Peripheral Blood Of Healthy Individuals Research Articles

IFN-α production by plasmacytoid dendritic cell associations with polymorphisms in gene loci related to autoimmune and inflammatory diseases.

The type I interferon (IFN) system is persistently activated in systemic lupus erythematosus (SLE) and many other systemic autoimmune diseases. Studies have shown an association between SLE and several gene variants within the type I IFN system. We investigated whether single-nucleotide polymorphisms (SNPs) associated with SLE and other autoimmune diseases affect the IFN-α production in healthy individuals. Plasmacytoid dendritic cells (pDCs), B cells and NK cells were isolated from peripheral blood of healthy individuals and stimulated with RNA-containing immune complexes (ICs), herpes simplex virus (HSV) or the oligonucleotide ODN2216. IFN-α production by pDCs alone or in cocultures with B or NK cells was measured by an immunoassay. All donors were genotyped with the 200K ImmunoChip, and a 5 bp CGGGG length polymorphism in the IFN regulatory factor 5 gene (IRF5) was genotyped by PCR. We found associations between IFN-α production and 18-86 SNPs (P ≤ 0.001), depending on the combination of the stimulated cell types. However, only three of these associated SNPs were shared between the cell-type combinations. Several SNPs showed novel associations to the type I IFN system among all the associated SNPs, whereas some loci have been described earlier for their association with SLE. Furthermore, we found that the SLE-risk variant of the IRF5 CGGGG-indel was associated with lower IFN-α production. We conclude that the genetic variants affecting the IFN-α production highlight the intricate regulation of the type I IFN system and the importance of understanding the mechanisms behind the dysregulated type I IFN system in SLE.

Monocyte-derived dendritic cells can induce autoreactive CD4(+) T cells showing myeloid lineage directed reactivity in healthy individuals.

T cells against self-antigens can be detected in peripheral blood of healthy individuals, although intrathymic negative selection removes most high-avidity T cells specific for self-antigens from the peripheral repertoire. Moreover, spontaneous T-cell proliferation following stimulation with autologous monocyte-derived dendritic cells (autoDCs) has been observed in vitro. In this study, we characterized the nature and immunological basis of the autoDC reactivity in the T-cell repertoire of healthy donors. We show that a minority of naive and memory CD4(+) T cells within the healthy human T-cell repertoire mediates HLA-restricted reactivity against autoDCs, which behave like a normal antigen-specific immune response. This reactivity appeared to be primarily directed against myeloid lineage cells. Although cytokine production by the reactive T cells was observed, this did not coincide with overt cytotoxic activity against autoDCs. AutoDC reactivity was also observed in the CD8(+) T-cell compartment, but this appeared to be mainly cytokine-induced rather than antigen-driven. In conclusion, we show that the presence of autoreactive T cells harboring the potential to react against autologous and HLA-matched allogeneic myeloid cells is a common phenomenon in healthy individuals. These autoDC-reactive T cells may help the induction of primary T-cell responses at the DC priming site.

Stimulation of bacterial biofilms on Th17 immune cells.

We investigated the role of bacterial biofilms in stimulating T helper 17 (Th17) cells in infected organisms. The formation of bacterial biofilms isolated from clinical lavage fluid samples was measured. Th17 cells and interleukin 17 (IL-17) levels in the peripheral blood of healthy individuals, people infected by biofilm bacteria, people infected by non-biofilm bacteria, and in the lavage fluid from people infected by bacteria were determined. Differences in those data were tested using the SPSS 17.0 statistical software. Th17 cells and IL-17 levels in the peripheral blood of biofilm bacteria-infected people, non-biofilm bacteria-infected people, and healthy controls were 0.59 ± 0.18% and 108.8 ± 20.5 pg/mL; 0.58 ± 0.18% and 100.1 ± 20.7 pg/mL; and 0.55 ± 0.17% and 100.0 ± 21.4 pg/mL, respectively; there were no statistically significant differences (P > 0.05). Th17 cells and IL-17 levels in the lavage fluid of biofilm bacteria-infected people and non-biofilm bacteria-infected people were 1.37 ± 0.34% and 157.4 ± 30.8 pg/mL; and 1.11 ± 0.21% and 136.2 ± 24.3 mg/mL, respectively; the differences were statistically significant (P < 0.05). Bacterial biofilms can increase the expression levels of Th17 cells and IL-17 in local infections; this may be the mechanism by which chronic injuries are caused by biofilm infections.

