Differential gene expression in peripheral blood lymphocytes of cancer patients treated with whole body hyperthermia and chemotherapy: a pilot study.

PURPOSE: The effect of whole body hyperthermia (WBH) at 41.8-42 degrees C on the cellular immune system is still poorly investigated. The aim of this study was to identify genes that become upregulated in peripheral blood lymphocytes (PBLs) of cancer patients during a combined treatment with WBH and chemotherapy by generating complex arrays of cDNA. METHODS: PBLs were obtained from four patients with different malignancies treated with WBH and varying cytostatic schedules before treatment and immediately thereafter. After constructing subtracted cDNA libraries, clones were screened for cDNA induction by dot-blot and semi-quantitative RT-PCR (sq-RT-PCR). RESULTS: Among 192 clones, 39 cDNAs were significantly upregulated. Sequencing revealed three groups of genes for which upregulation of mRNA was confirmed by sq-RT-PCR. The first group consisted of genes encoding for various heat shock proteins (HSP 60, 90a, 90b, 105). Further sq-RT-PCR demonstrated differential expression of HSP27 and HSP70 as well. The second group (calcyclin-binding-protein, haemoglobin-beta-chain) comprised genes without pre-specified association to hyperthermia. The cDNA encoding macrophage-inflammatory-protein-1-beta was also observed and may be associated with the pre-described activation of lymphocyte sub-populations during WBH. CONCLUSION: Treatment with WBH and chemotherapy elicits significant short-term effects on the expression of a variety of genes responsible for cellular integrity, stimulation and migration of immune effector cells. Further investigation is warranted to more clearly define the role of those genes for the clinical effect of WBH.

Clinical relevance of CD95 (Fas/Apo-1) on T cells of patients with B-cell chronic lymphocytic leukemia.

OBJECTIVE: Apoptosis mediated via CD95 (Fas/Apo-1) is a key regulator for the biology of normal and malignant lymphocytes. Although the function of CD95 on B-cell chronic lymphocytic leukemia cells (B-CLL cells) has been studied intensively, the clinical importance of CD95 expression on normal T cells in B-CLL has not been clarified. This study aimed to investigate whether expression of CD95 on peripheral blood T cells correlates with clinically relevant parameters of B-CLL disease. MATERIALS AND METHODS: Expression of CD95 (Fas/Apo-1) on peripheral blood T lymphocytes of patients with B-CLL was determined using flow cytometry and was correlated with expression of activation markers, sensitivity to apoptosis by anti-CD95, and clinical data, such as blood count, Binet stage, therapy, progression-free probability, and survival probability. RESULTS: Differential CD95 expression did not correlate with activation markers or with levels of apoptosis through anti-CD95. However, high levels of CD95 on T cells from B-CLL patients correlated significantly with low lymphocyte doubling time, increased Binet stages, and requirement for chemotherapeutic treatment. Furthermore, increased cell-surface CD95 on T cells was associated with reduced progression-free probability and poorer survival. CONCLUSIONS: CD95 levels on T cells correlate with the clinical course of B-CLL. Prospective studies appear warranted to investigate whether CD95 on T cells has a direct influence on B-CLL disease progression.

Selective internalization of monoclonal antibodies by B-cell chronic lymphocytic leukaemia cells.

B-cell chronic lymphocytic leukaemia (B-CLL) cannot be cured by conventional chemotherapy, therefore, toxin-linked therapeutic monoclonal antibodies (mAbs) are increasingly examined for their potential to improve clinical outcome. The current study aimed to identify mAbs that were internalized by the B-CLL cells of 14 patients, using both flow cytometry and confocal laser scanning microscopy. Anti-CD5, CD22 and CD40 mAbs were effectively taken up by B-CLL cells, whereas mAbs against CD19, CD20, CD23 and CD45 were not. This study may form a basis for further research to identify antibodies that may serve as carriers for toxins to treat B-CLL.

Interleukin-6 enhances whereas tumor necrosis factor alpha and interferons inhibit integrin expression and adhesion of human mast cells to extracellular matrix proteins.

Integrins are expressed on mast cells and constitute an essential prerequisite for the accumulation of the cells at sites of inflammation. In order to clarify a potential contribution of inflammatory cytokines to this process, we have studied the modulation of integrin expression and adhesion of immature human mast cells (HMC-1) to extracellular matrix proteins by interleukin-6, tumor necrosis factor alpha, interferon-alpha and interferon-gamma. Corticosteroids were used for comparison. On fluorescence-activated cell sorter analysis, preincubation of cells for 48 h with different concentrations of interleukin-6 induced a significant, up to 40%, increase of alpha v alpha 5, CD49b (alpha 2), CD49e (alpha 5), CD49f (alpha 6), and CD51 (alpha v). In contrast, different concentrations of tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone (10-8-10-10 M) inhibited expression of adhesion receptors by up to 60%, reaching significance for some but not all integrins. On semiquantitative polymerase chain reaction analysis, interleukin-6, the other cytokines, and corticosteroids significantly modulated expression of alpha1, alpha v and alpha 5 integrin chains at mRNA level. Functional significance of these findings was proven in adhesion assays using fibronectin, laminin, and vitronectin, with interleukin-6 causing significant enhancement of adhesion in all cases, tumor necrosis factor alpha and dexamethasone inducing significant reduction of adhesion to fibronectin and laminin, and interferon-gamma significantly inhibiting adhesion to fibronectin only. Specificity of interleukin-6-induced changes was demonstrated using antibodies against alpha1 and alpha 5 integrins in unstimulated and interleukin-6-prestimulated cells. These data show that interleukin-6 stimulates mast cell adhesion to extracellular matrix and thus allows for the accumulation of the cells at tissue sites by enhancing integrin expression, whereas tumor necrosis factor alpha, interferon-alpha, interferon-gamma, and dexamethasone downmodulate this process.

Increased expression of CD40 ligand on activated T cells of patients with colon cancer.

PURPOSE: Proper function of T lymphocytes is crucial for an effective destruction of cancer cells in vivo. Identifying the cell surface molecules on the T cells that may be involved in this antitumor response may help to elucidate immunological factors influencing the biology of human tumors. EXPERIMENTAL DESIGN: Differences in the antigen expression profile of unstimulated and stimulated peripheral blood T-lymphocytes from patients with colon cancer and from normal controls were determined using flow cytometry. RESULTS: Freshly isolated peripheral blood T cells of patients with colon cancer did not differ in their phenotype significantly from those of patients with nonmalignant diseases. In contrast, in vitro stimulated T cells of patients with colon cancer had a significantly increased expression of CD40 ligand (CD40L, CD154) compared with activated T cells of the control group; increased CD40L expression was also found in the CD4(+)- and CD8(+)-T-cell subpopulations. CONCLUSIONS: The data presented support additional studies investigating the role of CD40L in the immune response against colon carcinoma.