Peripheral Blood Of Volunteers Research Articles

ELISPOT assay of the SARS-CoV-2 specific T cells immune response

The COVID-19 pandemic has stimulated interest in the development of biotechnology, as well as in the search for new solutions in the diagnostics of immune processes. The response of immunoglobulins A, M and G had a significant role in the assessment of virus-specific immune responses. Later, it was understood that for a comprehensive assessment of adaptive immunity processes, it is reasonable to study its cellular component. One of the most affordable methods for assessing T cell immunity, which has proven itself in the diagnosis of other infectious diseases, such as latent tuberculosis infection, is IGRA ELISPOT.The aim of the study. To determine SARS-СoV-2 specific immune response of T lymphocytes in vitro in the peripheral blood of volunteers from various groups using IGRA ELISPOT method. We evaluated the applicability of the method to assess T cell immune response to infection and vaccination. In addition, we determined the duration of the maintenance period of the SARS-CoV-2 specific T cells immune response induced by vaccination.Materials and methods. The study was carried out on venous blood samples of volunteers from three groups: 1) hospital patients with COVID-19; 2) COVID-19 convalescents; 3) vaccinated against COVID-19. The T cell immune response was assessed using the TigraTest® SARS-CoV-2 test system, which determines in vitro the number of T cells secreting interferon-gamma in response to stimulation with SARS-СoV-2 peptides in two antigens panels: 1) peptides of the spike protein (S); 2) peptides of N, M, Orf3a and Orf7a proteins.Conclusion. The IGRA ELISPOT assay is a specific and sensitive tool in the assessment of T cell immunity to the SARS-CoV-2 virus. The method makes it possible to assess SARS-CoV-2 specific T cell responses induced both by natural encounter with the pathogen and by vaccination. It is advisable to use the method in routine practice for comprehensive assessment of immunity to SARS-CoV-2.

Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells.

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.

Ixazomib Impairs Dendritic Cell Function and T Cell Proliferation and Affects the Development of GvHD in a Schedule-Dependent Fashion

Background: Targeting T cells alone has yielded limited success in the prevention of graft-versus-host disease (GvHD) following allogeneic blood and marrow transplantation (BMT). Dendritic cells (DCs) play a central role in alloreactivity and therefore represent a suitable target. Proteasome inhibitors (PI), with their ability to inhibit the function and maturation of DC, have prompted investigators to examine their potential role in the prevention of GvHD. The investigational PI, ixazomib (IXZ), dissociates rapidly from 20S and is therefore truly reversible. It is also orally bioavailable. Our aim in this study was to explore its effect on healthy volunteer peripheral blood dendritic and T cells and in a pre-clinical GvHD mouse model.Methods: To characterize the effects of IXZ on healthy volunteer peripheral blood DCs, DCs were isolated using EasySep Pan-DC Pre-Enrichment Cocktail with purity over 90% (STEMCELL Technologies). DCs were then treated with IXZ at different concentrations (10-40nM) for 4 hrs and then stimulated with lipopolysaccharide (LPS) for 16 hrs. After this treatment, DCs were surface stained with antibodies against maturation markers and analyzed by flow cytometry. DC survival was evaluated with 7AAD staining and FACS analysis. To assess the effect of IXZ on the production of pro-inflammatory cytokines, DCs were incubated with IXZ at increasing concentration before or after the addition of LPS. Total pro-inflammatory cytokines in the supernatant of tissue culture were measured using EMD Millipore cytokine arrays. Standard mixed lymphocyte reaction and T cell proliferation assays were used to evaluate T cell function. At a minimum, all experiments were done in triplicate. Unpaired t test was used for statistical analysis. A p-value < 0.05 was considered significant. The B6 → BALB/c pre-clinical GvHD model was adopted to evaluate the effect of IXZ on GvHD development. Mice were transplanted in 3 groups. Group 1 received a lethal dose of total body irradiation (TBI), donor bone marrow (BM) cells, and IXZ, group 2 received TBI, donor BM cells donor splenocytes, and a vehicle, and group 3 received TBI, donor BM cells, donor splenocytes, and IXZ. The dose of BM cells and splenocytes was 5 X 106 each. IXZ was given at 1.5 mg/kg subcutaneously. Two dosing schedules were tested in 2 separate experiments: day-1 and +2 or day +1 and +4.Results: IXZ inhibited the expression of 6 DC maturation markers including CD40, CD54, CD80, CD83, CD86 and CD197 (CCR-7). The inhibition started at a concentration of 10nM and was dose-related. IXZ also decreased the percentage of total DCs simultaneously expressing multiple markers. DCs viability remained unchanged in comparison to control at a concentration of 10nM and dropped to 68% and 43%, on average with concentrations of 20nM and 40nM, respectively. IXZ significantly decreased DC production of IL-6, IL-12, and IL-23 starting at the concentration of 20nM. IL-1β was decreased at the concentration of 40 nM. Importantly, there was no significant change in the cytokine production by DCs when IXZ was added 4 hrs after LPS except for IL-1β which increased at 30nM. Starting at the concentration of 10nM, IXZ dose-dependently inhibited T cell proliferation. At 40nM IXZ abolished T cells. In our in vivo study IXZ improved GvHD scores on days +7 and +11 in group 3 in comparison to group 2 when it was given on days -1 and +2. Conversely, when IXZ was given on day +1 and +4, group 3 mice had higher scores of GvHD and worse survival outcomes when compared to group 2. There was no noticeable drug toxicity in group 1 mice.Conclusion: In summary: 1) IXZ inhibits DC maturation with relative preservation of cell viability and inhibits pro-inflammatory cytokine production in DCs when added before LPS stimulation; 2) IXZ inhibits T-cell proliferation; 3) IXZ affects GvHD development in a schedule-dependent fashion with early administration improving and late administration worsening GvHD. Additional analysis of tissue and serum samples is in progress. These results provide background for careful design of clinical trials using IXZ for the prevention of GvHD. DisclosuresAl-Homsi:Millennium Pharmaceuticals: Research Funding.