Relationship between phosphatidylserine exposure and serum-dependent opsonization in phagocytes

AIM: To investigate the relationship between the exposure of phosphatidylserine(PS) and serum-dependent opsonization in peripheral blood mononuclear cells(PBMCs), to lay a foundation for exploring the influence of physiological microenvironment on the function of phagocytes.MET HODS: PBMCs were isolated from the peripheral blood of healthy individuals using Ficoll-paque and treated by RPMI 1640 medium with or without serum after washing with PBS. The phosphatidylserine(PS) exposure was detected by annexin V staining before and after in vitro serum incubation. Malondialdehyde(MDA) in treated PBMCs was examined by spect rophotometry to evaluate the interference of lipid peroxidation. The PS exposure in different subsets of PBMCs was analyzed by flow cytometry with antiCD3, anti-CD56 and anti-HLA-DR staining. Phagocytosis was detected by FCM after incubation with bacteria expressing green fluorescent protein(GFP). RESULTS: PS+ cells significantly increased in PBMCs after serum incubation, and shortterm serum incubation immediately induced the increase of PS+ cells. Serum incubation had no influence on the membrane MDAcontent, which excluded the interference by the products of lipid peroxidation. Cell subset analysis showed that HLA-DR+ mononuclear phagocytes were the main cells exposing PS in PBMCs and significantly increased in Annexin V+ cells. Bacterial phagocytosis showed that the exposed PS was involved in the serumdependent opsonization. CONCLUSION: The exposure of PS is serumdependent and is involved in the opsonization of bacteria by PBMCs.

AUTOANTIBODY LEVELS INFLUENCES THE NUMBER AND PHENOTYPE OF INFILTRATING NEUTROPHILS IN INFLAMED JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

AUTOANTIBODY LEVELS INFLUENCES THE NUMBER AND PHENOTYPE OF INFILTRATING NEUTROPHILS IN INFLAMED JOINTS OF PATIENTS WITH RHEUMATOID ARTHRITIS

Fluorinated Fe(III) salophene complexes: optimization of tumor cell specific activity and utilization of fluorine labeling for in vitro analysis.

Fluorine-substituted iron(III) salophene complexes (salophene = N,N'-bis(salicylidene)-1,2-phenylenediamine) were synthesized and evaluated for biological activity. All complexes showed growth inhibitory effects with IC50 values ranging from 0.05 to 2.45 μM against HT-29 colon carcinoma as well as MCF-7 and MDA-MB-231 mammary carcinoma cells (cisplatin: 5.75, 12.72, 5.81 μM, respectively). HR-CS MAS investigations revealed that the complexes were highly protein-bound already after an incubation period of 10 min and accumulated more effectively in tumor cells than cisplatin. Interestingly, the ligands were enriched in the cells too, indicating that the salophene moiety acts as a carrier ligand and mediates the uptake of the complexes. Furthermore, induction of apoptosis proved to be dependent on the substitution pattern as well as on the tumor cell line, as evidenced from the annexin V-FITC/PI assay. Most of the complexes, especially the highly active 5-Fe, showed tumor cell specific effects and no/less influence on the proliferation of T-cells generated from the peripheral blood of healthy individuals.

Methylation of the nonhomologous end joining repair pathway genes does not explain the increase of translocations with aging.

