Biomedical subjects

Andrew J Kassianos

Publications and source records attributed to Andrew J Kassianos.

4 recordsLinked to original sources

PubMed2006

Numerical and functional assessment of blood dendritic cells in prostate cancer patients.

BACKGROUND: Prostate cancer is one of the leading causes of cancer deaths in males and there are currently no effective treatments available for metastatic disease. Although recent clinical trials using dendritic cell (DC) based immunotherapy treatments have demonstrated safety, immunological responses, and some clinical efficacy, better vaccine delivery strategies need to be developed. We have undertaken the first detailed analysis of blood DC (BDC) subsets and their function in prostate cancer patients, with a view to utilizing immunoselected BDC for immunotherapy. METHODS: We enumerated the CD11c+CD1c+, CD11c+CD16+, and CD11c-CD123+ BDC subsets in whole blood of prostate cancer patients using a single platform TruCOUNT assay. These subsets were identified and purified using flow cytometry and immunomagnetic selection, and their functional capacity analyzed by costimulatory molecule expression, cytokine secretion, and antigen presenting ability. RESULTS: There were no significant differences in the number or composition of these subsets compared to healthy donors and these cells could be purified with equal efficiency from both groups. The prostate cancer patients BDC had similar levels of key costimulatory molecules and cytokine expression profiles, compared to healthy donors, and these were upregulated to the same extent, in response to exogenous stimuli. BDC from both groups were capable of eliciting allogeneic proliferative responses and inducing autologous CD4+ responses to naïve and recall antigens, and antigen-specific CD8+ responses to influenza matrix protein and prostate specific antigen. CONCLUSIONS: These results indicate that an immunoselected CD1c+ BDC preparation could provide a suitable vaccine delivery vehicle for future prostate cancer immunotherapy trials.

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PubMed2005

Simple, robust strategies for generating DNA-directed RNA interference constructs.

We describe two complementary strategies for preparing DNA-directed RNA interference (ddRNAi) constructs designed to express hpRNA. The first, oligonucleotide assembly (OA), uses a very simple annealing protocol to combine up to 20 short nucleotides. These are then cloned into appropriately designed restriction sites in expression vectors. OA can be used to prepare simple hairpin (hp)-expressing constructs, but we prefer to use the approach to generate longer constructs. The second strategy, long-range cloning (LRC), uses a novel adaptation of long-range PCR protocols. For LRC, entire vectors are amplified with primers that serve to introduce short sequences into plasmids at defined anchor sites during PCR. The LCR strategy has proven highly reliable in our hands for generating simple ddRNAi constructs. Moreover, LCR is likely to prove useful in many situations in which conventional cloning strategies might prove problematic. In combination, OA and LRC can greatly simplify the design and generation of many expression constructs, including constructs for ddRNAi.

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PubMedDate not supplied

Immunoselection of functional CMRF-56+ blood dendritic cells from multiple myeloma patients for immunotherapy.

Dendritic cells (DCs) loaded with tumor-associated antigens are a promising treatment to prevent disease relapse in patients with multiple myeloma (MM). Early-phase clinical trials have shown safety, efficacy, and immunologic responses in MM, but a key issue now is the isolation of a functional, clinically relevant DC preparation. The authors have described a unique blood DC (BDC) isolation platform based on positive immunoselection with the CMRF-56 antibody. To validate this as a feasible source of BDCs for immunotherapy, the authors undertook a quantitative and functional analysis of BDCs in MM patients and healthy donors. These data show that MM patients have similar numbers of CD11c+CD16+ and CD11c+CD16- BDCs but about half the number of CD11c-CD123+ BDCs in whole blood compared with healthy donors. BDCs could be isolated by CMRF-56+ immunoselection from all MM patients tested, with similar yields and purity to healthy donors. These BDCs could be activated ex vivo with poly I:C or LPS. Furthermore, CMRF-56+ preparations could induce potent CD4+ and CD8+ T-lymphocyte responses in both MM patients and healthy donors. These data suggest that BDCs with in vitro functional integrity can be isolated from MM patients in sufficient numbers to justify a clinical trial.

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PubMedDate not supplied

CD11c+ blood dendritic cells induce antigen-specific cytotoxic T lymphocytes with similar efficiency compared to monocyte-derived dendritic cells despite higher levels of MHC class I expression.

Dendritic cell (DC) immunotherapy for cancer has shown promising results in phase I and II clinical trials. Most studies have used monocyte-derived DCs (MoDCs) but their poor migratory capacity in vivo has emerged as a key issue. The natural circulating peripheral blood CD11c+ DC precursors (BDCs) may be an attractive alternative to MoDCs, as they can be isolated rapidly in sufficient quantities, and have superior migratory and T helper-1-inducing capacity in vitro. We performed the first comparative analysis of the ability of autologous BDCs and MoDCs in healthy donors to induce tumor-specific cytotoxic T lymphocytes (CTLs). BDCs expressed significantly higher levels of major histocompatibility complex class I and CD83 in the absence of exogenous stimuli compared with MoDCs. After activation with polyinosinic-polycytidylic acid, BDCs expressed higher levels of major histocompatibility complex class I, CD40, CD80, and CD83, and secreted higher levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, and IL-8 compared with MoDCs. Despite these differences, both preparations secreted similar levels of IL-12 in response to polyinosinic-polycytidylic acid and, importantly, induced CTL responses of similar magnitude and affinity against influenza matrix protein and MART-1. The ability of BDCs to induce efficient CTL responses, combined with their migratory capacity, makes them an appealing alternative to be investigated in clinical immunotherapy research protocols.

Antigens, CD↗