ACP-196 Is a Second Generation Inhibitor of Bruton Tyrosine Kinase (BTK) with Enhanced Target Specificity

Background: Signaling through the B-cell receptor (BCR) results in cell survival and proliferation, and this pathway is critical for the progression of many B-cell malignancies, such a chronic lymphocytic leukemia (CLL). BTK, a TEC family kinase, is essential to the BCR signaling cascade. The recent approval of the first-in-class BTK inhibitor for the treatment certain B-cell malignancies, ibrutinib, has validated BTK as a clinical target. ACP-196 is a novel, second generation inhibitor of BTK with an IC50of 3 nM against purified BTK and EC50 of 8 nM in a human whole-blood CD69 B-cell activation assay. In vivo, oral administration of ACP-196 inhibits BCR-induced CD69 expression in murine splenic lymphocytes with an ED50 of 1.3 mg/kg compared with 2.9 mg/kg for ibrutinib. ACP-196 also has improved target specificity over ibrutinib with 323-,94-, 19- and 9-fold selectivity over the other TEC kinase familymembers (ITK, TXK, BMX, and TEC, respectively) and no activity against EGFR (Covey AACR, 2015). TXK and ITK are critical for T-cell receptor activation and development. ITK is also necessary for natural killer (NK)-cell mediated antibody dependent cell cytotoxicity (ADCC), which is important for the efficacy of monoclonal antibody therapy, eg, obinutuzumab. Additionally, therapeutic blockade of EGFR signaling is employed for treatment of epithelial cancers and is associated with adverse events, such as diarrhea and skin rash. Here, we describe results from signaling and functional assays to further elucidate the differential effects of ACP-196 on primary CLL cells compared with T-cells, NK-cells and epithelial cells.Methods: CLL patient samples were evaluated for pathway activation in the presence and absence of ACP-196 using phospho-protein immunoblotting. Helper T-cell (Th) skewing assays were performed using purified naive CD4+ T-cells from C57BL/6, Itk knock out (KO) and Itk/Txk double KO mice in the presence ACP-196 or ibrutinib. CD8+ T-cell activity was assessed by cytolytic activity (CTL) assays using T-cells isolated from C57BL/6 mice. The CTL response was initiated with various effector:T-cell ratios using C57BL/6 T-cells and murine BALB/c lymphoblasts. To evaluate NK-cell ADCC function, standard chromium release assays were done using purified NK-cells isolated from healthy volunteer peripheral blood mononuclear cells and cultured with obinutuzumab-coated (10 µg/mL) CLL cells in the presence and absence of ACP-196. Immunoblotting of H460 lung cancer cells for phospho-EGFR was used to evaluate the effect of ACP-196 on EGFR signaling.Results: ACP-196 treatment of primary human CLL cells showed a dose-dependent (0.01μM-1μM) decrease in p-BTK (Y223), p-ERK (T202/Y204) and p-AKT (T308) and p-IκB-α (S32), without impact on total protein levels. These findings demonstrate ACP-196 inhibits autophosphorylation of BTK as well as down-stream effectors of BCR signaling.Phospho-protein immunoblotting of primary human T-cells and Jurkat cells showed that ACP-196 does not inhibit T-cell receptor signaling molecules downstream of ITK and TXK such as IκBα, NFAT, or JunB. In in vitro helper T cell skewing assays, ACP-196, unlike ibrutinib, showed no effect on differentiation of Th subsets (Th1/Th2/Th17) or regulatory T cells (Tregs). The effect of ibrutinib on Th and Treg differentiation was similar to that observed in cells from Itk KO and Itk/Txk KO mice. In addition, ACP-196 had no effect on CD8+ T-cell viability or cytotoxicity as compared with ibrutinib-treated CD8+ T cells (46% and 36% reduction, respectively).In the in vitro ADCC assay, ACP-196 had no effect on NK cell function (chromium release of 34.8% vs 39.6% for