Chromosome translocations are especially frequent in human lymphomas and leukemias but are insufficient to drive carcinogenesis. Indeed, several of the so-called tumor specific translocations have been detected in peripheral blood of healthy individuals, finding a higher frequency of some of them with aging. The inappropriate repair of DNA double strand breaks by the nonhomologous end joining (NHEJ) pathway is one of the reasons for a translocation to occur. Moreover, fidelity of this pathway has been shown to decline with age. Although the mechanism underlying this inefficacy is unknown, other repair pathways are inactivated by methylation with aging. In this study, we analyzed the implication of NHEJ genes methylation in the increase of translocations with the age. To this aim, we determined the relationship between translocations and aging in 565 Spanish healthy individuals and correlated these data with the methylation status of 11 NHEJ genes. We found higher frequency of BCL2-JH and BCR-ABL (major) translocations with aging. In addition, we detected that two NHEJ genes (LIG4 and XRCC6) presented age-dependent promoter methylation changes. However, we did not observe a correlation between the increase of translocations and methylation, indicating that other molecular mechanisms are involved in the loss of NHEJ fidelity with aging.

Exercise before Scuba Diving Ameliorates Decompression-Induced Neutrophil Activation

The goals of this study were to investigate the difference in responses between a scuba dive preceded by aerobic exercise (EX) and a nonexercise control dive (CON) and to further evaluate the potential relation between venous gas emboli (VGE) and microparticles (MP). We hypothesized that exercise would alter the quantity and subtype of annexin V-positive MP and VGE. Nineteen divers performed two dives to 18 m seawater for 41 min separated by at least 3 d, one of which was preceded by 60 min of treadmill interval exercise. Blood was obtained before exercise, before diving, and 15 min, 2 h, 4 h, and 24 h after surfacing. Intravascular bubbles were quantified by transthoracic echocardiography at 15, 40, 80, and 120 min. The median VGE remained unchanged between the two dives; however, there was a significant increase in VGE in the exercise dive at 40 and 80 min at rest. MP were significantly elevated by approximately 2 times at all time points after CON compared with those after EX. Markers of neutrophil and platelet activation were elevated by both dives, and these elevations were attenuated in the EX dive. We conclude that some of the differences observed between the EX and CON related to MP and platelet and neutrophil activation provide additional insight into the potential protective benefits of exercise; however, further study is needed to understand the mechanism and true potential of these benefits.

Abstract 22: Interferon-γ produced by tumor-infiltrating NK cells and CD4+ T cells downregulates TNFSF15 expression in endothelial cells

Abstract Endothelial cells in an established vasculature secrete tumor necrosis factor superfamily-15 (TNFSF15; VEGI; TL1A) that functions as a negative modulator of neovascularization to maintain blood vessel stability. TNFSF15 gene expression diminishes at angiogenesis and inflammation sites such as in cancers and wounds. Interferon-γ (IFNγ) is a pleiotropic cytokine expressed primarily by lymphocytes of T cell and NK cell lineages. IFNγ may mediate anti-tumor effects either directly on cancer cell growth or indirectly through immunomodulatory and antiangiogenic responses. However, it was also reported that IFNγ promotes angiogenesis under certain circumstances. Here we show that IFNγ suppresses TNFSF15 expression in human umbilical cord vein endothelial cells (HUVEC). This activity is mediated by IFNγ receptor and the transcription factor STAT1. Since tumor-infiltrating NK and CD4+ T cells are the main sources of IFNγ in tumor lesions, we isolated these cells from peripheral blood of healthy individuals, treated the cells with ovarian cancer OVCAR3 cell-conditioned media, and found a 1- and 10-fold increase of IFNγ production in NK and CD4+ T cells, respectively, compared with that in vehicle-treated cells. These findings support the view that tumor-infiltrating NK and CD4+ T cells under the influence of cancer cells significantly increase the production of IFNγ, which in turn inhibits TNFSF15 expression in tumor vascular endothelial cells, shifting the balance of pro- and anti-angiogenic factors toward escalated angiogenesis potential in the tumor. Citation Format: Xin Gu, Tao Cheng, Luyuan Li. Interferon-γ produced by tumor-infiltrating NK cells and CD4+ T cells downregulates TNFSF15 expression in endothelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 22. doi:10.1158/1538-7445.AM2014-22

Effect of CIK on multidrug-resistance reversal and increasing the sensitivity of ADR in K562/ADR cells.

