A multicentre verification study of the QuantiFERON®-TB Gold Plus assay.

OBJECTIVES: The aim of this verification study was to compare the QuantiFERON®-TB Gold Plus (QFT-Plus) to the QuantiFERON®-TB Gold In Tube (QFT-GIT). The new QFT-Plus test contains an extra antigen tube which, according to the manufacturer additionally elicits a CD8+ T-cell response above the CD4+ T-cell response. We assessed the value of this tube in detecting recent latent tuberculosis infections. METHODS: Between May 2015 and December 2016, 1031 subjects underwent QFT-Plus and QFT-GIT test. Overall agreement between both tests and performance for different test indications and/or immune states was assessed. A difference of >0.6 IU/mL interferon-γ release between the two antigen tubes of the QFT-Plus assay was considered a true difference and used as estimation for CD8+ T-cell response. RESULTS: Analysis of the QuantiFERON tests resulted in an overall agreement between assays of 95%. Subjects considered to be recently exposed to tuberculosis had significantly more often a true difference in interferon-γ release compared to all other subjects (p = 0.029). CONCLUSION: Results of QFT-Plus are highly comparable to QFT-GIT. Although there is an indication that a true difference in interferon-γ release between the antigen tubes is associated with recent latent tuberculosis infection, the QFT-Plus could not be used to exclude recent exposure.

Phase II controlled trial of post-exposure immunization with recombinant gp160 versus antiretroviral therapy in asymptomatic HIV-1-infected adults. VaxSyn Protocol Team.

OBJECTIVE: To alter the natural course of HIV-1 infection by inducing or potentiating immune responses to HIV-1 envelope glycoprotein. DESIGN: Multicentre, double-blind, three-arm, placebo-controlled study. SETTING: Outpatients attending clinics in two University Hospitals. PATIENTS: Ninety-nine asymptomatic HIV-1-infected adults with CD4+ T-cell counts > 400 and < 600 x 10(6)/l and no previous antiretroviral therapy were included. INTERVENTIONS: Patients were randomly assigned to three groups treated with: (i) gp160 in alum over a 2-year period in combination with placebo for the full study duration (n = 32); (ii) gp160 in alum over a 2-year period in combination with zidovudine for the full study duration (n = 34); and (iii) alum over a 2-year period in combination with zidovudine for the full study duration (n = 33). RESULTS: Immunotherapy was well tolerated and no significant differences in disease progression were seen in the treatment groups. The majority of patients (85%) receiving gp160 showed persistent lymphoproliferative responses to the immunogen and to a different Env antigen preparation. CD4+ cell count changes in patients receiving zidovudine alone were significantly higher than those seen in patients receiving immunotherapy alone after 1 year of treatment. Zidovudine administration was associated with initial transient reduction of plasma viraemia. CONCLUSIONS: Prolonged immunization with a soluble HIV-1 subunit provided no benefit to asymptomatic HIV-1-infected patients and was inferior to zidovudine monotherapy. Furthermore, immunization with gp160 shortened the duration of the transient viral load reduction induced by zidovudine.

Spectrotype of anti-gp120 antibodies remains stable during the course of HIV disease.

Human immunodeficiency virus (HIV) infection is characterized by a progressive decline in immune functions. The behavior of B-cell clones specifically engaged in the anti-HIV response could play a relevant role in the pathogenesis of such impairment. The spectrotype observed on isoelectric focusing and reverse blotting after antigen challenge is the serum image of antigen-specific B-cell activity and may provide some insight into Ag-dependent B-cell clone recruitment. In this study, we examined the spectrotype of anti-gp120 antibodies in a group of sera from 56 HIV-infected patients, belonging to groups II, III, and IV of the Centers for Disease Control classification, as well as in a group of 31 sera from 12 patients in a 21-month follow-up evaluation (range 7-36 months). All tested sera were positive for gp120 antibodies on Western blot. In the first group of 56 HIV-infected subjects, only 19 displayed well-focused banding patterns. Among these, the spectrotype was found to be consistently oligoclonal, thus confirming clonal restriction of anti-gp120 antibodies previously described by other investigators. No correlation could be established between a particular spectrotype and phase of the disease. The follow-up evaluation in the second group of 31 sera revealed the tendency in each patient to maintain the same spectrotype throughout the course of the disease. These findings confirm clonal restriction of anti-gp120 antibodies in HIV infection and suggest that the number of B-cell clones recruited in the anti-gp120 response remains stable over the course of the disease, at least in the time range explored by us.

