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1.
PLoS One ; 18(9): e0290793, 2023.
Article in English | MEDLINE | ID: mdl-37713423

ABSTRACT

The efficacy of PD-1 monoclonals such as pembrolizumab can be modulated by the signals delivered via their Fc region. Tumour/inflammation associated proteases can generate F(ab')2 fragments of therapeutic monoclonals, and subsequent recognition of F(ab')2 epitopes by circulating anti-hinge antibodies (AHA) can then, potentially, link F(ab')2 binding to the target antigen with novel Fc signalling. Although elevated in inflammatory diseases, AHA levels in cancer patients have not been investigated and functional studies utilising the full repertoire of AHA present in sera have been limited. AHA levels in pembrolizumab treated melanoma patients (n = 23) were therefore compared to those of normal donors and adalimumab treated patients. A subset of melanoma patients and the majority of adalimumab patients had elevated levels of AHA reactive with F(ab')2 fragments of IgG4 anti-PD-1 monoclonals (nivolumab, pembrolizumab) and IgG1 therapeutic monoclonals (rituximab, adalimumab). Survival analysis was restricted by the small patient numbers but those melanoma patients with the highest levels (>75% percentile, n = 5) of pembrolizumab-F(ab')2 reactive AHA had significantly better overall survival post pembrolizumab treatment (p = 0.039). In vitro functional studies demonstrated that the presence of AHA+ sera restored the neutrophil activating capacity of pembrolizumab to its F(ab')2 fragment. Neither pembrolizumab nor its F(ab')2 fragments can induce NK cell or complement dependent cytotoxicity (CDC). However, AHA+ sera in combination with pembrolizumab-F(ab')2 provided Fc regions that could activate NK cells. The ability of AHA+ sera to restore CDC activity was more restricted and observed using only one pembrolizumab and one adalimumab patient serum in combination with rituximab- F(ab')2. This study reports the presence of elevated AHA levels in pembrolizumab treated melanoma patients and highlight the potential for AHA to provide additional Fc signaling. The issue of whether tumour associated proteolysis of PD-1 mAbs and subsequent AHA recognition impacts on treatment efficacy requires further study.


Subject(s)
Immune Checkpoint Inhibitors , Melanoma , Humans , Adalimumab/therapeutic use , Rituximab , Melanoma/drug therapy , Immunoglobulin G
2.
Front Oncol ; 11: 709543, 2021.
Article in English | MEDLINE | ID: mdl-34497762

ABSTRACT

Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53. Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1Met, a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1Met-induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1Met.

3.
Cancer Immunol Immunother ; 69(12): 2453-2464, 2020 Dec.
Article in English | MEDLINE | ID: mdl-32556495

ABSTRACT

The PD-1-targeting IgG4 antibody pembrolizumab has significant anti-tumor activity in a proportion of stage IV melanoma patients. A subset of patients develop anti-drug antibodies (ADA) which can form immune complexes (IC) with pembrolizumab. Although IC can induce powerful, Fc-mediated, immune-regulatory effects, their functional impact during pembrolizumab treatment is unclear. The functional effects of IC generated in vitro using pembrolizumab and patient-derived ADA was, therefore, investigated. Screening identified a patient whose trough serum samples from three treatment cycles contained both ADA with neutralizing activity and low levels of pembrolizumab. This patient responded well to therapy over 2 years and had ongoing, infusion-related, hypersensitivity reactions despite the later absence of detectable ADA. The components of IC were mimicked by forming a complex of pembrolizumab by absorption onto a solid phase with or without subsequent exposure to the ADA+ patient sera. Complexes comprised of pembrolizumab alone significantly inhibited TLR4 (LPS)-driven IL-10 production by PBMC and stimulated the generation of reactive oxygen species by granulocytes. In contrast, soluble and solid-phase F(ab´)2 fragments of pembrolizumab had no effect demonstrating the requirement for cross-linked Fc regions. IC containing pembrolizumab and ADA could additionally induce complement and NK activation. The results of this study demonstrate that, when oligomerized, the Fc region of pembrolizumab alone can provide immuno-regulatory signals. Furthermore, IC containing both pembrolizumab and patient-generated ADA can induce additional signals. These Fc-mediated signals may modulate both hypersensitivity reactions and anti-tumor responses associated with pembrolizumab therapy.


Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Antibodies, Neutralizing/immunology , Antigen-Antibody Complex/immunology , Melanoma/drug therapy , Skin Neoplasms/drug therapy , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Neutralizing/blood , Antigen-Antibody Complex/blood , Drug Hypersensitivity/blood , Drug Hypersensitivity/immunology , Female , Humans , Leukocytes, Mononuclear , Male , Melanoma/blood , Melanoma/immunology , Melanoma/secondary , Middle Aged , Primary Cell Culture , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Skin Neoplasms/blood , Skin Neoplasms/immunology , Skin Neoplasms/pathology
4.
J Immunol Methods ; 474: 112672, 2019 11.
Article in English | MEDLINE | ID: mdl-31525368

ABSTRACT

Adalimumab is a TNF specific monoclonal widely used therapeutically. Monitoring adalimumab levels is important for guiding treatment strategies and is predominantly performed using an ELISA. The homogeneous mobility shift assay (HMSA) has many advantages over an ELISA for adalimumab monitoring but current HMSA methodologies do not discriminate between adalimumab and other TNF specific monoclonals such as infliximab. The development and validation of a competitive binding HMSA (cHMSA) specific for adalimumab is reported here. The cHMSA had a lower limit of quantitation of 1.25 µg/ml and the intra-assay and inter-assay coefficents of variation (CV) were <20%. No signal was detected in adalimumab naïve control serum including those containing rheumatoid factor or infliximab. The majority (14/20) of adalimumab patient samples containing anti-adalimumab antibodies gave a cHMSA signal >3 standard deviations lower than the controls. The performance of the cHMSA and an ELISA was compared using adalimumab patient samples (n = 82). There was a strong correlation between the assays (r = 0.91) and the intra-class correlation coefficient (0.88) was indicative of good-excellent inter-assay reliability. Bland-Altman plots showed little overall bias and comparison of the sub-groups defined using cut-points (1.25 or 7.3 µg/ml) gave percent agreement (>90%) and Cohens kappa (95% CI: 0.61-0.93) values indicative of substantial-almost perfect agreement. These results demonstrate that cHMSA provides an accurate and specific method for monitoring adalimumab levels and can additionally provide an initial screen for the presence of anti-adalimumab antibodies.


Subject(s)
Adalimumab/blood , Drug Monitoring/methods , Electrophoretic Mobility Shift Assay , Tumor Necrosis Factor Inhibitors/blood , Enzyme-Linked Immunosorbent Assay , Humans , Limit of Detection , Predictive Value of Tests , Reproducibility of Results
5.
Mol Nutr Food Res ; 63(18): e1900478, 2019 09.
Article in English | MEDLINE | ID: mdl-31216087

ABSTRACT

SCOPE: Blueberry polyphenols are thought to confer cardiovascular health benefits, but have limited bioavailability. They undergo extensive metabolism and their phenolic acid metabolites are likely to be the mediators of bioactivity. The effect of blueberry-derived phenolic acids on one aspect of inflammation, monocyte adhesion to vascular endothelial cells, is investigated. METHODS AND RESULTS: The major blueberry-derived phenolic acids in human plasma are identified and quantified. Three test mixtures representing compounds present at 0-4 h (Early), 4-24 h (Late), or 0-24 h (Whole) are used to investigate the effect on adhesion of monocytes to tumor necrosis factor alpha (TNFα)-activated endothelial cells. The Late mixture reduces monocyte adhesion, but there is no effect of the Early or Whole mixtures. Exclusion of syringic acid from each mixture results in inhibition of monocyte adhesion. Exposure to the phenolic acid mixtures has no effect on the endothelial surface expression of adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or E-selectin, suggesting that other molecular mechanisms are responsible for the observed effect. CONCLUSION: This study shows that physiological concentrations of blueberry polyphenol metabolites can help maintain cardiovascular health by regulating monocyte adhesion to the vascular endothelium.


Subject(s)
Blueberry Plants/chemistry , Hydroxybenzoates/blood , Hydroxybenzoates/pharmacology , Monocytes/drug effects , Cell Adhesion/drug effects , Cell Adhesion Molecules/metabolism , Flow Cytometry , Gallic Acid/analogs & derivatives , Gallic Acid/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Hydroxybenzoates/isolation & purification , Monocytes/cytology , Tumor Necrosis Factor-alpha/pharmacology
7.
Intern Med J ; 49(5): 598-606, 2019 05.
Article in English | MEDLINE | ID: mdl-30411451