the vehicle control). Lastly, at clinically relevant concentrations ACP-196 did not attenuate autophosphorylation of EGFR in H460 lung epithelial cells demonstrating no effect of ACP-196 on EGFR signaling.Conclusions: ACP-196 selectively inhibits BTK and BCR signaling at pharmacologic concentrations without off-target effects on T-cells, NK-cells and lung epithelial cells. ACP-196 is currently in Phase 3 trials for the treatment of CLL (ClinicalTrials.gov NCT0247568 and NCT02477696).This work was supported by the NIH NCI R01CA197870 and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Kaptein:Acerta Pharma: Employment. Van Lith:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Research Funding.

B7-H3 Silencing By RNAi Inhibits Tumor Progression and Enhances Chemosensitivity in U937 Cells

Background: Aberrant expression of the immunoregulatory protein B7-H3 in B7 family has been associated with more advanced disease and poor prognosis in a wide range of cancer. However, the role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated. In this study, we examined the gene expression and subcellular localization of B7-H3 in human peripheral blood cells and hematologic malignancy cell lines. Then, we determined the effects of down-regulating B7-H3 expression on U937 cells, which has the highest B7-H3 protein expression in the detected cell lines.Methods: B7-H3 expression in 12 healthy volunteer peripheral blood cell samples and 13 human hematologic malignancy cell lines was determined by RT-PCR, western blot and flow cytometry. B7-H3 knockdown in the U937 cell line was performed using shRNA lentivirus transduction. The effects on cell proliferation, cycle, migration and invasion were investigated by CCK-8 assay, methyl cellulose colony forming assay, PI staining, and transwell assays in vitro. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and Ki-67 and PCNA was detected through immunohistochemical. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using CCK-8, Annexin V-FITC/PI and Hoechst 33342 staining assays in vitro. The therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs were also studied by U937 xenograft models in vivo.Results: B7-H3 mRNA was widely expressed in the 12 hematologic malignancy cell lines except for CZ1 and PB MNCs of volunteers. But the protein level of B7-H3 was only abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. The down-regulation of B7-H3 in U937 cells significantly decreased cell growth and the rate of colony formation by 32.8% in 72 h and 70.3% in 14 d. Mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, U937 cell cycle was arrested at G0/G1 phase, and the cell cycle-related proteins Cyclin D1 and CDK4 were lower. Cell migration rate of B7-H3 knockdown cells was reduced more than five-fold, and invasion capacity was decreased by 86.7%. The rates of distant metastasis in B7-H3 knockdown xenografts were significantly decreased. The invasion-related proteins MMP-2 and MMP-9 were lower in both B7-H3 knockdown cells and xenografts. B7-H3 RNAi profoundly increased the anti-tumor effect of chemotherapy and enhanced the activity of caspase-3 in vitro and in vivo. At the end of observation (on day 19 after inoculation), inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine groups were 70.5%, 80.0%, and 90.0%, respectively (P =0.006, 0.004 and 0.016). The TUNEL positive cells were significantly increased in the B7-H3 shRNA combined with chemotherapy drugs groups.Conclusions: B7-H3 protein was abnormally overexpressed in 12 hematologic malignancy cell lines except for CZ1, with subcellular localizations in nucleus and cytoplasm mostly determined. B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia. DisclosuresNo relevant conflicts of interest to declare.