Leukemia is a leading cause of cancer-related mortality in children worldwide, and multidrug-resistance (MDR) is a main reason for tumor chemotherapy failure. The present study investigated the effects of ADR following incubation with cytokine-induced killer (CIK) cells on reversing MDR in K562/ADR cells. Mononuclear cells were isolated from the peripheral blood of healthy individuals and cultured in vitro in the presence of a combination of cytokines to generate CIK for K562/ADR cell treatment. A decreased level of P-glycoprotein expression and glutathione (GSH), an increased intracellular Rh-123 content, decreased mRNA and protein expression levels of MDR gene 1, MDR-associated protein 1, GSH S-transferase-π, B-cell lymphoma 2 and Survivin, and the decreased phosphorylation of AKT and the transcriptional activity of nuclear factor-κB and activator protein 1 were detected following ADR treatment in CIK co-cultured K562/ADR cells. Additionally, the level of ADR sensitivity and the apoptosis rate were increased in the CIK co-cultured K562/ADR cells. These results indicate that pre-treatment with CIK could reverse the MDR of K562/ADR cells, and that patients would be most likely to benefit from the combination of chemotherapy and CIK therapy.

Quest for Circulating Mesenchymal Stem Cells in Man.

Mesenchymal stem cells (MSC) are present in the bone marrow, from where they are thought to migrate via the bloodstream to sites of injury. On the other hand, virtually all tissues contain resident MSC that may contribute to local regenerative and immunomodulatory processes, thereby hypothetically pre-empting the need for recruitment of MSC via the bloodstream. Although there is some indication from animal models, the question remains whether there is solid evidence for the mobilization and migration of MSC in human. In the present study we investigated whether circulating MSC were present in the peripheral blood of healthy individuals and patients with organ injury. We were unable to detect MSC in the blood of healthy individuals by flow cytometry and cell culture techniques. We then analyzed the presence of MSC in the blood of patients with end-stage renal disease (n=10), end-stage liver disease (n=10) and in heart transplant patients with biopsy proven rejection (n=8), by culturing of mononuclear cells under MSC-supporting culture conditions. In none of these patients MSC were identified in the blood. In the stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we were able to detect MSC, indicating that the methods used enabled the detection of MSC. The conclusion of this study is that MSC are not detectable in the circulation in patients with injured solid organs and during aggressive immune responses.

Comparison of the Proportion of Non‐Classic (CD14+CD16+) Monocytes/Macrophages in Peripheral Blood and Gingiva of Healthy Individuals and Patients With Chronic Periodontitis

Monocyte subsets with low CD14 expression that coexpress CD16 (CD14+CD16+) are called non-classic or hyperinflammatory monocytes. Previous studies have reported an increase in the percentage of CD14+CD16+ monocytes in the peripheral blood of patients with chronic periodontitis (CP). To our knowledge, there are no reports demonstrating the presence of CD14+CD16+ monocyte-derived macrophages (MDMs) in the gingival tissue. The objective of this study is to identify the proportion of non-classic (CD14+CD16+) monocytes/macrophages in peripheral blood and gingiva of healthy individuals and patients with CP. A total of 60 individuals (n = 30 per group) were recruited for the study. Group 1 included 30 individuals with healthy gingiva, and group 2 included 30 patients with CP. Direct immunofluorescent staining was done in 200 μL whole-blood and single-cell suspensions obtained from gingival tissue, with fluorochrome-conjugated monoclonal antibodies against CD14, CD16, and human leukocyte antigen-DR (HLA-DR), and subjected to flow cytometric analysis. The mean percentage of CD14+CD16+ monocytes in the peripheral blood of healthy individuals was 9.10% ± 1.39%, and for patients with CP it was 14.18% ± 2.69% (P <0.05). The mean percentage of CD14+CD16+ MDMs in the gingival tissue of healthy individuals was found to be 0.93% ± 0.33%, whereas in patients with CP, it was 1.92% ± 0.78% (P <0.01). Non-classic monocytes/macrophages showed a high median fluorescent intensity for HLA-DR (DR++). This study demonstrates an increased proportion of CD14+CD16+HLA-DR++ monocytes/macrophages in the peripheral blood and gingiva of patients with CP.

