Diagnosis of renal allograft rejection and acute tubular necrosis by 99mTc-mononuclear leukocyte imaging.

One hundred kidney transplant recipients were evaluated on the first and fifth days after transplantation by Tc-99m mononuclear cell scintigraphy. We have developed a quantitative method to diagnose rejection and acute tubular necrosis (ATN) by comparing regions of interest drawn on allograft scintigraphs at different times after endovenous administration of the labeled cells. We suggest that the use of Tc-99m-WBC may be useful for the early diagnosis of rejection and the differential diagnosis of ATN.

Diagnosis of acute hepatitis E infection utilizing enzyme immunoassay.

Acute hepatitis E infection was diagnosed in a Pakistani immigrant admitted to the University of Illinois Hospital. Utilizing enzyme immunoassay (EIA) tests, specific IgG and IgM class antibodies to three different epitopes of hepatitis E virus (HEV) were detected 12 weeks after the onset of illness and in the early convalescent stage. Sixteen months after the onset of hepatitis, IgM anti-HEV was no longer detectable. Low levels of IgG class anti-HEV antibodies continued to be detected. We demonstrate the utility of the EIA HEV assay to diagnose prospectively acute HEV infection.

Frequency of cells positive for HIV-1 p24 antigen assessed by flow cytometry.

OBJECTIVE: Markers of HIV disease progression such as soluble p24 antigen detection and CD4 lymphocyte depletion are most useful in the later stages of HIV disease and are relatively insensitive as therapeutic monitors. Flow cytometric detection of HIV-1 replication in CD4 lymphocytes was evaluated for use as a marker in predicting disease progression earlier in the course of HIV disease. DESIGN: To determine whether the number of HIV-1-infected CD4 cells, as measured by p24 antigen detection, can be correlated with disease progression, we used flow cytometry to detect intracellular HIV-1 p24 in CD4 lymphocytes from HIV-1-seropositive subjects at all stages of HIV disease. METHODS: Mononuclear cells from HIV-1-seropositive subjects and uninfected control subjects were permeabilized and stained with anti-HIV-1 p24 monoclonal antibodies. The cells were then stained with a fluorescein isothiocyanate-conjugated goat antimurine immunoglobulin G followed by a phycoerythrin-conjugated monoclonal anti-CD4 antibody. The percentage of p24-positive CD4 lymphocytes was compared with absolute CD4 counts, soluble p24 detection and Walter Reed classification. RESULTS: CD4 lymphocyte absolute counts and the percentage of CD4 lymphocytes declined as the Walter Reed classification indicated disease progression. The mean percentage of p24 antigen-positive CD4 lymphocytes increased with disease progression. Only 30% of Walter Reed stage 6 subjects were soluble p24 antigen-positive, whereas 68% were cellular p24 antigen-positive. CONCLUSION: The percentage of p24 antigen-positive CD4 lymphocytes increased as HIV disease progressed. Flow cytometric quantitation of p24 antigen-positive CD4 cells is a useful method of monitoring in vivo HIV replication and disease progression.

Human neutrophil oxidative response and phagocytic killing of clinical and laboratory strains of Enterococcus faecalis.

Many clinical isolates of Enterococcus faecalis produce a hemolysin/bacteriocin that is plasmid mediated. Recent human epidemiologic studies and animal research suggest that this hemolysin/bacteriocin may enhance the pathogenicity of hemolysin-producing enterococci compared with non-hemolysin-producing strains. These studies determined that clinical strains that produce hemolysin/bacteriocin differed from non-hemolysin-producing clinical and laboratory strains in their ability to induce the production of reactive oxygen intermediates in human peripheral blood neutrophils and in their susceptibility to phagocytic killing in vitro. The induction of superoxide anion generation by neutrophils was demonstrated to be directly proportional to the presence of the hemolysin/bacteriocin plasmid and was transferable to a non-hemolysin-producing laboratory strain by transconjugation. The presence of the plasmid, however, did not effect killing by phagocytic cells in vitro. It is proposed that hemolysin/bacteriocin-producing strains of enterococcus may be more pathogenic due to reactive oxygen product-induced tissue injury in vitro.