ABSTRACT

BACKGROUND: The impact of changes in novel agent (NA) usage on the survival of multiple myeloma (MM) patients in real-world hospital settings is unclear. In New Zealand (NZ) in 2011, frontline bortezomib became available and thalidomide availability was expanded. AIM: This retrospective study analyses the impact these change had on the survival of MM patients treated at a NZ hospital. METHODS: Clinical and overall survival (OS) data were collected on MM patients who were treated at Christchurch Hospital during 2000-2009 (pre-cohort, n = 337) and 2011-2017 (post-cohort, n = 343). Outcomes were compared using pre-cohort data truncated at 2011. RESULTS: Patients in the post-cohort had significant increases (P < 0.001) in not only NA usage (85 vs 55%) and OS (median = 56 vs 44 months) but also the proportion (74 vs 49%) of young patients (age < 70) who received an autologous stem cell transplant (ASCT). Separate analysis of older patients demonstrated that those in the post-cohort had significantly longer OS (median OS 28 vs 17, P < 0.001) although 5-year relative survival remained less than 50%. Separate analysis of young patients demonstrated that those in the post-cohort had significantly increased initial OS with the survival curves converging at 5 years. Although ASCT-treated patients had similar OS in each cohort, their progression-free survival (PFS) was significantly increased in the post-cohort (median 40 vs 20 months, P < 0.0001). CONCLUSION: In the setting of a NZ hospital the increased availability of NA was associated with a significant improvement in both the OS of older patients and the PFS of ASCT patients.


Subject(s)
Health Services Accessibility/trends , Hematopoietic Stem Cell Transplantation/trends , Hospitalization/trends , Multiple Myeloma/epidemiology , Multiple Myeloma/therapy , Progression-Free Survival , Adult , Aged , Aged, 80 and over , Antineoplastic Agents/administration & dosage , Antineoplastic Agents, Alkylating/administration & dosage , Bortezomib/administration & dosage , Female , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunosuppressive Agents/administration & dosage , Induction Chemotherapy/methods , Induction Chemotherapy/trends , Male , Melphalan/administration & dosage , Middle Aged , Multiple Myeloma/diagnosis , New Zealand/epidemiology , Retrospective Studies , Thalidomide/administration & dosage , Transplantation, Autologous/methods , Transplantation, Autologous/trends , Treatment Outcome
8.
Ther Drug Monit ; 40(6): 705-715, 2018 12.
Article in English | MEDLINE | ID: mdl-30439789

ABSTRACT

BACKGROUND: The measurement of anti-drug antibody (ADA) levels in adalimumab (ADAL)-treated and infliximab (IFX)-treated patients is critical for guiding therapeutic strategies. The homogeneous mobility shift assay (HMSA) and affinity capture elution (ACE) assay provide effective, drug-tolerant formats for measuring total ADA levels. However, their ability to discriminate between ADA from samples with or without neutralizing capacity is unclear and therefore was analyzed in this study. METHODS: Sera from ADAL and IFX patients with low drug levels (<1 mcg/mL) were analyzed by ACE, HMSA, and bridging assay. Neutralizing capacity was determined by competitive ligand-binding assay. RESULTS: HMSA and ACE detected high ADA levels in all ADAL (19/42) and IFX (27/64) samples with neutralizing capacity. ADA was also detected in most of the samples without neutralizing capacity, but levels were significantly lower (P < 0.0001). Receiver operator characteristic curve analysis demonstrated that for both assays, ADA levels were a strong discriminatory marker of neutralizing ADA (area under the curve > 0.9, P < 0.0001). Using a signal >8× background as a cut-point, neutralizing ADA could be identified with high specificity (HMSA > 95%, ACE > 85%) and sensitivity (HMSA > 70%, ACE > 80%). The detection of multimeric drug-ADA complexes after HMSA was also a highly specific marker (specificity > 95%) of neutralizing ADA in both ADAL and IFX patients. Results using ACE and HMSA were highly correlated. CONCLUSIONS: Results obtained after HMSA and ACE analysis are strongly correlated, and in both assays, high ADA levels are a specific marker of neutralizing capacity. The detection of multimeric complexes by HMSA also selectively identifies sera with neutralizing capacity. These data support the use of these assays as quantitative rather than simple qualitative measures of ADA.