Hypomethylating Agent Azacitidine Induces FoxP3 Negative HLA-G Expressing Immunoregulatory T Cells

The major obstacles in using FOXP3+ T regulatory cells as T-cell based immunotherapy against GVHD after allogeneic hematopoietic cell transplantation are their low numbers in the circulation and the lack of specific cell surface markers for efficient purification. DNA methylation has been considered to play a role in the regulation of T-cell effector function and cytokine gene expression, indicating a promising role of hypomethylating agents for immunomodulation. Recently it was shown that in-vitro treatment of conventional T-cells with hypomethylating agent azacitidine (aza) induced FoxP3 expression and converted CD4+CD25- cells into immunosuppressive T-cells the suppressor function of which is independent of FOXP3 expression (Choi et al Blood 116:129;2010), suggesting that aza induced suppressor function depends on the modification of other hypomethylated genes. Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule, shown to exert immunoregulatory functions, the expression of which is epigenetically regulated. In this study we investigated whether hypomethylating agent aza can induce HLA-G+ immunoregulatory T cells. We used CD3+ T cells from peripheral blood of healthy individuals isolated by MACS negative selection. T cells were stimulated with anti-CD3+ plus anti-CD28+ coated magnetic beads and then were treated for 72 hours with aza (0.5-15 mM) in the presence of 50U/ml inteleukin-2 (IL-2). Detailed phenotypical characterization of in-vitro aza treated cells was performed with flow cytomentry. Aza-induced FACS sorted HLA-G+ T cells, were irradiated and then used as third party cells in allogeneic mixed lymphocyte cultures (MLC). For the analysis of the in-vivo effect of aza on HLA-G expression, peripheral blood of patients with myelodysplastic syndrome (MDS) was obtained at baseline and after Vidaza treatment. In-vitro treatment of CD3+ T cells with aza increases the percentage of HLA-G+ cells, with maximum induction at a concentration of aza 7.5 mM in comparison to control (aza-0mM) (n=4, 9,11±0,6% vs 0,76±0,74%, p&lt;0,0001). However optimum aza concentration for the maximum HLA-G induction with the lowest toxicity in CD3 T cells was determined at 5 mM (HLA-G+:6,88±3,9%, p=0,0022). Aza treatment increases the percentage of CD4+CD25highFoxP3+ cells in culture. Maximum HLA-G induction was observed on CD4+CD25+ population (n=2, 9,21±5,5%). Aza treatment of FACS-sorted CD4+CD25negHLA-Gneg cells induced CD4lowCD25+HLA-G positive cells, revealing that aza induced HLA-G positive cells are not the result of a selectively expanded proexisting HLA-G+ population. Strickingly aza induced CD4lowCD25+HLA-G+ are FoxP3 negative. Aza induced HLA-G+ cells showed suppressive function (20% inhibition of proliferation) in allo MLCs, whereas HLA-G- selected cells had no effect.The percentage of peripheral blood HLA-G+ lymphocytes of healthy donors was 1,33±0,36% (n=4) in comparison to MDS patients which was 2,08±0,99% (n=2). In accordance to the in- vitro studies, preliminary data from MDS patients under Vidaza show up to 10 fold increase in CD4+CD25highHLA-G+ cells on day 28 post treatment. In conclusion, aza induces de-novo HLA-G expression on CD4+ T cells in-vitro and in-vivo. By using hypomethylating agent aza in-vitro, we generated a CD4lowHLA-G+ FoxP3neg immunoregulatory population, that is highly possible to be the aza induced FoxP3 independent T regulatory population.This strategy may provide an easy methodology to generate ex-vivo immunoregulatory T cells for adoptive immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Interferon-γ produced by tumor-infiltrating NK cells and CD4+ T cells downregulates TNFSF15 expression in vascular endothelial cells

