JC virus-specific immune responses in human immunodeficiency virus type 1 patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a frequently fatal disease caused by uncontrolled polyomavirus JC (JCV) in severely immunodeficient patients. We investigated the JCV-specific cellular and humoral immunity in the Swiss HIV Cohort Study. We identified PML cases (n = 29), as well as three matched controls per case (n = 87), with prospectively cryopreserved peripheral blood mononuclear cells and plasma at diagnosis. Nested controls were matched according to age, gender, CD4(+) T-cell count, and decline. Survivors (n = 18) were defined as being alive for >1 year after diagnosis. Using gamma interferon enzyme-linked immunospot assays, we found that JCV-specific T-cell responses were lower in nonsurvivors than in their matched controls (P = 0.08), which was highly significant for laboratory- and histologically confirmed PML cases (P = 0.004). No difference was found between PML survivors and controls or for cytomegalovirus-specific T-cell responses. PML survivors showed significant increases in JCV-specific T cells (P = 0.04) and immunoglobulin G (IgG) responses (P = 0.005). IgG responses in survivors were positively correlated with CD4(+) T-cell counts (P = 0.049) and negatively with human immunodeficiency virus RNA loads (P = 0.03). We conclude that PML nonsurvivors had selectively impaired JCV-specific T-cell responses compared to CD4(+) T-cell-matched controls and failed to mount JCV-specific antibody responses. JCV-specific T-cell and IgG responses may serve as prognostic markers for patients at risk.

IL1B and DEFB1 Polymorphisms Increase Susceptibility to Invasive Mold Infection After Solid-Organ Transplantation.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in immune genes have been associated with susceptibility to invasive mold infection (IMI) among hematopoietic stem cell but not solid-organ transplant (SOT) recipients. METHODS: Twenty-four SNPs from systematically selected genes were genotyped among 1101 SOT recipients (715 kidney transplant recipients, 190 liver transplant recipients, 102 lung transplant recipients, 79 heart transplant recipients, and 15 recipients of other transplants) from the Swiss Transplant Cohort Study. Association between SNPs and the end point were assessed by log-rank test and Cox regression models. Cytokine production upon Aspergillus stimulation was measured by enzyme-linked immunosorbent assay in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and correlated with relevant genotypes. RESULTS: Mold colonization (n = 45) and proven/probable IMI (n = 26) were associated with polymorphisms in the genes encoding interleukin 1β (IL1B; rs16944; recessive mode, P = .001 for colonization and P = .00005 for IMI, by the log-rank test), interleukin 1 receptor antagonist (IL1RN; rs419598; P = .01 and P = .02, respectively), and β-defensin 1 (DEFB1; rs1800972; P = .001 and P = .0002, respectively). The associations with IL1B and DEFB1 remained significant in a multivariate regression model (P = .002 for IL1B rs16944; P = .01 for DEFB1 rs1800972). The presence of 2 copies of the rare allele of rs16944 or rs419598 was associated with reduced Aspergillus-induced interleukin 1β and tumor necrosis factor α secretion by PBMCs. CONCLUSIONS: Functional polymorphisms in IL1B and DEFB1 influence susceptibility to mold infection in SOT recipients. This observation may contribute to individual risk stratification.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Circulating FABP4 Is a prognostic biomarker in patients with acute coronary syndrome but not in asymptomatic individuals.

OBJECTIVE: Blood-borne biomarkers reflecting atherosclerotic plaque burden have great potential to improve clinical management of atherosclerotic coronary artery disease and acute coronary syndrome (ACS). APPROACH AND RESULTS: Using data integration from gene expression profiling of coronary thrombi versus peripheral blood mononuclear cells and proteomic analysis of atherosclerotic plaque-derived secretomes versus healthy tissue secretomes, we identified fatty acid-binding protein 4 (FABP4) as a biomarker candidate for coronary artery disease. Its diagnostic and prognostic performance was validated in 3 different clinical settings: (1) in a cross-sectional cohort of patients with stable coronary artery disease, ACS, and healthy individuals (n=820), (2) in a nested case-control cohort of patients with ACS with 30-day follow-up (n=200), and (3) in a population-based nested case-control cohort of asymptomatic individuals with 5-year follow-up (n=414). Circulating FABP4 was marginally higher in patients with ST-segment-elevation myocardial infarction (24.9 ng/mL) compared with controls (23.4 ng/mL; P=0.01). However, elevated FABP4 was associated with adverse secondary cerebrovascular or cardiovascular events during 30-day follow-up after index ACS, independent of age, sex, renal function, and body mass index (odds ratio, 1.7; 95% confidence interval, 1.1-2.5; P=0.02). Circulating FABP4 predicted adverse events with similar prognostic performance as the GRACE in-hospital risk score or N-terminal pro-brain natriuretic peptide. Finally, no significant difference between baseline FABP4 was found in asymptomatic individuals with or without coronary events during 5-year follow-up. CONCLUSIONS: Circulating FABP4 may prove useful as a prognostic biomarker in risk stratification of patients with ACS.