Kinetics of immune functions and virus replication during HIV-1 infection.

INTRODUCTION: Kinetics of human immunodeficiency virus type 1 (HIV-1) cytotoxic T lymphocyte (CTL) responses and viral load were evaluated in HIV-1 infected homosexual men who progressed to AIDS within 3-6 years after seroconversion and in long-term survivors who remained AIDS-free for > 9 years with normal CD4+ T cell counts. METHODS: CTL against four major HIV-1 gene products (i.e. Gag, reverse transcriptase (RT), Nef and Env) were expanded in vitro under limiting dilution conditions using antigen specific stimulation. CTL activity was measured in standard split-well 51Cr-release assay. Viral load was measured both as serum HIV-1 RNA levels and frequency of circulating CD4+ T cells productively infected with HIV-1. Polyclonal T cell function in vitro was determined in whole blood lymphocyte cultures, measuring lymphoproliferative responses to CD3 monoclonal antibody. RESULTS: Long-term survival was associated with either persistently high or stable low HIV-1 specific CTL responses, accompanied by preserved in vitro polyclonal T cell reactivity and low viral load. In progressors, HIV-1 specific CTL responses were initially generated with similar kinetics as compared to long-term survivors. However, with progression to AIDS antiviral CTL activity and T cell function deteriorated simultaneously, while viral load increased. CONCLUSIONS: Our results are consistent with the hypothesis that HIV-1 specific CTL are beneficial through control of viremia to the virologic set-point and contribute to maintenance of the asymptomatic phase. However, loss of HIV-1 specific immune control as part of a more general loss of T cell function is the precipitating event in AIDS pathogenesis.

Antigen-specific T-lymphocyte proliferative responses during highly active antiretroviral therapy (HAART) of HIV-1 infection.

To evaluate functional T-cell recovery during combination therapy with ritonavir, lamivudine (3TC), and zidovudine (ZDV), peripheral blood mononuclear cells (PBMC) were obtained from 4 HIV-1 infected patients (baseline values: 40 403 CD4+ T-cells/microl; 4.6-6.4 log HIV-1 RNA copies/ml) before HAART administration (week -1) and after 5, 20, and 37 weeks of treatment on average. In vitro lymphoproliferative responses (LPR) to C. albicans, tetanus toxoid, and M. tuberculosis protein purified derivative (PPD), as recall antigens (Ag), and to recombinant HIV-1 Gag-p24 and p17 were measured by 3H-Thymidine incorporation. LPR to recall Ag, almost undetectable before therapy, appeared in all four patients during HAART soon after maximal load reduction was achieved. LPR to Gag-p17, but not to p24, became also detectable in three patients, even though remaining weak. In conclusion, improved T-lymphocyte function during HAART was achieved probably mostly as a result of lower virus inhibitory factors and cytokines.

Lymphoproliferative response to HIV type 1 p24 in long-term survivors of HIV type 1 infection is predictive of persistent AIDS-free infection.

To establish immunologic correlates of progression to AIDS in long-term survivors of HIV-1 infection, HIV-1-specific T cell-mediated responses, together with T cell reactivity to recall antigens, were studied in frozen samples collected after 5 and 8 years of documented HIV-1 infection. Eight of 21 homosexual men, who remained asymptomatic and maintained CD4+ T cell numbers >400 cells/microl for 9 years of HIV-1 infection, progressed to AIDS (CDC 1993 definition) within 12.5 years of infection (late progressors, LPs). The remainders showed minimal deterioration of immune parameters (long-term nonprogressors, LTNPs). CD4+ T cell numbers and T cell function measured at years 5 and 8 of follow-up were comparable in the two groups. At both time points responses to recall antigens did not significantly differ between the two groups, although a significant decline of lymphoproliferative responses to Candida and tetanus toxoid was observed in LPs. Circulating HIV-1-specific cytotoxic T lymphocyte precursors were found in broad frequency ranges in both LPs and LTNPs and, similarly, no significant differences were found in comparing the breadth of serum neutralizing activity against heterologous HIV-1 primary isolates. In contrast, lymphoproliferative responses to p24gag, but not p17gag or gp160env, were detected only in LTNPs and were totally absent in LPs at both time points (p < 0.01). Our data suggest that the presence of circulating p24-specific CD4+ T cells may reflect effective viral control and be predictive of subsequent favorable clinical course in long-term asymptomatic individuals.