Endothelial cells in an established vasculature secrete tumor necrosis factor superfamily-15 (TNFSF15; VEGI; TL1A) that functions as a negative modulator of neovascularization to maintain blood vessel stability. TNFSF15 gene expression diminishes at angiogenesis and inflammation sites such as in cancers and wounds. We reported previously that vascular endothelial growth factor and monocyte chemotactic protein-1 contribute to TNFSF15 downmodulation in ovarian cancer. Here we show that interferon-γ (IFNγ) suppresses TNFSF15 expression in human umbilical vein endothelial cells. This activity is mediated by IFNγ receptor and the transcription factor STAT1. Immunohistochemical analysis of ovarian cancer clinical specimens indicates that TNFSF15 expression diminishes while tumor vascularity increases in specimens with high-grades of IFNγ expression. Since tumor-infiltrating NK and CD4(+) T cells are the main sources of IFNγ in tumor lesions, we isolated these cells from peripheral blood of healthy individuals, treated the cells with ovarian cancer OVCAR3 cell-conditioned media, and found a onefold and tenfold increase of IFNγ production in NK and CD4(+) T cells, respectively, compared with that in vehicle-treated cells. These findings support the view that tumor-infiltrating NK and CD4(+) T cells under the influence of cancer cells significantly increase the production of IFNγ, which in turn inhibits TNFSF15 expression in vascular endothelial cells, shifting the balance of pro- and anti-angiogenic factors toward escalated angiogenesis potential in the tumor.

Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+regulatory T cells, evoking antitumor immune responses in humans

CD4(+) Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show that CCR4 was specifically expressed by a subset of terminally differentiated and most suppressive CD45RA(-)FOXP3(hi)CD4(+) Treg cells [designated effector Treg (eTreg) cells], but not by CD45RA(+)FOXP3(lo)CD4(+) naive Treg cells, in peripheral blood of healthy individuals and cancer patients. In melanoma tissues, CCR4(+) eTreg cells were predominant among tumor-infiltrating FOXP3(+) T cells and much higher in frequency compared with those in peripheral blood. With peripheral blood lymphocytes from healthy individuals and melanoma patients, ex vivo depletion of CCR4(+) T cells and subsequent in vitro stimulation of the depleted cell population with the cancer/testis antigen NY-ESO-1 efficiently induced NY-ESO-1-specific CD4(+) T cells. Nondepletion failed in the induction. The magnitude of the responses was comparable with total removal of FOXP3(+) Treg cells by CD25(+) T-cell depletion. CCR4(+) T-cell depletion also augmented in vitro induction of NY-ESO-1-specific CD8(+) T cells in melanoma patients. Furthermore, in vivo administration of anti-CCR4 mAb markedly reduced the eTreg-cell fraction and augmented NY-ESO-1-specific CD8(+) T-cell responses in an adult T-cell leukemia-lymphoma patient whose leukemic cells expressed NY-ESO-1. Collectively, these findings indicate that anti-CCR4 mAb treatment is instrumental for evoking and augmenting antitumor immunity in cancer patients by selectively depleting eTreg cells.

Abstract 1240: A flexible antibody-based strategy targeting CD71 for the treatment of AIDS-related non-Hodgkin's lymphoma.