Safety of human immunisation with a live-attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled phase I trial.

BACKGROUND: Tuberculosis remains one of the world's deadliest transmissible diseases despite widespread use of the BCG vaccine. MTBVAC is a new live tuberculosis vaccine based on genetically attenuated Mycobacterium tuberculosis that expresses most antigens present in human isolates of M tuberculosis. We aimed to compare the safety of MTBVAC with BCG in healthy adult volunteers. METHODS: We did this single-centre, randomised, double-blind, controlled phase 1 study at the Centre Hospitalier Universitaire Vaudois (CHUV; Lausanne, Switzerland). Volunteers were eligible for inclusion if they were aged 18-45 years, clinically healthy, HIV-negative and tuberculosis-negative, and had no history of active tuberculosis, chemoprophylaxis for tuberculosis, or BCG vaccination. Volunteers fulfilling the inclusion criteria were randomly assigned to three cohorts in a dose-escalation manner. Randomisation was done centrally by the CHUV Pharmacy and treatments were masked from the study team and volunteers. As participants were recruited within each cohort, they were randomly assigned 3:1 to receive MTBVAC or BCG. Of the participants allocated MTBVAC, those in the first cohort received 5 × 10(3) colony forming units (CFU) MTBVAC, those in the second cohort received 5 × 10(4) CFU MTBVAC, and those in the third cohort received 5 × 10(5) CFU MTBVAC. In all cohorts, participants assigned to receive BCG were given 5 × 10(5) CFU BCG. Each participant received a single intradermal injection of their assigned vaccine in 0·1 mL sterile water in their non-dominant arm. The primary outcome was safety in all vaccinated participants. Secondary outcomes included whole blood cell-mediated immune response to live MTBVAC and BCG, and interferon γ release assays (IGRA) of peripheral blood mononuclear cells. This trial is registered with ClinicalTrials.gov, number NCT02013245. FINDINGS: Between Jan 23, 2013, and Nov 6, 2013, we enrolled 36 volunteers into three cohorts, each of which consisted of nine participants who received MTBVAC and three who received BCG. 34 volunteers completed the trial. The safety of vaccination with MTBVAC at all doses was similar to that of BCG, and vaccination did not induce any serious adverse events. All individuals were IGRA negative at the end of follow-up (day 210). After whole blood stimulation with live MTBVAC or BCG, MTBVAC was at least as immunogenic as BCG. At the same dose as BCG (5×10(5) CFU), although no statistical significance could be achieved, there were more responders in the MTBVAC group than in the BCG group, with a greater frequency of polyfunctional CD4+ central memory T cells. INTERPRETATION: To our knowledge, MTBVAC is the first live-attenuated M tuberculosis vaccine to reach clinical assessment, showing similar safety to BCG. MTBVAC seemed to be at least as immunogenic as BCG, but the study was not powered to investigate this outcome. Further plans to use more immunogenicity endpoints in a larger number of volunteers (adults and adolescents) are underway, with the aim to thoroughly characterise and potentially distinguish immunogenicity between MTBVAC and BCG in tuberculosis-endemic countries. Combined with an excellent safety profile, these data support advanced clinical development in high-burden tuberculosis endemic countries. FUNDING: Biofabri and Bill & Melinda Gates Foundation through the TuBerculosis Vaccine Initiative (TBVI).