Antiviral phospholipids. Anti-HIV drugs conjugated to the glycerobackbone of phospholipids.

Heteroatom fatty acid analogs of myristic acid containing oxygen or sulfur substituted for the alkyl methylene groups inhibit replication of the human immunodeficiency virus (HIV) in infected cells by acting as alternative substrates during the viral protein myristoylation event. In this class of compounds, 12-methoxydodecanoic acid is the most potent compound but is approximately 10(3)-fold less active than azidothymidine. The antiviral activity of 12-methoxydodecanoic acid can be enhanced > 40-fold by preparing L-alpha-phosphatidylethanolamine containing 12-methoxydodecanoic acid in both alkyl chains. In addition, the diacylated L-alpha-phosphatidylcholine analog containing 12-methoxydodecanoic acid in both alkyl chains (i) has a 15-fold better antiviral selectivity, (ii) is 7-fold more potent, and (iii) is 10-100-fold more synergistic with azidothymidine than 12-methoxydodecanoic acid. Because of potent synergism, the antiviral selectivity of the diacylated L-alpha-phosphatidylcholine analog is > 10(4) when coadministered with azidothymidine. Phospholipid conjugates are chiral at the C-2 carbon of the glycerol backbone and most interesting is the observation that both the D- and L-isomers of phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine have approximately equal antiviral activity. Phospholipase A2 stereospecifically hydrolyzes only the L isomer of phospholipids and similar activity for both the D- and L- phospholipid isomers suggests that phospholipase A2 is not the rate-limiting enzyme for release of the drugs in vivo.

Evaluation of a flow cytometric model for monitoring HIV antigen expression in vitro.

Using flow cytometry, monoclonal antibodies to the HIV proteins p24, gp41 and p17 were evaluated for their ability to detect HIV antigens associated with HIV-infected T cells. Mixtures containing varying ratios of HIV-infected and uninfected cells were subjected to analysis with these monoclonal antibodies. In most cases, the monoclonal antibodies identified the correct ratio of HIV-infected cells to uninfected cells in the mixtures tested. An HIV anti-p24 monoclonal antibody was selected for further studies. Flow cytometric analysis was performed on various populations of cells including uninfected, acutely infected and chronically infected cells. Based on cell population fluorescence intensity three distinct regions were identified. In the first region were cells having low level fluorescence that were considered negative for HIV antigens, a profile detected in uninfected cells, and in the majority of cells in the first days following acute HIV infection. In the second region were those cells exhibiting strong fluorescence such as chronically infected cells or acutely infected cells several days after infection. A third region was identified containing cells that were intermediate in fluorescence intensity. Cells exhibiting intermediate intensity fluorescence appeared to have low concentrations of HIV p24 antigen associated with them either through viral adsorption and uptake or through low level virus expression. These intermediate region cells appeared in the early stages following acute infection, and also when chronically infected cells and uninfected cells were permeabilized together, suggesting a 'leaching' of HIV proteins from highly infected cells to uninfected cells. This leaching type of phenomenon could present problems in determining gating parameters for positive cells since uninfected cells that have associated HIV antigens exhibit higher fluorescence intensity than uninfected cells.

Altered lymphocyte phenotypes and proliferative responses in chimpanzees infected with hepatitis C virus.

Peripheral blood mononuclear cells (MNC) from three chimpanzees infected with hepatitis C virus (HCV) and from two uninfected animals were analyzed by monoclonal antibody phenotyping using flow cytometry. Significant differences in numbers of MNC's expressing cluster designation (CD) phenotypes CD4, CD14, CD19, and CD45RA were found. Additionally, significant differences in MNC proliferation in response to mitogens were also found. This altered proliferative capacity and cellular phenotype profile may be important markers in studying the pathogenesis of chronic HCV disease.

Effect of glucocorticoids and interferon-gamma on the oxidative responses of monocytes from leprosy patients and normal donors.