Subject(s)
Adalimumab/immunology , Antibodies, Neutralizing/blood , Electrophoretic Mobility Shift Assay/methods , Enzyme-Linked Immunosorbent Assay/methods , Infliximab/immunology , Adolescent , Adult , Aged , Antibodies, Neutralizing/immunology , Child , Clinical Trials as Topic , Female , Humans , Male , Middle Aged , Sensitivity and Specificity , Young Adult
9.
Pathog Dis ; 75(4)2017 06 01.
Article in English | MEDLINE | ID: mdl-28430970

ABSTRACT

The modulation of T cell responses by Helicobacter pylori is thought to potentiate both H. pylori persistence and development of gastric pathologies including cancer. Release of outer membrane vesicles (OMV) by H. pylori provides a potential vehicle for modulation of the immune system. Although OMV are thought to have T cell suppressive activity, this has not yet been demonstrated. Their suppressive activity was investigated in this study using the responses of peripheral blood mononuclear cells (PBMC) to T cell stimuli as a readout. We demonstrate that addition of OMV to PBMC significantly inhibits subsequent T cell proliferation in a cyclo-oxygenase-2 (COX-2)-dependent manner. Addition of OMV did not significantly modulate PBMC apoptosis, but induced strong expression of COX-2 by the monocytes present and significantly increased levels of PGE2 and IL-10. These effects were independent of vacuolating cytotoxin expression. Together, these findings demonstrate that OMV can suppress human T cell responses and that the predominant mechanism is not through a direct effect on the T cells but results from the induction of COX-2 expression in monocytes. This increased COX-2 activity may modulate not only H. pylori-directed immune responses but also wider immune responses.


Subject(s)
Cell-Derived Microparticles/metabolism , Cyclooxygenase 2/metabolism , Helicobacter pylori/immunology , Host-Pathogen Interactions , Immune Tolerance , Monocytes/enzymology , T-Lymphocytes/immunology , Cells, Cultured , Humans , Immune Evasion
10.
PLoS One ; 12(3): e0172858, 2017.
Article in English | MEDLINE | ID: mdl-28257435

ABSTRACT

Chronic lymphocytic leukemia (CLL) is associated with T cell dysfunction. Activated CLL cells are found within the lymphoid tumor micro-environment and overcoming immuno-suppression induced by these cells may improve anti-CLL immune responses. However, the mechanisms by which activated CLL cells inhibit T cell responses, and reagents targeting such mechanisms have not been identified. Here we demonstrate that the ability of in vitro activated CLL cells to suppress T cell proliferation is not reversed by the presence of ecto-nuclease inhibitors or blockade of IL-10, PD-1 and CTLA-4 pathways. Caffeine is both an adenosine receptor antagonist and a phosphatidylinositol-3-kinase, p110δ (PI3Kδ) inhibitor and, at physiologically relevant levels, significantly reversed suppression. Significant reversal of suppression was also observed with the PI3Kδ specific inhibitor Idelalisib but not with adenosine receptor specific antagonists. Furthermore, addition of caffeine or Idelalisib to activated CLL cells significantly inhibited phosphorylation of AKT, a downstream kinase of PI3K, but did not affect CLL viability. These results suggest that caffeine, in common with Idelalisib, reduces the immuno-suppressive activity of activated CLL cells by inhibiting PI3Kδ. These findings raise the possibility that these compounds may provide a useful therapeutic adjunct by reducing immuno-suppression within the tumor micro-environment of CLL.


Subject(s)
Caffeine/administration & dosage , Immunity, Cellular/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Purines/administration & dosage , Quinazolinones/administration & dosage , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Phosphatidylinositol 3-Kinases/biosynthesis , Phosphorylation/drug effects , Protein Kinase Inhibitors/administration & dosage , Signal Transduction/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tumor Microenvironment/drug effects
11.
Transpl Immunol ; 32(3): 164-71, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25968653

ABSTRACT

BACKGROUND: Myeloid-derived suppressor cells (MDSC) are powerful suppressors of immune responses. MDSC numbers are increased in some renal transplant recipients (RTR) and there is increasing interest in their potential role in promoting both transplant tolerance and transplant associated malignancy. However the factors influencing MDSC mobilisation are unknown, and the relative temporal changes in the granulocytic (G-MDSC) and monocytic (Mo-MDSC) subsets of MDSC have not been defined. METHODS: The circulating frequencies of MDSC and dendritic cells (DC) were analysed by multicolour flow cytometry in RTR (n = 8) prior to transplant and at regular intervals out to 1 year post-transplant. RESULTS: In RTRs, numbers of both G-MDSC and Mo-MDSC increased rapidly following transplant and peaked within 8 days, whilst DC numbers decreased. A second peak in G-MDSC numbers was observed in 7/8 patients within 3 months and 2 patients had a further 3rd peak in G-MDSC numbers which, in one patient, corresponded to a rejection event. Mo-MDSC numbers underwent less fluctuation and a subsequent peak in numbers was observed in only 2/8 patients. Respective kidney donors (n = 5) underwent only small transient increases in MDSC following surgery. Overall, there was little correlation between increases in MDSC and the occurrence of detectable clinical events or treatment changes. CONCLUSIONS: In RTRs, MDSC numbers increase rapidly and peak following commencement of immunosuppression, but then fluctuate in response to as yet undefined stimuli. Further studies are required to identify the factors modulating MDSC mobilisation.