Enhancement of viability of radiosensitive (PBMC) and resistant (MDA-MB-231) clones in low-dose-rate cobalt-60 radiation therapy

AbstractObjective:In the present study, the authors investigated the in vitrobehavior of radio-resistant breast adenocarcinoma (MDA-MB-231) cells line and radiosensitive peripheral blood mononuclear cells (PBMC), as a function of different radiation doses, dose rates and postirradiation time kinetics, with a view to the interest of clinical radiotherapy.Materials and Methods:The cells were irradiated with Co-60, at 2 and 10 Gy and two different exposure rates, 339.56 cGy.min–1 and the other corresponding to one fourth of the standard dose rates, present over a 10-year period of cobalt therapy. Post-irradiation sampling was performed at pre-established kinetics of 24, 48 and 72 hours. The optical density response in viability assay was evaluated and a morphological analysis was performed.Results:Radiosensitive PBMC showed decrease in viability at 2 Gy, and a more significant decrease at 10 Gy for both dose rates. MDAMB- 231 cells presented viability decrease only at higher dose and dose rate. The results showed MDA-MB-231 clone expansion at low dose rate after 48–72 hours post-radiation.Conclusion:Low dose rate shows a possible potential clinical impact involving decrease in management of radio-resistant and radiosensitive tumor cell lines in cobalt therapy for breast cancer.

Immunomodulation of allergic immune response during specific immunotherapy

Atopic, IgE-mediated allergies are one of the major public health problems in Finland and other Western countries. These diseases are characterized by type 2 T helper (Th2) cell predominated immune responses (interleukin-4 (IL-4), IL-5) against ubiquitous environmental allergens. Despite of adequate pharmacological treatment, more than 20% of the patients with allergic rhinitis develop asthma. Allergen specific immunotherapy (SIT) is the only treatment currently available to affect to the natural course of allergic diseases. This treatment involves repeated administration of allergens to the patients either via sublingual route (sublingual immunotherapy, SLIT) or by subcutaneous injections (subcutaneous immunotherapy, SCIT). Successful treatment with SCIT or SLIT has been shown to provide long-term remission in symptoms, and prevent disease progression to asthma, but the immunological mechanisms behind these beneficial effects are not yet completely understood. Increased knowledge of such mechanisms could not only help to improve SIT efficacy, but also provide tools to monitor the development of clinical response to SIT in individual patients, and possibly also, predict the ultimate therapeutic outcome. The aim of this work was to clarify the immunological mechanisms associated with SIT by investigating the specific allergen-induced immune responses in peripheral blood mononuclear cells (PBMC) of allergic rhinitis patients during the course of SLIT and SCIT. The results of this work demonstrate that both therapies induced increases in the protective, Th2-balancing Th1 type immune responses in PBMC, e.g. by up-regulating signaling lymphocytic activation molecule (SLAM) and interferon gamma (IFN-γ) expression, and augmented tolerogenic T regulatory (Treg) cell type responses against the specific allergens, e.g. by increasing IL-10 or Forkhead box P3 (FOXP3) expression. The induction of allergen-specific Th1 and Treg type responses during SLIT were dependent on the treatment dose, favoring high allergen dose SLIT. During SCIT, the early decrease in Th2 type cytokine production - in particular of IL-4 mRNA and IL-4/IFN-γ expression ratio - was associated with the development of good therapeutic outcome. Conversely, increases in both Th2 (IL-5) and Th1 (IFN-γ, SLAM) type responses and IL-10 mRNA production were seen in the patients with less effective outcome. In addition, increase in Th17 type cytokine (IL-17) mRNA production was found in the PBMC of patients with less effective outcome during both SLIT and SCIT. These data strengthen the current hypothesis that immunomodulation of allergen-specific immune responses from the prevailing Th2-biased responses towards a more Th1 type, and induction of tolerogenic Treg cells producing IL-10 represent the two key mechanisms behind the beneficial effects of SIT. The data also give novel insight into the mechanisms why SIT may fail to be effective in some patients by demonstrating a positive correlation between the proinflammatory IL-17 responses, Th2 type IL-5 production and clinical symptoms. Taken together, these data indicate that the analysis of Th1, Th2, Treg ja Th17-associated immune markers such as IL-10, SLAM, IL-4, IL-5 and IL-17 could provide tools to monitor the development of clinical response to SIT, and thereby, predict the ultimate clinical outcome already in the early course of the treatment.

IFN-g in human Chagas' disease: protection or pathology?