[Amplification Ex Vivo and Cytocidal Activity of Leukemia Tumor-Associated Antigen-Specific Cytotoxic T Lymphocytes].

To explore the feasibility of amplifying the leukemia tumor associated antigens-specific cytotoxic T lymphocytes (TAA-CTL) ex vivo and to evaluate the cytotoxicity of TAA-CTL. The peripheral blood mononuclear cells were enriched by density gradient centrifugtion; TAA-CTL were generated by stimulation of PBMNC with peptide-pulsed DC at an effector-to-target ratio of 10:1; immunophenotype of TAA-CTL was analyzed by flow cytometry; cytotoxicity assay was used to evaluate the cytotoxic activity of TAA-CTL against peptide-pulsed autologous target cells (PHA-Blasts). TAA-CTL expanded from volunteer showed a mean expansion of 3.81±1.61, the phenotyping of the TAA-CTL was predominantly CD3+ (97.22±0.71)% with varying content of CD4+ (41.47±27.08)% and CD8+ (56.40±11.15)% T cells, it also contained few nature killer cells (0.50±0.31)% and rare residual B cells (0.14±0.20)%; the subpopulations of TAA-CTL and CTL were not statisticaly significantly different in the proportion (P>0.05); the detection of intracellular cytokines after stimulation with peptide showed that the secretion rates of IFN-γ and TNF-α in CD8+ TAA-CTL were (27.67±2.21)% and (34.2±0.71)%, while the secretion rates were (21.6±2.55)% and (9.97±3.44)% in CD4+ TAA-CTL. Compared with the CD8+ TAA-CTL group, the secretion rates of IFN-γ and TNF-α were (1.36±0.04)% and (5.58±0.03)% in CD8+ CTL, the rates of IFN-γ and TNF-α were (0.91±0.06)% and (1.60±0.07)% in CD4+ CTL. The secretions of IFN-γ and TNF-α in CTL were both significantly lower than those in TAA-CTL (P<0.01); the specific killing efficiency of the TAA-CTL against TAA-pulesd target cells were (77.00±1.00)%, (67.40±3.60)%, (60.55±2.45)% and (26.85±5.25)%, when the effecto-target ratios were 40:1, 20:1, 10:1 and 5:1, and there was negligible lysis of TAA-CTL for PHA-blast (P<0.01). TAA-CTL can be successfully induced and generated ex vivo from the healthy volunteer peripheral blood, and the TAA-CTL possess a specific killing activity.

Effect of novel proteasome and immunoproteasome inhibitors on dendritic cell maturation, function, and expression of IκB and NFκB