Subject(s)
Dendritic Cells/immunology , Kidney Transplantation , Myeloid Cells/immunology , Adult , Allografts/immunology , Cell Count , Follow-Up Studies , Humans , Immune Tolerance , Immunity, Cellular , Male , Middle Aged , Prospective Studies , Tissue Donors , Transplant Recipients
12.
Immunol Lett ; 152(2): 151-2, 2013 May.
Article in English | MEDLINE | ID: mdl-23727069

ABSTRACT

T cell responses to CD3/CD28 antibodies are widely used to demonstrate the immunosuppressive activity of added human granulocytic myeloid-derived suppressor cells (G-MDSC). Granulocytic populations have the well established capability to chemically modify antibody structure and/or, phagocytose stimulatory CD3/CD28 antibody coated beads. However the possibility that the suppression observed in CD3/CD28 antibody based assays may result from the effects of the G-MDSC on the stimulatory antibodies rather than the T cells is not routinely controlled for experimentally. In the absence of controls to evaluate potential antibody associated artefacts considerable caution should be applied to the use and interpretation of this assay system as a means of defining suppressive G-MDSC populations.


Subject(s)
CD28 Antigens/immunology , CD3 Complex/immunology , Granulocytes/immunology , Antibody Formation/immunology , Humans , Lymphocyte Activation/immunology , Myeloid Cells/immunology , T-Lymphocytes/immunology
13.
Immunology ; 137(3): 249-58, 2012 Nov.
Article in English | MEDLINE | ID: mdl-23025756

ABSTRACT

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T-cell responses to CD3(+) CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN(act)) can, without any T-cell interaction, induce apparent T-cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell-directed suppression of T-cell proliferation was observed when PMN(act) were added to pre-activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin-2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN(act) , suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co-culture with PMN(act) also induced a significant decrease in T-cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N-hydroxy-nor-l-arginine induced both a further significant, catalase-sensitive, loss in T-cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T-cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.


Subject(s)
Cell Proliferation , Neutrophils/immunology , T-Lymphocytes/immunology , Cell Survival , Cells, Cultured , Coculture Techniques , Humans , Lymphocyte Activation , Neutrophils/cytology , T-Lymphocytes/cytology
14.
Nephrol Dial Transplant ; 27(1): 402-10, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21617199

ABSTRACT

BACKGROUND: Cancer, particularly cutaneous squamous cell carcinoma (SCC), is a major cause of mortality in renal transplant recipients (RTRs). Myeloid-derived suppressor cells (MDSC) play a central role in suppressing cancer immunosurveillance but their potential mobilisation in RTRs and levels relative to those of other immunoregulatory dendritic cell (DC) populations have not been analysed. METHODS: The circulating frequencies of MDSC and DC were analysed by multicolour flow cytometry in immunocompetent patients without (n = 13) or with (ICI-SCC(Pos), n = 14) current SCC, normal donors (NDs, n = 34), chronic kidney disease patients (CKD patients, n = 22) and RTRs (n = 31). RESULTS: Compared to NDs, RTRs had significantly elevated levels of both CD14(Neg) and CD14(Pos) MDSC subsets (P < 0.001), while CKD patients and ICI-SCC(Pos) had significantly elevated levels of only the CD14(Neg)-MDSC subset. DC frequencies were significantly decreased in RTRs and CKD patients but were at normal levels in ICI-SCC(Pos). The MDSC/DC ratio was significantly elevated (P < 0.05) in RTRs (median = 5.7), CKD patients (median = 3.2) and ICI-SCC(Pos) (median = 3.5) relative to NDs (median = 0.7). The use of immunosuppressive drugs in CKD patients and past/current occurrence of SCC in RTRs was associated with significantly increased CD14(Neg)-MDSC frequencies. MDSC enriched from RTRs, when co-cultured with activated NDs T cells significantly suppressed extracellular IL-10 levels and can, when activated with formyl-methionyl-leucyl-phenylalanine, inhibit T-cell proliferation. CONCLUSIONS: RTRs, CKD patients and ICI-SCC(Pos) have increased MDSC frequencies and MDSC/DC ratios. These changes may impact on cancer immunosurveillance. Therefore, MDSC represent both a potential therapeutic target and prognostic marker in these patients, with respect to the development of SCC and other malignancies.