An apparently paradoxical role for IFN-g in human Chagas' disease was observed when studying the pattern of cytokine production by peripheral blood mononuclear cells (PBMC) obtained from two groups of chagasic patients after specific stimulation with Trypanosoma cruzi-derived antigens. The groups studied were 1) patients treated with benznidazole during the acute phase of Trypanosoma cruzi infection and 2) chronically infected untreated patients. In the treated group, higher levels of IFN-g were produced by PBMC from individuals cured after treatment when compared to non-cured patients. In contrast, in the chronically infected group (not treated) higher levels of IFN-g were produced by PBMC from cardiac patients in comparison with asymptomatic (indeterminate) patients. This apparently paradoxical role for IFN-g in human Chagas' disease is discussed in terms of the possibility of a temporal difference in IFN-g production during the initial stages of the infection (acute phase) in the presence or absence of chemotherapy. The maintenance of an immune response with high levels of IFN-g production during the chronic phase of the infection may favor cure or influence the development of the cardiac form of the disease

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-g and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response

Cytokine profile and pathology in human leishmaniasis

The clinical spectrum of leishmaniasis and control of the infection are influenced by the parasite-host relationship. The role of cellular immune responses of the Th1 type in the protection against disease in experimental and human leishmaniasis is well established. In humans, production of IFN-g is associated with the control of infection in children infected by Leishmania chagasi. In visceral leishmaniasis, an impairment in IFN-g production and high IL-4 and IL-10 levels (Th2 cytokines) are observed in antigen-stimulated peripheral blood mononuclear cells (PBMC). Moreover, IL-12 restores IFN-g production and enhances the cytotoxic response. IL-10 is the cytokine involved in down-regulation of IFN-g production, since anti-IL-10 monoclonal antibody (mAb) restores in vitro IFN-g production and lymphoproliferative responses, and IL-10 abrogates the effect of IL-12. In cutaneous and mucosal leishmaniasis, high levels of IFN-g are found in L. amazonensis-stimulated PBMC. However, low or absent IFN-g levels were observed in antigen-stimulated PBMC from 50% of subjects with less than 60 days of disease (24 ± 26 pg/ml). This response was restored by IL-12 (308 ± 342 pg/ml) and anti-IL-10 mAb (380 ± 245 pg/ml) (P<0.05). Later during the disease, high levels of IFN-g and TNF-a are produced both in cutaneous and mucosal leishmaniasis. After treatment there is a decrease in TNF-a levels (366 ± 224 pg/ml before treatment vs 142 ± 107 pg/ml after treatment, P = 0.02). Although production of IFN-g and TNF-a might be involved in the control of parasite multiplication in the early phases of Leishmania infection, these cytokines might also be involved in the tissue damage seen in tegumentary leishmaniasis

Human_Leishmaniasis@cytokines.bahia.br

The cell-mediated immune response is critical in the resistance to and recovery from leishmaniasis. Cytokines are central elements in mounting an immune response and have received a great deal of attention in both human and experimental leishmaniasis. IFN-g is responsible for macrophage activation leading to leishmanicidal mechanisms. Understanding the balance of cytokines that lead to enhanced production of or synergize with IFN-g, and those cytokines that counterbalance its effects is fundamental for developing rational immunotherapeutic or immunoprophylactic approaches to leishmaniasis. Here we focus on the cytokine balance in human leishmaniasis, particularly IL-10 as an IFN-g opposing cytokine, and IL-12 as an IFN-g inducer. The effects of these cytokines were evaluated in terms of several parameters of the human immune response. IL-10 reduced lymphocyte proliferation, IFN-g production and cytotoxic activity of responsive human peripheral blood mononuclear cells. Neutralization of IL-10 led to partial restoration of lymphoproliferation, IFN-g production and cytotoxic activity in unresponsive visceral leishmaniasis patients. IL-12 also restored the responses of peripheral blood mononuclear cells from visceral leishmaniasis patients. The responses obtained with IL-12 are higher than those obtained with anti-IL-10, even when anti-IL-10 is combined with anti-IL-4

Monounsaturated fats and immune function

Animal studies suggest that olive oil is capable of modulating functions of cells of the immune system in a manner similar to, albeit weaker than, fish oils. There is some evidence that the effects of olive oil on immune function in animal studies are due to oleic acid rather than to trace elements or antioxidants. Importantly, several studies have demonstrated effects of oleic acid-containing diets on in vivo immune responses. In contrast, consumption of a monounsaturated fatty acid (MUFA)-rich diet by humans does not appear to bring about a general suppression of immune cell functions. The effects of this diet in humans are limited to decreasing aspects of adhesion of peripheral blood mononuclear cells, although there are trends towards decreases in natural killer cell activity and proliferation. The lack of a clear effect of MUFA in humans may be attributable to the higher level of monounsaturated fat used in the animal studies, although it is ultimately of importance to examine the effects of intakes which are in no way extreme. The effects of MUFA on adhesion molecules are potentially important, since these molecules appear to have a role in the pathology of a number of diseases involving the immune system. This area clearly deserves further exploration

