Expression Of MIP-1β Research Articles

Cytomegalovirus (CMV) Infection Suppresses The Expression Levels of Serum Cytokines in Follicular Lymphoma Patients

Follicular lymphoma (FL) is an indolent B-cell neoplasm. Increasing evidence suggests that chronic cytomegalovirus infection impacts the immunity of FL patients. This study was designed to unveil the impact of CMV infection on FL biology. Pretreatment serum samples from 42 FL patients recruited into a phase 3 FL clinical trial to compare two alternative frontline chemoimmunotherapy regimens were used. Twenty-one (21) of these patients had tested positive for CMV IgG antibodies, whereas 21 patients were negative. A multiplex assay was employed to measure the expression levels of 27 cytokines in each of these samples. Results were then compared between CMV-positive and CMV- negative groups using the Mann-Whitney U test using IBM SPSS Statistics version 20. Sera from CMV-positive FL patients showed decreased expression levels of most cytokines than those from the CMV-negative population. Data shows a statistically significant down-regulation of IL-6, IL-8, IL-9, IL-17A, FGF-basic, MIP-1α, and MIP-1β expression in CMV-positive FL patients compared to the CMV-negative group. Overall, our results suggest that CMV infection down-regulates the expression levels of serum cytokines in FL. Complementary studies to investigate immune cells in these patients' blood and tissue biopsies could provide a comprehensive perspective on the impact of CMV infection on FL.

Macrophages of different tissue origin exhibit distinct inflammatory responses to mycobacterial infection.

Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, invitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1β, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1β expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in invitro studies.

Characterization of Immune Phenotypes in Peripheral Blood of Adult Renal Transplant Recipients using Mass Cytometry (CyTOF)

Abstract Background Chronic immunosuppressive therapy in transplant patients can be associated with opportunistic infections and risks of cancer development. There is an important need to better measure the immune status of patients in a transplant setting to improve patient monitoring and guide development of appropriate immunosuppressive agents and regimen. Results In this study, CyTOF was performed to comprehensively characterize the immune differences between 10 renal transplant recipients and 11 age-matched healthy donors, longitudinally over 6 months. Overall, the immune cells of transplant patients showed signs of increased CD8+ T cell activation/differentiation. Higher levels of CD57 on CD8+ T cells were observed. CD8+ T cells and their memory subsets also showed increased production of MIP-1β and IFNγ. Further, CD107a and Granzyme B expression were also increased in CD8+ T cells and CD56hi NK cells, while Tregs had decreased IL-10 production. These changes are consistent with a more stimulated immune system, despite the administration of immune suppressants to the patients. However, there were also some signs of immune suppression in the transplant patients consistent with the treatment regimen. γδT cells showed decreased TNFα and IFNγ expression, while MIP-1β expression was reduced in NKT, CD56hi NK and γδT cells. CD8+ T memory cell subsets also showed decreased expression of IL-2, IL-17 and GM-CSF. Conclusions This mosaic pattern of functional changes highlights the importance of comprehensive immune monitoring of these patients. Such information will aid in optimizing individual treatment strategies and development of improved immunosuppressants that specifically target overly activated populations.

Schistosoma mansoni soluble egg antigen (SEA) and recombinant Omega-1 modulate induced CD4+ T-lymphocyte responses and HIV-1 infection in vitro

Parasitic helminths evade, skew and dampen human immune responses through numerous mechanisms. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we analyzed the effects that soluble egg antigen (SEA) from Schistosoma mansoni had on modulating HIV-1 infection and cytokine/chemokine production in vitro. We determined that SEA, specifically through kappa-5, can potently bind to DC-SIGN and thereby blocks DC-SIGN mediated HIV-1 trans-infection (p<0.05) whilst not interfering with cis-infection. DCs exposed to SEA whilst maturing under Th2 promoting conditions, will upon co-culture with naïve T-cells induce a T-cell population that was less susceptible to HIV-1 R5 infection (p<0.05) compared to DCs unexposed to SEA, whereas HIV-1 X4 virus infection was unaffected. This was not observed for DCs exposed to SEA while maturing under Th1 or Th1/Th2 (Tmix) promoting conditions. All T-cell populations induced by SEA exposed DCs demonstrate a reduced capacity to produce IFN-γ and MIP-1β. The infection profile of T-cells infected with HIV-1 R5 was not associated with down-modulation of CCR5 cell surface expression. We further show that DCs maturing under Tmix conditions exposed to plant recombinant omega-1 protein (rω-1), which demonstrates similar functions to natural ω-1, induced T-cell populations that were less sensitive for HIV-1 R5 infection (p<0.05), but not for X4 virus infection. This inhibition associated again with a reduction in IFN-γ and MIP-1β expression, but additionally correlated with reduced CCR5 expression. We have shown that SEA parasite antigens and more specifically rω-1 can modulate HIV-1 infectivity with the potential to influence disease course in co-infected individuals.