Dendritic cells (DC) play a central role in the pathophysiology of graft versus host disease (GvHD). Their antigen presenting capacity is nuclear factor κB- (NF-κB) dependent. Consequently, DC have emerged as a potential target for the prevention of GvHD and clinical trials with bortezomib are underway. We explored the activity of novel proteasome and immunoproteasome inhibitors on healthy volunteer peripheral blood DC. After incubation with the drug or drug combination, DC were stimulated with lipopolysaccharide, stained for maturation surface markers and then analyzed by flow cytometry. We found that the different molecule(s) inhibited DC maturation marker expression to variable degrees, with the constitutive proteasome-selective agent being the least active. In a DC and allogeneic CD4+ mixed lymphocyte reaction, DC incubation with the studied proteasome and immunoproteasome inhibitor(s), impeded T cell proliferation as measured by BrDU incorporation. Finally, we found that DC incubation with the drug(s) enhanced IκB expression and that oprozomib inhibited NF-κB expression. We concluded that based on its activity and oral bioavailability, oprozomib merits further investigation in an animal GvHD prevention model. We also suggest that altering IκB and NF-κB expressions may, in DC, represent a new mechanism of action of proteasome and immunoproteasome inhibitors.

Dendritic Cell Depletion in Burn Patients Is Regulated by MafB Expression

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23–68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.

Relationship between the phenotypes and functions of peripheral blood dendritic cells and the different spleen deficiency syndrome types in patients with chronic hepatitis B

To study the phenotypes and functions of dendritic cells (DCs) derived from peripheral blood monocytes of chronic hepatitis B (CHB) patients with different traditional Chinese medicine (TCM) syndrome types, and to explore the relationship between TCM syndrome type and DC functions. Sixty CHB patients were included in this study. All the CHB patients were divided into spleen deficiency and liver stagnation, spleen deficiency and dampness-heat and deficiency of both spleen and kidney groups according to TCM syndrome diagnosis standard. There were 20 cases in each group, and ten healthy people were included as normal control. The volunteer's peripheral blood was collected for monocyte separation, biochemical test and hepatitis B virus DNA loads detection. DCs were induced and isolated from peripheral blood monocytes, and then the expressions of surface markers CD80, CD86, CD1a and HLA-DR were detected by flow cytometric analysis method. Interleukin-10 (IL-10) production of the DCs was quantified by enzyme-linked immunosorbent assay. The proliferation of DCs in the CHB patients was slower than that in the healthy volunteers (P<0.05). The expressions of DC surface molecules such as CD80, CD86, and CD1a were obviously decreased in the CHB patients as compared with those in the healthy volunteers (P<0.05). More over, expressions of DC surface molecules were different among CHB patients with different TCM syndrome types. The positive expressions of CD80, CD1a, and HLA-DR in the CHB patients with spleen deficiency and liver stagnation were obviously higher than those in the CHB patients with deficiency of both spleen and kidney (P<0.05), and the CD1a expression in the CHB patients with spleen deficiency and dampness-heat was higher than that in the CHB patients with deficiency of both spleen and kidney (P<0.05). In DC culture supernatant, the IL-10 concentration of the CHB patients with deficiency of both spleen and kidney was higher than that of the CHB patients with spleen deficiency and liver stagnation (P<0.05), and the IL-10 concentrations of the CHB patients with different TCM syndrome types were higher than that of the healthy volunteers (P<0.05). During the pathogenic course of CHB, the phenotypes and functions of DCs are different in CHB patients with different TCM syndrome types. It suggests that there is a correlation between TCM syndrome type and body immunity function.

BAX UTR G (-248) A Polymorphism Has No Impact on Survival in Patients with Multiple Myeloma.