Response to hepatitis B vaccine in a cohort of Gambian children.

In a cohort of 1000 Gambian children immunized with four doses of 10 micrograms of plasma-derived hepatitis B virus vaccine, 44 subjects (4.4%) showed no response (< 10 mIU/ml; 6 subjects) or low specific antibody response (10 to 99 mIU/ml; 38 subjects) to hepatitis B surface antigen. Serologic indices, potentially correlated with low immunologic response, were investigated in sera obtained from these children and in sex-, age- and village-matched controls who showed a normal response. The presence of circulating immune complexes in similar proportion of responding and poorly responding children together with a low prevalence of rheumatoid factors suggested that polyclonal B cell activation was not correlated with the subnormal humoral response. Concentrations of serum immunoglobulin (Ig) and IgG subclasses did not differ in the two groups. Some of the African prevalent Ig allotypes were determined, but no significant differences in the two groups were found. The humoral response to hepatitis B surface antigen did not correlate with the response to tetanus toxoid.

T-lymphocyte subpopulations in insulin-dependent (type I) diabetes mellitus.

Initial reports of blood T cell subsets in insulin-dependent (type I) diabetes mellitus (IDDM) are conflicting and, consequently, difficult to relate to animal models of the disease. To minimize technical artefacts, which may have contributed to previous results, we used direct immunofluorescence on whole blood and counted 3,000 lymphocytes by flow cytometer. Forty-two IDDM patients divided in three groups of 14 according to the disease duration and 12 age and sex matched controls were studied for T3, T4, T8 and HLA-DR expression. No statistically significant differences were found in their total blood lymphocyte counts or in the percentage of T3, T4 and T8 positive cells, although mild lymphopenia was found in the group of long-standing diabetics. The percentage of activated T cells, identified as T3+/DR+ cells, was significantly increased in the groups of patients studied more than a month after diagnosis and in four of 14 patients studied within a month from diagnosis. Seven new onset IDDM patients were studied for co-expression of T8 and Leu 15 antigens (putative suppressor cell phenotype), but no significant differences was found compared with controls. We conclude that T4/T8 ratio abnormalities previously reported in Ficoll separated cells are not reproduced when unseparated cells are analysed by flow cytometry, although the presence of HLA-DR+ T cells is confirmed.

Desferoxamine blocks IL 2 receptor expression on human T lymphocytes.

Thymidine uptake by PHA-stimulated human lymphocytes is reduced in the presence of 100 microM or greater concentrations of the iron-chelating agent desferoxamine (DF). We assessed expression of IL 2 receptor, 4F2 and Ia antigens, IL 2 production, and cell cycle progression by blood mononuclear cells (MNC) stimulated by PHA in the presence or absence of DF to determine whether the lack of T cell proliferation was a manifestation of inhibition of an earlier activation event. Tac antigen expression on PHA-stimulated MNC was inhibited by DF throughout 8 days of culture, and those cells which were positive had a low density of Tac antigen as compared with controls without DF. Expression of other activation antigens, 4F2 and Ia, was not impaired by DF. The supernatants of the DF-containing and control cultures contained equivalent IL 2 activity, as measured on the HT-2 cell line. Cell cycle analysis of these cultures shows that the addition of DF at the beginning of culture blocks most cells from undergoing G0 to G1 transition, whereas later addition of DF arrests the progression of the T cell blasts through the cell cycle. Separation of cells cultured with PHA and DF into Tac+ and Tac- subsets showed that progression from G0 to G1 was restricted to the former subset. These results suggest that interference with IL 2 receptor expression might contribute to the block in mitogen-induced proliferation caused by DF.

Specific lysis of varicella zoster virus-infected B lymphoblasts by human T cells.