Leprosy patients suffering from erythema nodosum leprosum are frequently treated with glucocorticosteroids. The role glucocorticosteroids and interferon-gamma (IFN-gamma) play in regulating the interaction of phagocytic cells with Mycobacterium leprae was examined. Monocytes from leprosy patients receiving prednisone therapy responded to lower concentrations of IFN-gamma in vitro with enhanced superoxide anion release when challenged with M. leprae or M. bovis BCG than did monocytes from healthy subjects and other leprosy patients. Although the number of patients was small and the population heterogeneous, the data suggested that prednisone could alter IFN-gamma efficacy and led to the examination of the effect of glucocorticosteroids on IFN-gamma activation of monocytes. IFN-gamma treatment following in vitro dexamethasone pretreatment of monocytes from healthy subjects resulted in a greater enhancement of superoxide anion generation than that observed with IFN-gamma treatment alone. These findings are important considerations in evaluating patient immune function because IFN-gamma is being used in a number of clinical trials with leprosy patients.

Seroprevalence of HTLV-I/II and HIV-1 infection among male intravenous drug abusers in Chicago.

We surveyed for serologic evidence of either HIV-1 or HTLV-I/II infection in 387 male veterans who entered into an inpatient drug treatment center. Serum was obtained after receiving written informed consent. Serum specimens were tested by enzyme-linked immunosorbent assay for antibody to HIV-1 and for antibody to HTLV-I/II; sera that were repeatedly reactive were then tested by Western blot (HIV-1/HTLV-I/II) and radioimmunoprecipitation assay (HTLV-I/II). Sixty-five of 387 (16.79%) patients were tested and confirmed as positive for HTLV-I/II only antibodies and 30 of the 387 (7.75%) were positive for HIV-1 only antibodies. An additional nine patients (2.32%) were seropositive for antibodies to both viruses. A statistically significant difference in the CD4/CD8 lymphocyte ratio was associated with HIV-1 seropositivity. HTLV-I/II seropositivity was strongly associated with black race, age, and duration of i.v. drug use, but not with sexual intercourse as determined by lifetime history of number of sexual partners, incidence of sexually transmitted diseases, type of drug used, or needle-sharing practices.

The effect of interleukin 4 (BSF-1) on infection of peripheral blood monocyte-derived macrophages with HIV-1.

The effects of various cytokines were examined in an in vitro model of human immunodeficiency virus type 1 (HIV-1) infection of human peripheral blood monocyte-derived macrophages (MDM). Monocytes were obtained from blood of normal donors by Ficoll/hypaque gradient centrifugation and adherence. These cells were allowed to mature in the presence of varying concentrations of cytokines. After five days in culture, cells were harvested, counted, and inoculated with S5G7, an HTLV-IIIB subclone. The cells were replated in the presence of the same concentrations of cytokines. Culture supernatants were sampled over 28 days for p24 antigen (Ag) as measured by Ag capture assay. In repeat experiments, the following observations were made: 1. MDM from some donors could be infected only in the presence of tumor necrosis factor-alpha (TNF-alpha), granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 4 (IL-4); 2. The effect of GM-CSF was variable; TNF alpha also enhanced HIV replication above controls; 3. IL-4 was the most potent enhancer of HIV-1 replication in MDM of the cytokines tested, inducing p24 Ag levels 75-230 times those seen in control cultures run simultaneously. This effect was dose dependent. Ag production was not observed until Day 14 postinfection in most experiments. Multinucleated giant cell formation was observed only in the presence of IL-4.

Effect of Mycobacterium leprae's phenolic glycolipid-I on interferon-gamma augmentation of monocyte oxidative responses.

Peripheral blood monocytes were pretreated with phenolic glycolipid-I (PGL-I), dimycocerosyl phthiocerol (DIM), or mycoside A, then cultured in the presence or absence of interferon-gamma (IFN-gamma). Their oxidative responses to Mycobacterium leprae, phorbol myristate acetate (PMA), and opsonized zymosan were evaluated. In response to M. leprae, monocytes pretreated with PGL-I released less O2- than nonlipid-treated control cells. The IFN-gamma augmentation of oxidative responses was suppressed only when in PGL-I-pretreated monocytes and only when the stimulus was M. leprae. This suggests that PGL-I, by affecting the IFN-gamma enhancement of phagocytic cell oxidative responses, aids further the intracellular survival of M. leprae.