Abstract AIDS related non-Hodgkin's lymphoma (AIDS-NHL) remains a significant clinical problem in the era of effective multiple-agent highly active anti-retroviral therapy (HAART). While an overall decrease in the incidence of AIDS-NHL has been seen in the HAART era, the risk for AIDS-NHL remains elevated, and not all AIDS-NHL subtypes have decreased in incidence, with the incidence of Burkitt's lymphoma (BL) remaining unchanged. In fact, AIDS-NHL is now the most common AIDS-related cancer in developed countries, where NHL accounts for 23-30% of all AIDS-related deaths. We have previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1/CD71), a receptor that is overexpressed on cancer cells due to their high demand for iron. This fusion protein exhibits an increased in vitro cytotoxicity against malignant hematopoietic cells compared to the parental antibody without avidin (ch128.1). This cytotoxicity is due to the ability of ch128.1Av to decrease cell surface TfR1, triggering its intracellular sequestration and degradation, with the subsequent induction of lethal iron deprivation. However, both ch128.1Av and ch128.1 confer protection in murine models of human disseminated multiple myeloma. ch128.1Av is also a universal delivery system and its cytotoxicity can be further enhanced by its conjugation with biotinylated drugs making it a unique molecule capable of a two-pronged attack against malignant cells through drug delivery and direct cytotoxic activity through iron starvation. The main goal of this study is to evaluate the potential use of ch128.1Av alone and as a delivery vehicle as a possible AIDS-NHL therapy. We found that, when used alone, ch128.1Av exhibits significant cytotoxic activity against two representative AIDS-NHL cell lines: 2F7 (AIDS-Burkitt's NHL) and RRBL (AIDS-diffuse large B-cell lymphoma). Importantly, the level of cytotoxicity was similar to that observed using the highly sensitive human B lymphoblastoid cell line IM-9. In addition, the cytotoxicity dramatically increases when this fusion protein is conjugated to biotinylated saporin 6 (b-SO6), a plant ribosome inactivating protein that blocks protein synthesis, resulting in an immunotoxin. We also found that non-activated (resting) B cells isolated from the peripheral blood of healthy individuals are resistant to ch128.1Av, but sensitive to ch128.1Av complexed to b-SO6, which can be explained by the different mechanisms of action of the fusion protein alone (iron starvation) and that of the immunotoxin (protein synthesis inhibition). We have previously reported ch128.1Av alone, or complexed to b-SO6, are not toxic to normal human hematopoietic stem cells, which can be explained by the lack of TfR1 expression on such cells. Our results suggest the potential use of anti-TfR1 antibody-mediated approaches as therapeutic interventions against AIDS-NHL. Citation Format: Tracy R. Daniels-Wells, Lai Sum Leoh, Daniel Widney, Dharma R. Thapa, Jose Leon Merino, Larry Magpantay, Otoniel Martínez-Maza, Manuel L. Penichet. A flexible antibody-based strategy targeting CD71 for the treatment of AIDS-related non-Hodgkin's lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1240. doi:10.1158/1538-7445.AM2013-1240

Peripheral Helper Lymphocytes Produce Interleukin 12 in Cancer Patients

The aim of the study was to seek evidence for the production of IL-12 by CD4+ T lymphocytes in in vitro and ex vivo trials. We performed in vitro trials with spleen cells from mice subjected to carcinogenesis, as well as ex vivo trials with cells obtained from the peripheral blood of healthy individuals and cancer patients. We were able to verify a significantly increased expression of IL-12 in CD4+ T lymphocytes from mice and patients with tumors, compared to controls. Follow-up studies are needed to clarify whether this difference is related to being in a chronic disease state or whether it is an attempt by the immune system to produce an anti-tumor response, since T lymphocytes from healthy donors were not able to produce IL-12 when in contact with polyclonal stimuli. We concluded that, in cancer, T helper cells are capable of synthesizing IL-12, raising the question of whether we are faced with another profile, Th12.

Polymorphonuclear Neutrophils in Osteomyelitis: Link to Osteoclast Generation and Bone Resorption