Epstein-Barr virus-transformed human B cells expressed cell surface varicella-zoster virus (VZV) antigens after superinfection with VZV although they did not form infectious centers in a plaque assay. The VZV-superinfected cells were lysed by autologous VZV-stimulated T-cell lines and their derivative clones. The effector cells were specific for VZV and an HLA DR antigen and were T4+. The specificity of lysis of Epstein-Barr virus-transformed, VZV-superinfected targets by prestimulated mononuclear cells in this system contrasted with the unrestricted lysis seen when the targets were VZV-infected fibroblasts.

A cytotoxic monoclonal islet cell surface antibody from the NOD mouse.

Monoclonal antibody (Mab) 1.93B7 was obtained by fusion of spleen cells from a diabetic NOD mouse with P3X63Ag8.653 myeloma cells and screening for complement mediated lysis of rat insulinoma (RIN) cells. Immunofluorescence studies revealed that this Mab binds to RIN cells but not to the rat pituitary tumour line GH3. The binding of Mab 1.93B7 to RIN cells was abolished by trypsin but not by neuraminidase treatment of the cells, suggesting that the antigen recognized is a protein. Mab 1.93B7 bound to approximately 30% of mouse (BALB/c) and rat islet cells which had been subjected to trypsin digestion and incubated as a single cell suspension for 12h to allow reexpression of trypsin sensitive antigens. Since Mab 1.93B7 is potentially pathogenic, as suggested by its reactivity to primary islet cells and its complement fixing capacity, we injected it into BALB/c and NOD mice. Cytotoxic activity against RIN cells was detected in the serum of the animals injected with Mab 1.93B7, but the Mab did not exert a diabetogenic action and failed to reverse diabetes when administered at onset in NOD mice. No modification of the course of spleen cell mediated transfer of diabetes in NOD mice was observed when the Mab was administered from the time of spleen cell inoculation to the appearance of glycosuria. The implications of the lack of an effect in vivo of Mab 1.93B7 under the conditions employed are discussed.

Cow's milk hypersensitivity in an elderly woman: clinical and immunologic findings.

We report a case of allergy to cow's milk diagnosed in a 79-year-old woman, who had the habit of drinking large amounts of milk and other dairy products for several years. She had been an asthmatic since the age of 40. At age 65, she noticed a correlation between cheese and milk intake and onset of asthma and two episodes of shock, and she has been avoiding milk for the past 15 years. Positive skin prick tests for cow's milk proteins, grass pollens, and house dust mite were observed. Serum IgE levels were above 3,000 U/mL, with high molecular weight IgE detected by gel chromatography. RAST results confirmed the presence of sensitizations detected by skin prick tests. The patient never suffered from atopic eczema. This unusual case of food allergy with onset after the age of 40 may indicate that prolonged exposure to food antigens in predisposed individuals may lead to the development of allergy even in adult age, lasting for decades after partial elimination of the allergen from the diet.

Processing and presentation of cell-associated varicella-zoster virus antigens by human monocytes.

To determine whether viral antigens associated with infected cells were processed for presentation to T cells, we cultured human blood mononuclear cells (MNC) from varicella-zoster virus (VZV) immune donors with VZV-infected fibroblasts of known HLA type which had been fixed in 0.05% glutaraldehyde. After 7-8 days of culture thymidine uptake by T4+ cells exceeded that of T8+ cells. Stimulated cells were depleted of adherent cells and restimulated with VZV-infected fibroblasts from donors matched or unmatched with the responder for HLA type in the presence or absence of fresh adherent cells. Proliferation of the VZV-specific blasts required the presence of adherent cells matched with the responder lymphocytes for HLA-DR; conversely, the VZV specific response was not restricted by the MHC of the fibroblasts used in the restimulation assay. Preincubation of the adherent cells with chloroquine inhibited the proliferative response in a dose-dependent manner. These results suggest that VZV antigens on infected cells may be processed by monocytes for presentation to T cells.

Circulating lymphocyte populations and autoantibodies in non-obese diabetic (NOD) mice: a longitudinal study.