Subject(s)
Carcinoma, Squamous Cell/immunology , Kidney Failure, Chronic/immunology , Kidney Transplantation/immunology , Myeloid Cells/cytology , Myeloid Cells/immunology , Skin Neoplasms/immunology , Tumor Escape/immunology , Adult , Aged , Aged, 80 and over , Carcinoma, Squamous Cell/blood , Cross-Sectional Studies , Dendritic Cells/cytology , Dendritic Cells/immunology , Female , Flow Cytometry , Follow-Up Studies , Glomerular Filtration Rate , Humans , Immunocompetence , Kidney Failure, Chronic/blood , Male , Middle Aged , Prognosis , Skin Neoplasms/blood
15.
Am J Physiol Heart Circ Physiol ; 291(4): H1952-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16973826

ABSTRACT

This study investigated whether gene expression profiles of myofibroblasts derived from infarcted myocardium differ from normal cardiac fibroblasts. We compared the expression of cytoskeletal proteins in cultured ovine cardiac fibroblasts derived from infarcted (ID) and noninfarcted ovine myocardium (NID) and the levels of expression of the natriuretic peptide receptors (NPR)-A and NPR-B in response to treatment with transforming growth factor (TGF)-beta1 and/or platelet-derived growth factor (PDGF). Transformation of cultured cardiac fibroblasts to myofibroblasts, as indicated by alpha-smooth muscle actin and vimentin expression, was independent of the presence of TGF-beta1, PDGF, or cell origin. ID fibroblasts had higher basal levels than NID fibroblasts of NPR-A (ID: 58.0 +/- 32.2 arbitrary density units, NID: undetectable), NPR-B (ID: 780 +/- 155, NID: 330 +/- 38 arbitrary density units) and collagen I (ID: 17.2 +/- 0.5, NID: 10.5 +/- 1.7 pg mRNA/mug total RNA, P < 0.05) but lower levels of alpha-SMa expression (ID: 50.2 +/- 7.9, NID: 76.9 +/- 3.2 fluorescence units, P < 0.05). NPR-A mRNA in ID fibroblasts showed a rapid fourfold increase in response to TGF-beta1 and/or PDGF at 4 and 2 h, respectively, followed by a profound decline; in NID cells, NPR-A mRNA was undetectable. In ID fibroblasts, cytokines reduced NPR-B mRNA below control levels; in NID fibroblasts, TGF-beta1 and PDGF elicited prompt increments in expression: a fourfold increase with TGF-beta1 at 8 h and a twofold increase with PDGF at 4 h (P < 0.05). In summary, transformation of cardiac fibroblasts to myofibroblasts in culture is independent of cytokine treatment. Moreover, whether the cultured cardiac fibroblasts are from infarct tissue is a major determinant of NPR expression levels and cytokine responses, even after four to five passages.


Subject(s)
Cytokines/pharmacology , Fibroblasts/metabolism , Myocardial Infarction/metabolism , Myocardium/metabolism , Receptors, Atrial Natriuretic Factor/metabolism , Actins/genetics , Actins/metabolism , Animals , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression Regulation/drug effects , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardium/cytology , Myocardium/pathology , Platelet-Derived Growth Factor/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Atrial Natriuretic Factor/genetics , Sheep , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1
16.
Cancer ; 106(10): 2148-57, 2006 May 15.
Article in English | MEDLINE | ID: mdl-16598754

ABSTRACT

BACKGROUND: CD40 plays a critical role in immunoregulation, and CD40 ligation is being investigated as a therapy for hematologic malignancies. Although soluble CD40 (sCD40) is a potential modulator of both antitumor responses and CD40-based therapies, the levels and significance of sCD40 in patients with hematologic malignancies are unknown. METHODS: The authors evaluated serum/plasma sCD40 levels using an enzyme-linked immunoassay in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and multiple myeloma (MM). RESULTS: Levels of sCD40 were elevated in serum (>1.697 ng/mL) or plasma (>0.649 ng/mL) from 73% of patients with CLL, 80% of patients with MCL, 40% of patients with AML, 43% of patients with MDS, and 33% of patients with MM. Multivariate analysis of patients with MM demonstrated that elevated sCD40 was a significant, independent predictor of poor survival. In multivariate analysis of patients with AML, sCD40 was a significant prognostic factor when the interaction of age and sCD40 was included as a variable. Further analysis demonstrated that elevated sCD86 levels were associated with significantly shorter survival only in AML patients younger than age 64 years. Release of sCD40 by CLL cells was induced by cross-linking with CD40 monoclonal antibody. CONCLUSIONS: Many patients with hematologic malignancies have elevated circulating levels of sCD40, and these elevated levels are associated with a poor prognosis at least in patients with MM and AML, suggesting that sCD40 may have a role in modulating antitumor responses and also may be a useful prognostic marker. In addition, the findings suggested that further studies will be required to determine the effect of circulating sCD40 on the clinical effectiveness of CD40-ligating reagents used in the treatment of hematologic malignancies.