Chronic and persistent inflammatory processes in bones may lead to severe erosions with consequent functional impairment sometimes requiring amputation of the limb. To explore the relationship between inflammation and bone erosion, biopsies of patients with osteomyelitis due to arterial occlusive disease or to diabetes mellitus were examined (n=31). Histologically, inflammation and bone erosion were confirmed. In the eroded bones the number of osteoclasts correlated with the abundance of infiltrated polymorphonuclear neutrophils (PMN), which were highly activated as shown by expression of MHC class II. For functional characterisation of the infiltrating PMN, patients with implant-associated osteomyelitis, a condition associated with persistent bacterial infection and bone destruction, were recruited. The cells were recovered from infected sites and examined ex vivo. These PMN expressed MHC class II and produced interleukin (IL)-8, a further indication of PMN activation. To assess a possible link between infiltrating PMN and bone erosion, we tested the effect of IL-8 on osteoclast generation in vitro. CD14+ monocytes derived from the peripheral blood of healthy individuals were cultivated with monocyte colony stimulating factor (M-CSF) and IL-8. Within 3 days, a translocation of the transcription factor NFATcl into the nucleus was seen, and by 10 to 20 days multinucleated cells with typical osteoclast morphology appeared that expressed tartrate-resistant acid phosphatase (TRAP) and cathepsin K. Moreover, the cells were able to resorb bone, proving that IL-8 was able to induce the differentiation of monocytes to osteoclasts. Because IL-8 is a major cytokine produced by activated PMN, we propose that in the course of persistent infection infiltrating PMN contribute to induction of osteoclast formation, thus providing a link between inflammation and bone erosion.

Improved accuracy and reproducibility of enumeration of platelet–monocyte complexes through use of doublet‐discriminator strategy

Platelet-monocyte complex (PMC) formation is a marker of in vivo platelet activation and may be readily measured by flow cytometry. Due to the high frequency of free platelets relative to monocytes and PMCs, false-positive identification through coincidence remains a significant technical problem.To overcome this problem, we evaluated the use of a doublet-discriminator strategy (DDM) to allow faster sample acquisition whilst significantly reducing aberrant coincidence. Fourteen healthy volunteers and 20 patients with coronary artery disease (CAD) gave arterial and/or peripheral venous blood samples (NaCit). Whole blood was labelled in duplicate with anti-CD61 and anti-CD14 using a standard lyse/wash protocol. One of each paired sample was serially diluted before analysis; the second was analyzed at full concentration but using FL1-width to exclude co-incident platelet and monocyte events. Control experiments were performed with ex vivo thrombin activated samples. With the DDM use PMC frequencies in the peripheral blood of healthy individuals and in CAD patients fell significantly [6.27% ± 1.77 (mean ± sd) to 2.57% ± 0.99 (P = 0.02)] and from 16.04% (± 11.26) to 7.66% (± 5.18) (P < 0.01), respectively. DDM use significantly reduced the percentage of PMCs in the ex vivo thrombin activated samples (P < 0.05). Use of DDM effectively reduces the coincidence and enumerates true PMC in the samples of normal individuals and in patients with CAD and in ex vivo thrombin activated samples.

IL-7R low memory CD8+ T cells are significantly elevated in patients with systemic lupus erythematosus

Human effector memory (EM) CD8(+) T cells include IL-7Rα(high) and IL-7Rα(low) cells with distinct cellular characteristics, including the expression of cytotoxic molecules. Both NK cells and the NK cell-associated molecule 2B4 that is expressed on CD8(+) T cells promote cytotoxicity. Here we analysed the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells and its contribution to cytotoxicity. We also analysed the frequency of IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells in patients with SLE or lupus and in healthy individuals given the potential role of cytotoxic CD8(+) T cells in the pathogenesis of lupus. We used flow cytometry to measure the expression of 2B4 on IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells as well as the frequency of these cell populations in the peripheral blood of healthy individuals and patients with SLE. Also, 2B4-mediated cytotoxicity was quantitated in IL-7Rα(high) and IL-7Rα(low) EM CD8(+) T cells using target cells with CD48 antigen. We found that IL-7Rα(high) EM CD8(+) T cells had higher levels of 2B4 expression compared with IL-7Rα(low) EM CD8(+) T cells. Triggering 2B4 enhanced the cytotoxic function of IL-7Rα(low) EM CD8(+) T cells against target cells. We also noticed that patients with SLE had an increased frequency of IL-7Rα(low) EM CD8(+) T cells that correlated with disease manifestation. Our findings show that SLE patients have increased IL-7Rα(low) EM CD8(+) T cells, possibly contributing to tissue damage through 2B4-mediated cytotoxicity.