HHV-8 infection in patients with AIDS-related Kaposi's sarcoma in Brazil

The aims of the present study were to determine the prevalence of human herpesvirus type 8 (HHV-8) in HIV-positive Brazilian patients with (HIV+/KS+) and without Kaposi's sarcoma (HIV+/KS-) using PCR and immunofluorescence assays, to assess its association with KS disease, to evaluate the performance of these tests in detecting HHV-8 infection, and to investigate the association between anti-HHV-8 antibody titers, CD4 counts and staging of KS disease. Blood samples from 66 patients, 39 HIV+/KS+ and 27 HIV+/KS-, were analyzed for HHV-8 viremia in peripheral blood mononuclear cells by PCR and HHV-8 antigenemia for latent and lytic infection by immunofluorescence assay. Positive samples for latent nuclear HHV-8 antigen (LNA) antibodies were titrated out from 1/100 to 1/409,600 dilution. Clinical information was collected from medical records and risk behavior was assessed through an interview. HHV-8 DNA sequences were detected by PCR in 74.3% of KS+ patients and in 3.7% of KS- patients. Serological assays were similar in detecting anti-LNA antibodies and anti-lytic antigens in sera from KS+ patients (79.5%) and KS- patients (18.5%). HHV-8 was associated with KS whatever the method used, i.e., PCR (odds ratio (OR) = 7.4, 95% confidence interval (CI) = 2.16-25.61) or anti-LNA and anti-lytic antibodies (OR = 17.0, 95%CI = 4.91-59.14). Among KS+ patients, HHV-8 titration levels correlated positively with CD4 counts (rho 0.48, P = 0.02), but not with KS staging. HHV-8 is involved in the development of KS in different geographic areas worldwide, as it is in Brazil, where HHV-8 is more frequent among HIV+ patients. KS severity was associated with immunodeficiency, but no correlation was found between HHV-8 antibody titers and KS staging.

Comparison of the quantitative competitive and semiquantitative RT-PCR methods for the determination of interferon-gamma mRNA levels in AIDS-free HIV-infected individuals

IFN-gamma mRNA expression was evaluated in nonstimulated peripheral blood mononuclear cells (PBMC) of HIV-infected and seronegative individuals using quantitative competitive and semiquantitative RT-PCR and the sensitivity of these methods was compared. A significant correlation was found between quantitative competitive and semiquantitative RT-PCR in samples of both HIV-seronegative (P = 0.004) and HIV-infected individuals (P = 0.0004). PBMC from HIV-infected individuals presented a remarkable increase of IFN-gamma mRNA expression, as determined by both types of RT-PCR methods. Semiquantitative RT-PCR even without an internal standard is also acceptable for measuring cytokine mRNA expression, but less reliable if small amounts are quantified. Moreover, we found that increased IFN-gammamRNA expression is independent of CD4+ cell count in AIDS-free HIV-infected patients.

Immune response to respiratory syncytial virus in young Brazilian children

We have evaluated the cellular and humoral immune response to primary respiratory syncytial virus (RSV) infection in young infants. Serum specimens from 65 patients <=12 months of age (39 males and 26 females, 28 cases <3 months and 37 cases > or = 3 months; median 3 ± 3.9 months) were tested for anti-RSV IgG and IgG subclass antibodies by EIA. Flow cytometry was used to characterize cell surface markers expressed on peripheral blood mononuclear cells (PBMC) from 29 RSV-infected children. There was a low rate of seroconversion in children <3 months of age, whose acute-phase PBMC were mostly T lymphocytes (63.0 ± 9.0%). In contrast, a higher rate of seroconversion was observed in children >3 months of age, with predominance of B lymphocytes (71.0 ± 17.7%). Stimulation of PBMC with RSV (2 x 10(5) TCID50) for 48 h did not induce a detectable increase in intracellular cytokines and only a few showed a detectable increase in RSV-specific secreted cytokines. These data suggest that age is an important factor affecting the infants' ability to develop an immune response to RSV.