Subject(s)
Biomarkers, Tumor/blood , CD40 Ligand/metabolism , Hematologic Neoplasms/blood , Hematologic Neoplasms/pathology , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Hematologic Neoplasms/mortality , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Leukemia, Myeloid, Acute/blood , Leukemia, Myeloid, Acute/mortality , Leukemia, Myeloid, Acute/pathology , Lymphoma, Mantle-Cell/blood , Lymphoma, Mantle-Cell/mortality , Lymphoma, Mantle-Cell/pathology , Male , Multivariate Analysis , Myelodysplastic Syndromes/blood , Myelodysplastic Syndromes/mortality , Myelodysplastic Syndromes/pathology , Prognosis , Proportional Hazards Models , Risk Assessment , Sampling Studies , Sensitivity and Specificity , Severity of Illness Index , Solubility , Statistics, Nonparametric , Survival Analysis
17.
Cancer Immunol Immunother ; 54(9): 880-90, 2005 Sep.
Article in English | MEDLINE | ID: mdl-15926080

ABSTRACT

The cellular products obtained following electrofusion (EF) of dendritic cells (DC) and tumour cells have shown promise as cancer vaccines. The immunogenicity of these preparations has been attributed to the presence of small numbers of DC-tumour hybrids and the contribution of the non-hybrid tumour cells present has received little attention. In this report, we investigated the effect of the EF process on the immunogenicity of allogeneic human cells, in particular the colorectal cell line, SW620. EF conditions were optimised to yield the maximum number of DC-SW620 hybrids co-expressing tumour associated antigen (TAA) and DC associated antigens. Exposure of SW620 to EF induced significant increases (P < 0.05) in apoptosis and necrosis. Pre-exposure of SW620 to the EF buffer alone [0.3 M glucose, 0.1 mM Ca(CH3COO)2 and 0.5 mM Mg(CH3COO)(2)] resulted in significant increases in TAA uptake by DC during co-culture (P < 0.05). DC phenotype was, however, not altered by exposure to EF treated tumour cells. In co-cultures of PBMC responders with SW620, the levels of IFNgamma release and cytotoxic activity were significantly increased (P < 0.05) by pre-exposure of the SW620 to EF. Pre-exposure of allogeneic non-T cells, the colorectal cell line Lovo and a breast cancer cell line (MCF7) to EF also significantly (P < 0.05) increased the levels of IFNgamma release by responding PBMC. These results demonstrate that the EF process itself can increase the immunogenicity of at least some human cell types independently of hybrid formation. These findings suggest that EF protocols should be evaluated with regard to the possibility that DC-tumour hybrids may not contribute all, or even most, of the immunostimulatory capacity present in preparations of EF treated cells.


Subject(s)
Antigen-Presenting Cells/immunology , Breast Neoplasms/immunology , Cancer Vaccines/immunology , Colonic Neoplasms/immunology , Dendritic Cells/immunology , Antigens, Neoplasm/immunology , Apoptosis/immunology , Breast Neoplasms/pathology , Cell Fusion , Cell Survival/immunology , Colonic Neoplasms/pathology , Electroporation/methods , Humans , Interferon-gamma/metabolism , Necrosis , T-Lymphocytes, Cytotoxic/immunology , Tumor Cells, Cultured/immunology , Tumor Cells, Cultured/metabolism
18.
Leuk Res ; 28(3): 237-41, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14687618

ABSTRACT

Recombinant forms of soluble CD83 (sCD83) inhibit anti-tumor responses. In this analysis of circulating sCD83 levels we report that although >95% of acute myeloid leukemia (AML) and multiple myeloma (MM) patients have normal or only weakly elevated sCD83 levels, 20% of chronic lymphocytic leukemia (CLL) and 5/7 mantle cell lymphoma (MCL) patients have significantly elevated levels (>1 ng/ml). Isolated CLL cells both weakly expressed membrane CD83 (mCD83), and released sCD83 during in vitro culture. We conclude that malignant cells are a potential source of sCD83 and that it may have functional and/or prognostic significance in hematological malignancies, particularly CLL and MCL.