Multiple myeloma is a malignant tumour of plasma cells that remains incurable for the vast majority of patients with a median survival of 2–3 years. The bax protein is a member of the Bcl-2 family and is an important pro-apoptotic protein. Low BAX expression has been shown to be associated with prolonged survival in patients with myeloma(1). A novel polymorphism, G(-248)A in the BAX promoter in patients with Chronic lymphocytic leukaemia (CLL) has shown to be associated with reduced Bax protein expression, progressive disease and treatment failure(2, 3). We report here the impact of this polymorphism on survival in 304 (M: 168; F: 136) patients with myelomaMaterials and MethodsThis study included 158 (M: 89; F: 69) patients from MRC Myeloma VII trial and 146 (M: 79; F: 67) under our care from January 1992 and February 2006. DNA was extracted from myeloma patients and healthy volunteer's peripheral blood using a standard salting out method. Analysis of the 5-UTR polymorphism was undertaken using method already published by our centre(3). Patients received either intensive or non intensive antimyeloma therapy as per standard practice. Statistical analysis was carried out using SAS software.ResultsThe frequency of GG and GA/AA genotypes was 81.6% and 18.4% in myeloma patients as compared to 85.2% and 14.8% in normal controls. Median age at diagnosis was 58 yr (range 34–87 yrs). (GA/AA Group: 58 yrs (42–85); GG Group: 58 yr (34–87)). There were no significant differences in the sex distribution, β2 microglobulin, haemoglobin, bone lesions, calcium and age between the GA/AA & GG groups with p values 0.72, 0.46, 0.95, 0.61, 0.49 and 0.46 respectively. Abnormal creatinine levels were significantly few in GA/AA group (p value: 0.002). Median survival for the AA/GA group was 48 months (95% CI 36 – 61 months) compared with 54 months in the GG group (95% CI 46 – 63 months)[p=0.5 log-rank test] with a median follow up of 62 months (range 1 to 141).ConclusionsOur results show that the GA/AA polymorphism has no impact on the overall survival in patients with myeloma. Further work is ongoing in order to assess the impact of this polymorphism on response in various treatment groups.

Transcriptosome and Serum Cytokine Profiling of B-CLL Identifies Oncogenic Pathways in Pathogenesis and Survival.

Background: B-cell chronic lymphocytic leukemia (B-CLL) the most common leukemia in the Western world accounts for 25% of all newly diagnosed leukemias. Despite new therapeutic advances, B-CLL is currently not curable. Potential oncogenic signaling pathways in B-CLL require elucidation. We identified ROR-1 receptor tyrosine kinase (RTK) in the WNT/Planar Cell Polarity non-canonical pathway as a possible mechanism of oncogenesis by gene expression profiling (GEP).Methods: Mononuclear cells from 8 low stage CLL patients were obtained through an IRB approved protocol and analyzed utilizing the HG-U133A 2.0 Affymetrix array (~18,400 transcripts, 22,000 probe sets) after isolating and purifying total RNA (Qiagen, RNAeasy). The control RNA samples were isolated from 8 normal volunteer peripheral blood (PB) B-cells (AllCell, CA) and a normal reactive lymph node. Tumor lineage was confirmed by immunohistochemistry (IHC). Quantitative real time RT-PCR was performed on 20 selected genes to validate the microarray GEP. Twenty one B-CLL patients (includes the 8 patients from the GEP) were evaluated by RT-PCR for several key components of oncogenic signaling pathways based on the GEP with gene specific probes. A serum cytokine profile (120 cytokines) on 12 CLL patients (8 for which GEP is available and 4 additional patients) and 4 normal volunteers were performed for identification of a signature for diagnostic and prognostic value.Results: Data are represented as “robust” increases or decreases of relative gene expression common to 8 patients. ROR-2, a close member of ROR-1, has been shown to bind Wnt-5A via the cysteine rich domain (CRD) and to activate the JNK signaling pathway. Our findings identify over-expression of members of the non-canonical WNT/PCP- ROR-1- signaling pathway genes that were validated by RT-PCR. A serum cytokine profile of 12 patients provides a signature that may be useful in CLL diagnosis and prognosis.Conclusions: GEP identified WNT/PCP-ROR-1 as key components of an autocrine pathway that helps B-CLL avoid apoptosis. Several serum cytokines are elevated and require validation as potential diagnostic and prognostic markers. GEP of B-CLL in combination with quantitative real time RT-PCR has identified several novel targets for therapy. The identification of ROR-1 RTK has led to the development of a molecular target for future therapeutic application. Several lead compounds have been identified and are being evaluated as potential therapies in B-CLL.