Tu1846 – Higher TNFα and Mip-1β Expression in Pretreatment Colonic Mucosa Have Potential to Predict of Achieving Mucosal Healing by Granulocyte and Monocyte Adsorptive Apheresis Therapy in Ulcerative Colitis Patients

Tu1846 – Higher TNFα and Mip-1β Expression in Pretreatment Colonic Mucosa Have Potential to Predict of Achieving Mucosal Healing by Granulocyte and Monocyte Adsorptive Apheresis Therapy in Ulcerative Colitis Patients

Role of STAT signaling and autocrine action of chemokines during H2 O 2 induced HTR-8/SVneo trophoblastic cells invasion.

During pregnancy, regulated generation of reactive oxygen species (ROS) is important for activation of signaling pathways and placentation. In the current study, the effect of H2 O2 on invasion of HTR-8/SVneo cells, a human extravillous trophoblast cell line, is investigated. Treatment of HTR-8/SVneo cells for 24 hr with H 2 O2 (25 µM) leads to a significant increase in invasion without affecting cell proliferation, viability, and apoptosis. Concomitantly, a significant increase in the matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases-1 (TIMP-1) ratio is observed. Further, significant increase in phosphorylation of signal transducer and activator of transcription 1 (STAT-1) and STAT-3 (both at ser727 residue) is observed on treating HTR-8/SVneo cells with 25 µM of H2 O2 accompanied by an increase in the secretion of interleukin-8 (IL-8) and macrophage inflammatory protein-1β (MIP-1β). A significant decrease in H2 O2 -mediated invasion of HTR-8/SVneo cells and reduced expression of IL-8 and MIP-1β accompanied by decrease in MMP-9/TIMP-1 ratio are observed on inhibiting STAT-1 and STAT-3 by small interfering RNA (siRNA). Inhibition of STAT-1 activity by fludarabine and STAT-3 activity by Stattic also leads to a decrease in H2 O2 -mediated increase in HTR-8/SVneo cell invasion. Inhibition of IL-8 and MIP-1β by siRNA also leads to a significant decrease in both basal and H2 O2 -mediated invasion. Interestingly, inhibition of MIP-1β by siRNA leads to a significant reduction in H2 O2 -mediated increase in IL-8. However, no significant effect of IL-8 silencing on H2 O2 -mediated MIP-1β expression was observed. From the above results, it can be concluded that H2 O2 activates STAT signaling, MIP-1β & IL-8 secretion and increases MMP-9/TIMP-1 ratio leading to an increased invasion of HTR-8/SVneo cells without affecting their viability.

Poorly cytotoxic terminally differentiated CD56negCD16pos NK cells accumulate in Kenyan children with Burkitt lymphomas

AbstractNatural killer (NK) cells are critical for restricting viral infections and mediating tumor immunosurveillance. Epstein-Barr virus (EBV) and Plasmodium falciparum malaria are known risk factors for endemic Burkitt lymphoma (eBL), the most common childhood cancer in equatorial Africa. To date, the composition and function of NK cells have not been evaluated in eBL etiology or pathogenesis. Therefore, using multiparameter flow cytometry and in vitro killing assays, we compared NK cells from healthy children and children diagnosed with eBL in Kenya. We defined 5 subsets based on CD56 and CD16 expression, including CD56negCD16pos. We found that licensed and terminally differentiated perforin-expressing CD56negCD16pos NK cells accumulated in eBL children, particularly in those with high EBV loads (45.2%) compared with healthy children without (6.07%) or with (13.5%) malaria exposure (P = .0007 and .002, respectively). This progressive shift in NK cell proportions was concomitant with fewer CD56dimCD16pos cells. Despite high MIP-1β expression, CD56negCD16pos NK cells had diminished cytotoxicity, with lower expression of activation markers NKp46, NKp30, and CD160 and the absence of TNF-α. Of note, the accumulation of poorly cytotoxic CD56negCD16pos NK cells resolved in long-term eBL survivors. Our study demonstrates impaired NK cell–mediated immunosurveillance in eBL patients but with the potential to restore a protective NK cell repertoire after cancer treatment. Characterizing NK cell dysfunction during coinfections with malaria and EBV has important implications for designing immunotherapies to improve outcomes for children diagnosed with eBL.

