical name [3,6] cyclohemiketal of 2-(5-methyl-2-furyl)-3-keto-L-butyrolactone. Two crystalline forms were produced by a reaction of the MFBL parent molecule with either succinic anhydride or succinimide, to create MFBL-SA (Nafocare B2) and MFBL-S (Nafocare B3) dimers, respectively. The structure of these compounds has been confirmed by modern methods of analytical chemistry, including x-ray crystallography. All three forms of the MFBLs showed negligible toxicity in single-dose acute LD-50s in mice. Also, the MFBLs did not demonstrate genotoxic activity at 800 mg/kg in the mouse micronucleus assay. The MFBLs are immunostimulatory in assays involving T- and B-lymphocytes, but not in immunoassays on macrophages derived from resident- or thioglycollate-elicited peritoneal exudate cells (PEC). Spleen cells from mice treated 4 days via the intraperitoneal, intravenous, or the oral routes responded significantly over controls to suboptimal stimulatory concentrations of polyclonal mitogens in the lymphocyte stimulation assay. The MFBLs were not mitogenic, since they did not increase DNA synthesis in resting spleen cells from MFBL-treated mice. Antibody production is also amplified by the MFBLs. Mice immunized with sheep erythrocytes, a T-cell-dependent antigen, and treated with MFBLs had a 200-800% increase in the numbers of antibody-producing splenic lymphocytes detected by the Jerne hemolytic plaque assay. Also, mice immunized with soluble bovine serum albumin (BSA), and treated with a MFBL, demonstrated at least a fourfold increase in IgG-specific antibodies to BSA, when compared with controls. To demonstrate effects of MFBLs on macrophages, we used the Fc receptor (FcR) surface marker and superoxide anion assays. Only at the highest in vitro dose of MFBL (16 micrograms/ml) was a significant increase in erythrocyte antibody rosette formation detected, using resident macrophages isolated from PEC. In the superoxide anion release assay neither resident- nor thioglycollate-elicited PECs, obtained from in vivo-treated mice or macrophages treated in vitro, showed increased production of superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune complexes, serum proteins, cell-mediated immunity, and immune regulation in patients with squamous cell carcinoma of the head and neck.

A collaborative study of the humoral and cellular immune status of patients with carcinoma of the Head and Neck (H&N) was conducted at the West Virginia University (WVU) hospital. In addition, blind-coded serum panels were supplied on H&N cancer patients being treated at the National Cancer Institute (NCI). Serum protein analysis of the WVU study groups revealed that at the pretreatment sampling, the alpha-1 acid glycoprotein (AGP), total complement, and IgA levels were significantly elevated. The AGP levels and total complement levels declined to normal levels in the post-treatment period, whereas the IgA levels remained elevated throughout the entire observation period. Levels of serum immune complexes (SIC) were measured in both the WVU and NCI H&N cancer populations using the polyethylene glycol (PEG) precipitation method. In both survey populations all cancer groups had significantly elevated levels of SIC when compared to any of the control populations. The SIC levels never returned to comparative normal values even in cases after successful treatment. A subpopulation of the WVU-H&N cancer study group underwent a short course of intravenous hyperalimentation prior to their treatment regimen. These patients demonstrated a transient decrease in their SIC levels as well as a concomitant increase in their in vitro cell-mediated immune (CMI) correlates. The analysis of in vitro CMI correlates of the WVU study group using both polyclonal mitogens and specific antigens demonstrated a significant depression in these parameters pretreatment and post-treatment. In addition, it was observed that the time course for elevation of selected serum proteins (i.e., IgA and SIC) correlated with concomitant drops in CMI activity. Investigations were also conducted into the effects of immune complex-rich serum fractions upon selected in vitro CMI correlates. Significant blockage of a normal donor leukocyte migration-inhibition assay was demonstrated. Also, a similar inhibition of the ability of normal human lymphocytes to form high affinity rosettes was accomplished with serum from H&N cancer patients.

Purification of haptoglobin and its effects on lymphocyte and alveolar macrophage responses.

Clinical observations made on a patient with acute hypersensitivity pneumonitis revealed that the patient's serum contained a mitogenic inhibitor and an extremely high haptoglobin (Hp) level; this led to an investigation of the role of Hp in lymphocyte function. Hp was isolated and purified from acute phase rabbit serum by a new method using DE-52 anion exchange chromatography and preparative isoelectric focusing in a range of pH 3.0-5.0. This technique produced a homogenous, biologically active product in fewer steps and in higher yields than existing techniques. Purified rabbit Hp significantly inhibited polyclonal lymphocyte mitogenic responses to PHA or Con A in a dose-response fashion. Hp also significantly inhibited B-cell mitogenesis at a high concentration (200 mg/100 ml) in response to LPS while it enhanced B-cell mitogenesis at lower concentrations (50 and 100 mg/100 ml). Purified Hp had no effect on monoclonal, antigen-specific (BSA) mitogenesis. Rabbit alveolar macrophages produced significant amounts of prostaglandin E in vitro in response to LPS but Hp had no effect on this production. However, Hp alone caused approximately the same amount of stimulation of PGE production by alveolar macrophages as did LPS alone. The ability of Hp to modulate lymphocyte as well as macrophage function seems to indicate that Hp plays a role in the moderation of inflammation. This ability to moderate inflammation, especially in the lungs, may play an important role in regulating tissue damage and disease following inhalation of inflammatory aerosols.