Differentiation and immune function of human dendritic cells following infection by respiratory syncytial virus

RSV causes annual epidemics of bronchiolitis in winter months resulting in the hospitalization of many infants and the elderly. Dendritic cells (DCs) play a pivotal role in coordinating immune responses to infection and some viruses skew, or subvert, the immune functions of DCs. RSV infection of DCs could alter their function and this could explain why protection after natural RSV infection is incomplete and of short duration. In this study, this interaction between DCs and RSV was investigated using a human primary culture model. DCs were generated from purified healthy adult volunteer peripheral blood monocytes. Effects of RSV upon DC phenotype with RSV primed DCs was measured using flow cytometry. Changes to viability and proliferation of cocultured DCs and T-cells were determined using microscopy with fluorescent dyes (Hoechst 33342 and propidium iodide). DC maturation was not prevented by the RSV challenge. RSV infected a fraction of DCs (10-30%) but the virus replicated slowly in these cells with only small reduction to cell viability. DCs challenged with RSV stimulated T-cell proliferation less well than lipopolysaccharide. This is the first study to demonstrate RSV infection of human monocyte derived DCs and suggests that the virus does not significantly interfere with the function of these cells and potentially may promote cellular rather than humoral responses.

Optimized Flow Cytometric Analysis of Endothelial Progenitor Cells in Peripheral Blood

Although flow cytometry is a rapid and convenient way to measure the number of circulating endothelial progenitor cells (EPC), there is no standard technique for preparation and measurement. The aim of this study was to present an optimized preparation method for EPC measurement which should serve as a standard to facilitate the comparison of the results in stem cell investigations by different research groups. We have looked for the preparation method which delivered the best immunostaining with the directly conjugated antibodies against VEGF R2, CD133, CD34, and CD45. In order to test the sensitivity of the method, we determined the number of EPC in the peripheral blood of volunteers by flow cytometry and by cell culture assay. Furthermore, we have evaluated the influence of different durations of conservation on the EPC cell count. The pre‐treatment of blood samples with 0.2% formaldehyde for 30 minutes delivers the best immunostaining, and blood samples can be stored overnight at 4°C without loss of counting rate for EPC. We found an excellent correlation (r=0.98) between the flow cytometric measurement and the cell count of the cell culture method. The presented protocol for the flow cytometric measurement of EPC in the peripheral blood can be used as a diagnostic or prognostic tool; we propose this protocol as the standard for EPC quantification.

Major upper gastrointestinal surgery is associated with an antigen-dependent proinflammatory T cell response

T cells play a central role in the immune response to injury. Oesophageal and pancreatic resections are associated with significant risk of systemic inflammatory response syndrome and sepsis. This study involved a detailed analysis of T cell function in a consecutive cohort of patients undergoing such surgery. Twenty-five patients undergoing major oncological upper gastrointestinal surgery were investigated for T cell expression of Fas and the activation markers CD69 and CD25, as well as interleukin (IL) 2, IL-10 and interferon (IFN) gamma responses to stimulation with staphylococcal enterotoxin B (SEB). T cell activation in healthy volunteer peripheral blood mononuclear cells was studied in co-culture with patients' serum, either alone or with cytotoxic T lymphocyte-associated antigen (CTLA) 4, an inhibitor of antigen presentation. T cells expressed significantly raised levels of CD69 and CD25 after surgery, but no change in Fas expression was evident. There was a significant increase in the production of IL-2 after surgery without a concomitant increase in IFN-gamma or IL-10 in response to SEB. Postoperative serum activated healthy volunteer T cells, a response that was inhibited (P = 0.053) by co-incubation with CTLA-4. Major surgery results in pan-T cell activation via a serum-mediated antigenic mechanism that is independent of Fas expression. Postoperative T cells are primed for an augmented proinflammatory response to superantigen, sustained for at least 1 week, implicating the adaptive immune system in the development of the postoperative systemic immunoinflammatory state.

The Effect of IVIG on Fcγ Receptor-Dependent Monocyte Phagocytosis and FcγR Gene Expression.