Mo1730 - Higher Expression of MIP-1β and IL23 in Colonic Mucosa of Pretreatment may Predict Therapeutic Efficacy of Granulocyte and Monocyte Adsorptive Apheresis in Elderly Patients with Ulcerative Colitis

Mo1730 - Higher Expression of MIP-1β and IL23 in Colonic Mucosa of Pretreatment may Predict Therapeutic Efficacy of Granulocyte and Monocyte Adsorptive Apheresis in Elderly Patients with Ulcerative Colitis

Expression and clinical significance of serum chemokines in patients with lung cancer

Objective To detect the expression levels of multiple serum chemokines including IFN-inducible T cell chemoattractant (ITAC), Fractalkine, macrophage inflammatory protein(MIP)-3α, IL-8, MIP-1α, MIP-1β in patients with lung cancer and explore their association with the clinical characteristics of lung cancer as well as the correlations among these chemokines. Methods Forty newly diagnosed patients with lung cancer and thirty healthy controls were enrolled for detection of the serum levels of 6 kinds of chemokines by Luminex technology. The correlations of clinical characteristics of lung cancer with these chemokines and the correlations among these chemokines were analyzed by SPSS 17.0 software. Results The serum levels [M(QR)] of IL-8, Fractalkine and MIP-3α in patients with lung cancer were 5.16(4.74), 128.45(141.89), 10.31(8.88) respectively, and 2.01(0.95), 61.46(74.81), 8.08(5.87) respectively in control group, with significant differences (Z=-4.783, P<0.001; Z=-4.046, P<0.001; Z=-3.105, P=0.002). The expression of MIP-1β in lung adenocarcinoma was significantly higher than that in squamous carcinoma [18.32(12.27) vs. 13.72(7.31), Z=-2.212, P=0.027], and of ITAC in squamous carcinoma was significantly higher than that in small cell lung cancer [24.51(22.48) vs. 9.28(4.85), Z=-2.460, P=0.014]. The expressions of MIP-3α and Fractalkine were positively correlated in the two groups (r=0.619, P<0.001; r=0.766, P<0.001). Conclusion The expressions of IL-8, Fractalkine and MIP-3α increase significantly in lung cancer patients, and they are may play important roles in metastatic lung cancer. Key words: Lung neoplasms; Chemotactic factors; Receptors, chemokine

Maturation and Mip-1β Production of Cytomegalovirus-Specific T Cell Responses in Tanzanian Children, Adolescents and Adults: Impact by HIV and Mycobacterium tuberculosis Co-Infections.

It is well accepted that aging and HIV infection are associated with quantitative and functional changes of CMV-specific T cell responses. We studied here the expression of Mip-1β and the T cell maturation marker CD27 within CMVpp65-specific CD4+ and CD8+ T cells in relation to age, HIV and active Tuberculosis (TB) co-infection in a cohort of Tanzanian volunteers (≤16 years of age, n = 108 and ≥18 years, n = 79). Independent of HIV co-infection, IFNγ+ CMVpp65-specific CD4+ T cell frequencies increased with age. In adults, HIV co-infection further increased the frequencies of these cells. A high capacity for Mip-1β production together with a CD27low phenotype was characteristic for these cells in children and adults. Interestingly, in addition to HIV co-infection active TB disease was linked to further down regulation of CD27 and increased capacity of Mip-1β production in CMVpp65-specific CD4+ T cells. These phenotypic and functional changes of CMVpp65-specific CD4 T cells observed during HIV infection and active TB could be associated with increased CMV reactivation rates.

Targeting angiogenic pathway for chemoprevention of experimental colon cancer using C-phycocyanin as cyclooxygenase-2 inhibitor.