Several previous observations indicate a role for the immune system in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) in non-obese diabetic (NOD) mice. In order to assess the status of the immune system in this model of spontaneous diabetes we studied the phenotype of circulating lymphocytes and the humoral autoimmunity to islet cells in non-diabetic NOD mice at various ages. Lymphocyte numbers were low in young NOD mice (age less than 160 days) as compared with other strains of mice and increased later to reach values in or above the range of controls. The percentages of circulating T lymphocytes and their L3T4+ and Lyt2+ subsets were higher in NOD mice of all ages and both sexes than in controls; however, no imbalance of the L3T4+ and Lyt2+ subpopulations was found. Anti-insulin autoantibodies were detected by an ELISA assay in all the NOD mice studied throughout the entire period of observation. Autoantibodies reacting with the cytoplasm of islet cells in Bouin's fixed pancreas sections, likely to be anti-insulin antibodies, were found in 47 to 58% of the samples from NOD mice aged 75 to 150 days. Antibodies to surface antigens of rat insulinoma cells were virtually absent in young NOD mice (75-100 days) and appeared in 33 to 43% of the samples from 150 to 185 days old NOD mice. The autoantibodies and the quantitative lymphocyte abnormalities reported here, although not predictive of the appearance of overt diabetes, are likely to be involved in the pathogenesis of the disease and therefore may indicate directions for future investigations.

The role of CD4+ and CD8+ T cells in the destruction of islet grafts by spontaneously diabetic mice.

Spontaneous development of diabetes in the nonobese diabetic (NOD) mouse is mediated by an immunological process. In disease-transfer experiments, the activation of diabetes has been reported to require participation of both CD4+ and CD8+ T-cell subsets. These findings seem to indicate that the CD4+ cells are the helper cells for the activation of cytotoxic CD8+ cells that directly destroy islet beta cells in type I diabetes. In this report we challenge this interpretation because of two observations: (i) Destruction of syngeneic islet grafts by spontaneously diabetic NOD mice (disease recurrence) is CD4+ and not CD8+ T-cell dependent. (ii) Disease recurrence in islet tissue grafted to diabetic NOD mice is not restricted by islet major histocompatibility complex antigens. From these observations we propose that islet destruction depends on CD4+ effector T cells that are restricted by major histocompatibility complex antigens expressed on NOD antigen-presenting cells. Both of these findings argue against the CD8+ T cell as a mediator of direct islet damage. We postulate that islet damage in the NOD mouse results from a CD4+ T-cell-dependent inflammatory response.

In vitro effect of ketoconazole on human neutrophil chemotaxis and Candida albicans killing.

Ketoconazole, an antifungal agent, was investigated to discover its possible enhancing role on polymorphonuclear chemotactic and Candida killing functions in vitro. Polymorphonuclear leukocytes from 10 healthy subjects and two different strains of Candida albicans were used. At concentrations of 1 microgram/ml Ketoconazole showed no distinct synergistic effect on either of the above-mentioned polymorphonuclear cellular functions, but, on the other hand it operated directly on Candida cells. In addition, this drug did not demonstrate any chemotactic activity on granulocytes. These data could suggest that host cellular defence and Ketoconazole exert parallel rather than synergistic activity.

Complement activation is variably affected by fibronectin preparations obtained through different procedures.

Human plasma fibronectin (FN) containing preparations (FNCP) were prepared by means of gelatin-affinity chromatography of normal plasma cryoprecipitate (FNCP-a), and by affinity chromatography of cryoprecipitate on a rabbit anti-human FN antiserum Sepharose column (FNCP-b) or of normal citrated plasma (FNCP-c). These preparations were then tested for their ability to influence certain complement (C')-dependent polymorphonuclear phagocyte activities. FNCP-a consistently inhibited humoral chemotaxis, whereas FNCP-b and -c did not. Measurement of C' activation by the C3 conversion assay revealed that only FNCP-a displayed inhibitory activity on classical and alternative pathways. Ouchterlony and immunoelectrophoretic analyses indicated substantial identity of all FNCPs. FNCP-a contained a consistent amount of lower MW contaminants (between 40 and 60 KD) which were shown not to be immunologically related to FN by Western blot analysis. Immunoblotting also revealed a single band in FNCP-a (MW approx. 210 KD) instead of the anticipated two bands which were evident in the other FNCPs. Thus, a slight modification of the FN molecule and/or the presence of low MW contaminants in the gelatin-purified preparation seem able to induce inhibitory effects on C'-dependent polymorphonuclear phagocytic activities.