BACKGROUNDThe mechanism(s) of intravenous immunoglobulin (IVIG) towards inhibition of monocyte phagocytic activity involves the function and/or the expression of inhibitory FcγRIIb in a murine model. To confirm these findings in human monocytes, we used a human monocyte phagocytic model in vitro to study the effects of IVIG on the phagocytic activity and the expression of FcγR genes.METHODSPart A: Monolayer Monocyte Phagocytosis AssayNormal volunteer's peripheral blood mononuclear cells (PBMC) were isolated from heparin anticoagulated blood by Ficoll-Hypaque (Pharmacia Biotech) density separation. The PBMCs were washed and the monocytes were purified using a magnetic bead-positive selection method with anti-CD14 antibody (Miltenyl Biotec). 105 monocytes were incubated in a microtiter plate at 37°C for 1 hour before exposed to IVIG 0.5 g/L. Anti-D (WinRho) sensitized Rh positive (R2R2) red cells were added to the monocytes at 0.5 hour and 18 hour post-IVIG treatment. After 1 hour incubation with sensitized RBC, monocytes phagocytic activity is measured by chemiluminescence detection with a LumiCount (Packard). The readings were normalized with maximal chemiluminescence signal achieved by the monocytes without prior exposure to IVIG (positive control). Part B: RT-PCR of FcγRIIa and FcγRIIbAfter 18 hours of exposure to two different concentrations of IVIG (0.5 and 5 gm/L), monocytes were collected and total RNA was isolated with TRIzol reagent (Invitrogen). 1 μg of RNA was used to generate first strand cDNA using Superscript II RT kit (Invitrogen). FcγRIIa and IIb were amplified with AmpliTaq Gold DNA polymerase system (Applied Biosystems). The PCR products were evaluated by polyacrylamide gel electrophoriesis.RESULTSPart A:Dose-response curves were generated by plotting normalized chemiluminescence against the concentration of anti-D used to sensitize the red cells. Anti-D sensitized red cells were phagocytosed by monocytes in a dose-dependent manner. There is a time-dependent inhibition of monocyte phagocytosis when monocytes were incubated with IVIG at 0.5 gm/L. (Fig. 1)Part B:There is no significant difference in the gene expression of FcRγIIb and FcγRIIa in the adherent monocytes after incubating with either low dose (0.5 gm/L) or high dose (5 gm/L) of IVIG for 18 hours. (Fig 2)CONCLUSIONDelayed inhibition of phagocytic activity with18-hour exposure to IVIG is not directly mediated via the modulation of FcγRIIb gene expression in human monocytes. Other mechanisms, such as intracellular signalling or receptor coupling, might be involved in the delayed inhibitory effects of IVIG. [Display omitted] [Display omitted]

The voltammetric behavior of bone marrow of leukaemia and its clinical application

The electrochemical voltammetric behavior of bone marrow of leukaemia has been investigated by a self-devised cytosensing system. The two anodic peaks of erythrocytes (red blood cells, RBC) in bone marrow of leukaemia appeared at 0.73±0.03 and 0.83±0.02V vs. SCE, respectively, on the first scan. The anodic peak of leukocytes (white blood cells, WBC) appeared at 0.32±0.03V vs. SCE. The anodic peak of RBC at 0.83V disappeared when the patients were cured. The experimental results show that the voltammetric behavior of erythrocytes is in constant contact with the initial stage of leukaemia. The cyclic voltammetric behavior of 40 cases of leukaemia including acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML) and 10 cases of healthy volunteer peripheral blood was studied. The cyclic voltammetric behavior of erythrocytes may provide a simple and specific marker for diagnosis of leukaemia.

THE EFFECTS OF SHORT-TERM TREATMENT WITH LEVAMISOLE ON CYTOKINES IN VOLUNTEERS AND CANCER-PATIENTS

In search for clues to the potential immunomodulating mechanism of action of levamisole which might be used as monitoring parameters, we have determined a variety of cytokines in the peripheral blood of volunteers and carcinoma patients before and after a single or a 3-day-treatment with 150 mg/day. In cancer patients no changes could be detected 4 days after a 3-day-treatment course in the levels of TNF-alpha, IL-1beta, IL-2 or IL-6. In a placebo-controlled volunteer study the same treatment did not affect the levels of beta2-microglobulin, IL-1beta, IL-1alpha, IL-2 or IL-6. However, 24hr after the last treatment the concentration of neopterin was slightly but significantly increased and the concentration of soluble IL-2 receptors decreased. A single treatment failed to produce such an effect. It is suggested that the measurement of neopterin and soluble IL-2 receptors may provide useful information in future trials.