An angiogenic pathway was studied that involved stromal tissue degradation with matrix metalloproteinases (MMPs), vesicular endothelial growth factor-A (VEGF-A), and hypoxia inducible factor-1α (HIF-1α) mediated growth regulation in a complex interaction with chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β). Gene and protein expression was studied with real-time PCR, Western immunoblot, and immunofluorescence. Morphological and histopathological analysis of tumor was done, as also the activity of MMPs and HIF-1α by gelatin zymography and ELISA. Binding interactions of proteins were studied by molecular docking. Piroxicam, a traditional NSAID and C-phycocyanin, a biliprotein from Spirulina platensis, were utilized in the chemoprevention of DMH-induced rat colon cancer. A significant number of tumors was evident in DMH treated animals, while with piroxicam and C-phycocyanin, the number and size of tumors/lesions were reduced. Colonic tissues showed severe dysplasia, tubular adenoma, and adenocarcinoma from DMH, with invasive features along with signet ring cell carcinoma. No occurrence of carcinoma was detected in either of the drug treatments or in a combination regimen. An elevated VEGF-A, MMP-2, and MMP-9 level was observed, which is required for metastasis and invasion into surrounding tissues. Drugs induced chemoprevention by down-regulating these proteins. Piroxicam docked in VEGF-A binding site of VEGF-A receptors i.e., VEGFR1 and VEGFR2, while phycocyanobilin (a chromophore of C-phycocyanin) docked with VEGFR1 alone. HIF-1α is up-regulated which is associated with increased oxygen demand and angiogenesis. MCP-1 and MIP-1β expression was also found altered in DMH and regulated by the drugs. Anti-angiogenic role of piroxicam and C-phycocyanin is well demonstrated.

Adjuvanted HLA-supertype restricted subdominant peptides induce new T-cell immunity during untreated HIV-1-infection

We investigated the potential of inducing additional T-cell immunity during chronic HIV-1 infection directed to subdominant HIV-1 epitopes from common HLA-supertypes. Ten treatment-naïve HIV-1-infected individuals were immunized with peptides in the adjuvant CAF01. One individual received placebo. T-cell immunogenicity was examined longitudinally by a flow cytometry (CD107a, IFNγ, TNFα, IL-2 and/or MIP1β expression) as well as IFNγ ELISPOT. Safety was evaluated by clinical follow up combined with monitoring of biochemistry, hematology, CD4 T-cell counts and viral load. New CD4 and CD8 T-cell responses specific for one or more vaccine epitopes were induced in 10/10 vaccinees. The responses were dominated by CD107a and MIP1β expression. There were no significant changes in HIV-1 viral load or CD4 T-cell counts. Our study demonstrates that the peptide/CAF01 vaccine is safe and that it is possible to generate new HIV-1 T-cell responses to defined epitopes in treatment-naïve HIV-1-infected individuals.

Tong Luo Jiu Nao injection, a traditional Chinese medicinal preparation, inhibits MIP-1β expression in brain microvascular endothelial cells injured by oxygen-glucose deprivation

Ethnopharmacological relevanceTong Luo Jiu Nao injection (TLJN), a modern Chinese formula based on Traditional Chinese Medicine theory, has been used to treat ischemic stroke and vascular dementia. TLJN belongs to the ethnopharmacological family of medicines. Aim of the studyTo investigate the protective effect of TLJN on oxygen-glucose deprivation (OGD) induced-injury of brain microvascular endothelial cells (BMECs). Materials and methodsThe model of OGD was established in the primarily cultured BMECs. TLJN was added to the OGD-injured BMECs for 6h. A series of assays were used to detect the effects of TLJN on: (i) MIP-1β content in BMECs conditioned media (CM) by ELISA; (ii) MIP-1β protein expression in BMECs by western blotting and immunocytochemistry; (iii) the expression of CCR5, receptor of MIP-1β, in BMECs by western blotting; (iv) the proliferative activity of microglial cells via the Cell Counting Kit-8 (CCK-8). ResultsOur results showed that the OGD-injured BMECs presented with large amounts of secretion and expression of MIP-1β and up-regulation of CCR5. Also, the CM of OGD-injured BMECs remarkably increased the proliferative activity of microglial cells. The TLJN-treated BMECs exhibited a reduction of MIP-1β content in BMECs-CM and a down-regulation of MIP-1β and CCR5 expression. In addition, an inhibitory effect of CM of OGD-injured plus TLJN injection-treated BMECs on microglial proliferation was also found. ConclusionTLJN reduced the expression of MIP-1β and CCR5 in OGD-injured BMECs, and the CM of OGD-injured plus TLJN injection-treated BMECs inhibited the proliferative activity of microglial cells, suggesting the therapeutic potential of TLJN on ischemic cerebral vascular disease.

IL-27 Enhances LPS-Induced Proinflammatory Cytokine Production via Upregulation of TLR4 Expression and Signaling in Human Monocytes

IL-27, which is produced by activated APCs, bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed, proinflammatory and anti-inflammatory activities are attributed to IL-27, and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however, the molecular mechanism behind this phenomenon is not understood. In this study, we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6, TNF-α, MIP-1α, and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming, we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore, IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge, this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections.

Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry

An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of β2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1β expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes.

Endothelin-1–Induced Macrophage Inflammatory Protein-1β Expression in Monocytic Cells Involves Hypoxia-Inducible Factor-1α and AP-1 and Is Negatively Regulated by microRNA-195

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1β or CCL4. ET-1-induced MIP-1β mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1β mRNA. Moreover, ET-1-induced MIP-1β mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.

Expansion of Functionally Skewed CD56-Negative NK Cells in Chronic Hepatitis C Virus Infection: Correlation with Outcome of Pegylated IFN-α and Ribavirin Treatment

NK cells are important innate immune effector cells, normally characterized as CD56(+)CD3(-) lymphocytes. In this study, we report that CD56(-)CD16(+) NK cells expand in many patients with chronic hepatitis C virus infection. These CD56(-) NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56(dim) and CD56(bright) NK cell subsets. In particular, CD56(-) NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1beta, IFN-gamma, TNF-alpha, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1beta alone dominated their response. CD56(-) NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56(dim) NK cells. Interestingly, pretreatment levels of CD56(-) NK cells correlated with the outcome of pegylated IFN-alpha and ribavirin treatment. In patients with CD56(-) NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56(-) NK cells functionally skewed toward MIP-1beta production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN-alpha and ribavirin.

Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge.

The rapid onset of massive, systemic viral replication during primary HIV/SIV infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation1–5. We hypothesized that vaccines designed to maintain differentiated “effector memory” T cell (TEM) responses5,6 at viral entry sites might improve efficacy by impairing viral replication at its earliest stage2, and have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM responses7–9. RhCMV vectors expressing SIV Gag, Rev/Nef/Tat, and Env persistently infected rhesus macaques (RM), regardless of pre-existing RhCMV immunity, and primed and maintained robust SIV-specific, CD4+ and CD8+ TEM responses (characterized by coordinate TNF, IFN-γ and MIP-1β expression, cytotoxic degranulation, and accumulation at extra-lymphoid sites) in the absence of neutralizing antibodies. Compared to control RM, these vaccinated RM showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated, limiting dose, intra-rectal challenge, including four animals that controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development: that vaccines capable of generating and maintaining HIV-specific TEM might decrease the incidence of HIV acquisition after sexual exposure.

Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

BackgroundMonitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals.MethodsWB (300 μl) was incubated at 37°C with specific antigens, in the form of peptides or commercial vaccines for 5–16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4°C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes.ResultsSpiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 μl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1β (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-γ gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination.ConclusionThis method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities.

Helioxanthin inhibits interleukin-1β-induced MIP-1β production by reduction of c-jun expression and binding of the c-jun/CREB1 complex to the AP-1/CRE site of the MIP-1β promoter in Huh7 cells

An elevated level of macrophage inflammatory protein-1β (MIP-1β) induced by IL-1β has been correlated with chronic hepatic inflammatory disease. However, molecular mechanism of IL-1β-induced MIP-1β expression in hepatic cells is obscure. Previously, we reported the mechanism of the anti-hepatitis B virus (HBV) activity of helioxanthin (HE-145). Here, we demonstrated that HE-145 inhibited IL-1β-induced MIP-1β expression in a dose-dependent manner in Huh7 cells. To understand the mode of action of HE-145, we first examined how IL-1β induced MIP-1β expression at the molecular level. Using selective inhibitors, we found that JNK and p38 pathways participated in IL-1β-induced MIP-1β expression. HE-145 specifically suppressed IL-1β-induced c-jun mRNA and protein expression and prevented c-jun-mediated AP-1 DNA-binding activity, whereas it had no effect on IL-1β-induced activation of JNK, p38 and ATF2. Further studies indicated that HE-145 may downregulate c-jun mRNA expression directly at transcriptional level without requirement of de novo protein synthesis. Mutational analysis and supershift assays indicated that IL-1β stimulated c-jun and CREB1 binding to the essential AP-1/CRE site of the MIP-1β promoter. The inhibitory effect of HE-145 on IL-1β-induced MIP-1β promoter activity was completely reversed by overexpressing c-jun. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay consistently revealed that HE-145 reduced c-jun binding to the AP-1/CRE site in vitro and in vivo. Our results established a major role for c-jun in IL-1β-induced MIP-1β expression in hepatic cells. The reduction in IL-1β-induced c-jun expression and subsequent binding of the c-jun/CREB1 complex to AP-1/CRE site mainly contributed to the inhibitory action of HE-145 on IL-1β-induced MIP-1β production.