Subject(s)
Antigens, Neoplasm/analysis , Hematologic Neoplasms/blood , Immunoglobulins/analysis , Membrane Glycoproteins/analysis , Acute Disease , Antigens, CD , Antigens, Neoplasm/physiology , Humans , Immunoglobulins/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Myeloid/blood , Lymphoma, Mantle-Cell/blood , Membrane Glycoproteins/physiology , Multiple Myeloma/blood , Neoplastic Stem Cells/metabolism , Prognosis , Solubility , CD83 Antigen
19.
Cancer ; 98(8): 1681-8, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14534885

ABSTRACT

BACKGROUND: Levels of the soluble form of CD86 (sCD86) are elevated in a proportion of patients with leukemia. Although it is a potential modulator of antitumor responses, the significance of sCD86 in patients with hematologic malignancies is unknown. METHODS: The authors evaluated sCD86 levels by enzyme-linked immunosorbent assay in patients with acute myeloid leukemia (AML) (n = 57 patients) and patients with myelodysplastic syndrome (MDS) (n = 40 patients) and analyzed the relation between sCD86 levels and various clinical parameters. RESULTS: Levels of sCD86 were elevated (> 2.32 ng/mL) relative to normal donors (0.22-2.32 ng/mL; n = 51 patients) in 25% of patients with AML and in 27% of patients with MDS. Patients with AML who had elevated sCD86 levels had significantly lower complete remission (CR) rates compared with patients with AML who had normal sCD86 levels. In multivariate analysis using sCD86 as a continuous variable and including the interaction of age and sCD86 as a variable, sCD86 was a significant prognostic factor (P = 0.014) independent of cytogenetics. Further analysis demonstrated that, in patients with AML age 60 years and younger, but not in patients older than 60 years, elevated sCD86 levels were associated with significantly shorter survival (P = 0.04). There was no correlation between sCD86 levels and CR rates or survival in patients with MDS. CONCLUSIONS: The presence in patients with AML of elevated levels of circulating sCD86 were associated with lower CR rates and poor survival. The prognostic significance of sCD86 was independent of cytogenetics but was modulated by age, in that it was independently significant only in younger patients. The results suggest that sCD86 may play a role in modulating immune responses associated with the progression of AML.


Subject(s)
Antigens, CD/blood , Leukemia, Myeloid, Acute/blood , Membrane Glycoproteins/blood , Myelodysplastic Syndromes/blood , Adolescent , Adult , B7-2 Antigen , Female , Humans , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Myelodysplastic Syndromes/mortality , Prognosis , Survival Rate
20.
Vaccine ; 21(9-10): 883-90, 2003 Feb 14.
Article in English | MEDLINE | ID: mdl-12547598

ABSTRACT

This study investigated whether formulation of antigen in mannosylated liposomes enhanced uptake and activation of dendritic cells (DC) and increased the ability of DC to induce primed T cell proliferation compared to formulation of antigen in unmodified liposomes or in solution. Immature human DC were generated from peripheral blood monocytes cultured with GM-CSF and IL-4. Uptake of antigen by DC and the degree of expression of the cell surface markers MHC class II, CD80, CD86 and the DC maturation marker CD83, was investigated by flow cytometry following incubation with liposomes or solution containing FITC-conjugated antigen. Exposure to liposomes containing FITC-ovalbumin resulted in enhanced expression of cell surface markers when compared to exposure to antigen in solution. Expression was highest following exposure to mannosylated liposomes. Mannosylated liposomes containing tetanus toxoid (TT) stimulated primed T cell proliferation more effectively than TT-neutral liposomes or TT-solution. This work suggests that mannosylated liposomes provide a versatile delivery vehicle for initiating enhanced immune responses to encapsulated peptide or protein vaccines.


Subject(s)
Antigens/administration & dosage , Dendritic Cells/immunology , Antigen Presentation , Antigens, CD/metabolism , B7-1 Antigen/metabolism , B7-2 Antigen , Cell Differentiation , Cells, Cultured , Dendritic Cells/cytology , Drug Delivery Systems , Fluorescein-5-isothiocyanate/administration & dosage , Histocompatibility Antigens Class II/metabolism , Humans , Immunoglobulins/metabolism , In Vitro Techniques , Liposomes/chemistry , Lymphocyte Activation , Membrane Glycoproteins/metabolism , Ovalbumin/administration & dosage , Ovalbumin/immunology , Solutions , T-Lymphocytes/immunology , Tetanus Toxoid/administration & dosage , CD83 Antigen
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