Discrimination of HIV-2 infection from HIV-1 infection by western blot and radioimmunoprecipitation analysis.

Serum and plasma samples were collected from blood donors who were confirmed positive for antibodies to HIV-1 in the United States, and from blood donors and individuals in West Africa and Portugal who were positive for antibodies to HIV-1, HIV-2, or both. Western blots and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) radioimmunoprecipitation assays (RIPA) utilizing native HIV-1 and HIV-2 proteins were performed on these specimens to determine the ability of these procedures to discriminate between HIV-1 and HIV-2 infections. Extensive serologic cross reactivity between HIV-1 and HIV-2 p24 was found in both populations. Antibody reactivity to the envelope protein gp120 was able to discriminate 20 of 20 (100%) U.S. specimens as HIV-1 infections. In specimens from West Africa and Portugal, Western blot and RIPA were in complete agreement on 33 of 42 samples (78.6%). Among these 33 specimens, 10 were found to be reactive for antibodies to HIV-1 only, 10 were reactive to HIV-2 only, and 13 were considered to be dually reactive, having antibodies reactive with both HIV-1 gp120 and HIV-2 gp120. Nine of the 42 specimens were discordant by Western blot and RIPA classification, being dually reactive by one procedure and reactive with only one viral gp120 by the other technique. Because of the serological cross reactivities between HIV-1 and HIV-2, in certain populations it is difficult to ascertain whether an individual is infected with HIV-1, HIV-2, a new viral type, or whether the individual is infected simultaneously with multiple viruses. More specific tests such as viral isolation or molecular probes may be necessary to distinguish between infections with these viruses in certain populations.

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae.

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.

Effect of Mycobacterium tuberculosis-derived sulfolipid I on human phagocytic cells.

Experiments were performed to determine the effects of Mycobacterium tuberculosis-derived sulfolipid I on phagocytic cells. Sulfolipid I was taken up in significant amounts by human neutrophils and in lesser amounts by monocytes and lymphocytes. Superoxide (O2-) production by neutrophils was significantly increased by sulfolipid I, but the rate of production was slower than that reported previously for other stimuli. The optimal concentration of sulfolipid I for stimulation of O2- production was 27 micrograms/ml, while higher concentrations produced less. At substimulatory levels sulfolipid I caused enhancement of O2- release from neutrophils when it was subsequently stimulated by other agents. Nonadherent monocytes from most normal donors failed to produce O2- when treated with sulfolipid I; however, adherent monocytes pretreated with gamma interferon did produce O2- with sulfolipid I stimulation. Priming for an enhanced oxidative response of activated monocytes was also observed. These sulfolipid I-induced changes in phagocytic cell function may be important in altering the ability of phagocytes to respond effectively to M. tuberculosis and may also cause exaggerated inflammatory responses.

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components.

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.

Mycobacterium leprae fails to stimulate phagocytic cell superoxide anion generation.

Mycobacterium leprae is an intracellular pathogen that is ingested by and proliferates within cells of the monocyte/macrophage series. Mechanisms by which intracellular pathogens resist destruction may involve failure to elicit a phagocyte "respiratory burst" or resistance to toxic oxygen derivatives and lysosomal enzymes. We have studied the ability of M. leprae and Mycobacterium bovis BCG to stimulate the generation of superoxide anion (O2-) in vitro by human blood neutrophils and monocytes and murine peritoneal macrophages. M. leprae bacteria failed to stimulate significant O2- release except at high bacteria-to-cell ratios (greater than 50:1) whether or not they were pretreated with normal serum or serum from patients with lepromatous leprosy. Either viable or irradiated BCG; on the other hand, stimulated the three cell types to release significant amounts of O2- when challenged with as few as 10 organisms per cell. Serum pretreatment enhanced the release of O2- by the three cell types. Preincubation for 18 h with viable M. leprae did not inhibit the ability of monocytes to respond with an oxidative burst to phagocytic stimuli. The failure of M. leprae to stimulate phagocyte O2- generation may be an important factor in its pathogenicity.