Selin Somersan

Primary Tumor Tissue Lysates Are Enriched in Heat Shock Proteins and Induce the Maturation of Human Dendritic Cells

Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.

A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy

Dendritic cells (DCs) can induce tumor- or pathogen-specific T cell responses in humans. We comprehensively compared the clinically available DC maturation stimuli for their ability to promote uniformly mature DCs that elicit higher levels of T cell responses. We compared the standard maturation stimulus, autologous monocyte-conditioned medium (MCM), with a synthetic double stranded RNA (poly I:C), soluble CD40 ligand trimer, and a defined cocktail of cytokines (TNF-alpha, IL-1 beta, IL-6) and PGE(2) to promote mature phenotype and function in human monocyte-derived DCs. The cocktail was the most efficient despite the lack of induction of IL-12p70. While these results support the use of the MCM-mimic cocktail in clinical DC immunotherapy trials, the roles of its individual constituents remain to be completely defined.

Requirement of Mature Dendritic Cells for Efficient Activation of Influenza A-Specific Memory CD8+ T Cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-γ production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-γ production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus’s known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.

Tethering and tickling a new role for the phosphatidylserine receptor

Several receptors are implicated in apoptotic cell (AC) uptake by phagocytic cells; however, their relative dominance in mammalian systems remains to be established. New studies shed light on the role of the phosphatidyl serine (PS) receptor (PSR). Ligation of PSR by PS on AC surfaces is considered essential for signaling uptake of ACs that are tethered to phagocytes via other receptors.

Generation of high quantities of viral and tumor-specific human CD4+ and CD8+ T-cell clones using peptide pulsed mature dendritic cells.

CD4+ and CD8+ T cells are key components of immune response against tumors and viruses. Many techniques have been used to clone and expand these cells in vitro for purposes of immunotherapy. Here, we describe an improved method to obtain large quantities of tumor and virus-specific human CD4+ and CD8+ T-cell clones. T cells derived from peripheral blood mononuclear cells (PBMCs) of healthy donors were stimulated several times by peptide pulsed monocyte-derived mature dendritic cells (DCs) in the presence of exogenous cytokines. T cells specific for influenza or melanoma antigens were detected by IFN-gamma intracellular staining and were cloned by limiting dilution. Specific polyclonal T-cell populations were derived for all epitopes presented by mature DCs. Nine different populations were cloned and clones were raised from eight of them. Clonality was verified by HLA/peptide tetramer staining. With additional rounds of stimulation after the cloning procedure, it was possible to obtain from 10(9) to 10(12) of each clone. Furthermore, clones could be maintained in culture in the presence of IL-2 for at least 1 month without losing their antigen-specific reactivity (e.g. cytokine secretion, cytolytic activity and proliferation). Importantly, a majority of the CD8+ T-cell clones recognized endogenously processed antigens. This method is of value for the purposes of adoptive anti-virus or anti-tumor immunotherapy.

Nonsteroidal anti-inflammatory drug sensitizes Mycobacterium tuberculosis to endogenous and exogenous antimicrobials

Existing drugs are slow to eradicate Mycobacterium tuberculosis (Mtb) in patients and have failed to control tuberculosis globally. One reason may be that host conditions impair Mtb’s replication, reducing its sensitivity to most antiinfectives. We devised a high-throughput screen for compounds that kill Mtb when its replication has been halted by reactive nitrogen intermediates (RNIs), acid, hypoxia, and a fatty acid carbon source. At concentrations routinely achieved in human blood, oxyphenbutazone (OPB), an inexpensive anti-inflammatory drug, was selectively mycobactericidal to nonreplicating (NR) Mtb. Its cidal activity depended on mild acid and was augmented by RNIs and fatty acid. Acid and RNIs fostered OPB’s 4-hydroxylation. The resultant 4-butyl-4-hydroxy-1-(4-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione (4-OH-OPB) killed both replicating and NR Mtb, including Mtb resistant to standard drugs. 4-OH-OPB depleted flavins and formed covalent adducts with N-acetyl-cysteine and mycothiol. 4-OH-OPB killed Mtb synergistically with oxidants and several antituberculosis drugs. Thus, conditions that block Mtb’s replication modify OPB and enhance its cidal action. Modified OPB kills both replicating and NR Mtb and sensitizes both to host-derived and medicinal antimycobacterial agents.

Cutting Edge: Identification of the Targets of Clonal Deletion in an Unmanipulated Thymus

Autoreactive thymocytes can be eliminated by clonal deletion during their development in the thymus. The precise developmental stage(s) at which clonal deletion occurs in a normal thymus has been difficult to assess, in large part because of the absence of a specific marker for TCR-mediated apoptosis. In this report, we reveal that Nur77 expression can be used as a specific marker of clonal deletion in an unmanipulated thymus and directly identify TCRintCD4+CD8+ and semimature CD4+CD8− thymocytes as the principal targets of deletion. These data indicate that clonal deletion normally occurs at a relatively late stage of development, as cells mature from CD4+CD8+ thymocytes to single-positive T cells.

Novel Cephalosporins Selectively Active on Nonreplicating Mycobacterium tuberculosis

We report two series of novel cephalosporins that are bactericidal to Mycobacterium tuberculosis alone of the pathogens tested, which only kill M. tuberculosis when its replication is halted by conditions resembling those believed to pertain in the host, and whose bactericidal activity is not dependent upon or enhanced by clavulanate, a β-lactamase inhibitor. The two classes of cephalosporins bear an ester or alternatively an oxadiazole isostere at C-2 of the cephalosporin ring system, a position that is almost exclusively a carboxylic acid in clinically used agents in the class. Representatives of the series kill M. tuberculosis within macrophages without toxicity to the macrophages or other mammalian cells.

Endocarditis caused by rhodotorula infection

ABSTRACT Rhodotorula is an emerging opportunistic fungal pathogen that is rarely reported to cause endocarditis. We describe a case involving a patient who developed endocarditis due to Rhodotorula mucilaginosa and Staphylococcus epidermidis, proven by culture and histopathology. The case illustrates the unique diagnostic and therapeutic challenges relevant to Rhodotorula spp.