CD40 Ligand--an anti-apoptotic molecule in Hodgkin's disease.

The expression of CD40L was investigated in HD involved lymph nodes by flow cytometry (FCM) and reverse transcriptase polymerase chain reaction (RT-PCR). Also an investigation of the role of CD40L in upregulation of the anti-apoptotic gene BclxL in a Hodgkin's disease (HD) derived cell line was undertaken. HD patients (n = 18) had significantly higher numbers of activated CD4+ and CD8+ T cells in the tumor microenvironment as compared to controls (n = 8). HD patients also demonstrated higher numbers of CD4+, CD8+ and CD19+ lymphocytes co-expressing CD40L as compared to controls. The CD40L signal was consistently and significantly upregulated in HD patients (n = 5) as compared to controls (n = 3) at the mRNA level. RT-PCR and FCM analysis revealed that soluble CD40L upregulated BclxL levels in the Fas-sensitive HD cell line HDLM2. We conclude that CD40L can act as an important anti-apoptotic molecule by upregulating BclxL expression in Reed-Sternberg cells of HD and may be partly responsible for their survival 'in-vivo'.

Circulating levels of TNF alpha and TNF receptor superfamily members in lymphoid neoplasia.

We have correlated the serum levels of TNF alpha and soluble TNF receptor superfamily members with clinico-pathologic parameters in patients of Hodgkin's disease (HD, N = 26) and non-Hodgkin's lymphoma (NHLs, N = 35). HD patients had significantly higher levels of TNF alpha, sTNFRI, and sTNFRII in serum while NHL patients had significantly higher levels of sTNFRI, sTNFRII, sCD27, and sFas as compared to controls. In NHL patients the levels of sCD27 correlated directly and significantly with the high-stage disease, bone marrow involvement, lymph nodal presentation, and serum LDH levels. Similarly in NHL patients, levels of sFas also correlated directly and significantly with the presence of high stage disease. HD patients with B symptoms had significantly higher levels of sTNFRII.

High cytosolic pool of p75 TNF receptors and delayed surface downmodulation by mononuclear cells from non-hodgkin's lymphoma patients.

Analysis of the expression of TNF-Rs on fresh peripheral blood mononuclear cells (PBMNC) by Scatchard analysis showed that Gr I (stages I and II) but not Gr II (stages III and IV) NHL patients have a significantly higher expression of surface TNF-Rs than normal controls. A rapid decrease in the binding of radiolabelled anti-p75 TNF-R Mab which gradually increased after 16 h was seen in normal controls, while NHL patients showed a rapid increase in the binding of the Mab after activation of cells and a decrease in binding was observed only after 24 h. Western blot analysis for normal controls showed a weak presence of the 42 kDa fragment only, while the cytosolic extracts from fresh PBMNCs of NHL patients showed presence of both intact p75 TNF-R, as well as a 42 kDa fragment corresponding to a soluble form of p75 TNF-R. Our results suggest that increased cytosolic pools of TNF-R in NHL patients might contribute to a rapid increase in its surface expression following activation of cells.

Ceramide-induced apoptosis in fas-resistant Hodgkin's disease cell lines is caspase independent.

We investigated whether cell-permeable, synthetic ceramide (C6 ceramide) could induce apoptosis in Fas-resistant Hodgkin's disease (HD)-derived cell lines. Despite strongly expressing the Fas-receptor, two of three HD-derived cell lines were resistant to Fas-mediated apoptosis. This resistance to Fas could not be attributed to differential Fas isoform generation patterns between the Fas-resistant and the Fas-sensitive cell lines. The Fas-resistant cell lines did not demonstrate the presence of Fas exon 8 deletion. Bcl-2 and BclxL levels were comparable between the Fas-resistant and the Fas-sensitive cell lines. C6 ceramide could induce apoptosis in both Fas-resistant cell lines and this was associated with a decrease in BclxL level. Caspase-1, caspase-3, or pan-caspase inhibitors could not prevent ceramide-induced apoptosis. Furthur, ceramide treatment did not lead to cleavage of caspase 3 or poly(ADP-ribose) polymerase, but caused a loss in mitochondrial transmembrane potential which could not be prevented by caspase inhibitors. Thus, we conclude that ceramide-induced apoptosis in Fas-resistant HD cell lines is caspase independent.

IL-12 and IL-2 potentiate the in vitro tumor-specific activity of peripheral blood cells from cervical cancer patients.

We have carried out a detailed analysis of the cellular immune functions of cervical cancer patients in comparison with healthy controls. It has been observed that the freshly isolated peripheral blood mononuclear cells (PBMC) exhibit natural cytotoxicity (NC) against a number of targets including tumor cells, mainly delivered by NK cells, which are non-adoptive and MHC unrestricted. Upon stimulation with cytokines like IL-2, IL-7, IL-12, IL-15 and interferons, PBMC acquire lymphokine activated killer (LAK) activity which enables them to lyse a wide range of targets including fresh tumor cells and virally infected cells. We compared the effect of IL-2 and IL-12 on enhancement of NC of PBMC from cervical cancer patients. IL-12 stimulated cultures (CD3+, CD56+) exhibited significant levels of tumoricidal activity. IL-2 stimulated lytic activity sustained even after 10 days while that of IL-12 stimulated cells declined after 6 days. Activation of PBMC was marked by increase in the expression of activation marker CD45RO and adhesion molecules LFA-1alpha, ICAM-1 and CD44. Addition of IL-12 to IL-2 stimulated cultures further enhanced the degree of lytic activity. Our data, thus, provide an evidence that PBMC from cervical cancer patients can be stimulated in response to cytokines and local or systemic treatment with low doses of cytokines may help to yield a better immune response against virus infected tumor cells in cervical cancer.

Immune functions, clinical parameters and hormone receptor status in breast cancer patients.

We have carried out a detailed analysis of the cellular immune functions of breast cancer patients in comparison with healthy controls. A possible correlation between immune and clinical parameters was analysed in 50 breast cancer patients. Immune parameters, natural killer cell and T lymphocyte functions and the numbers of circulating T lymphocytes were analysed against the clinical parameters comprising the tumour burden, the stage of the disease and the expression of hormone receptors on the tumour. In order to analyse the immune function data effectively, low responders were identified with stringent cut-off values. Considerably higher proportions of low responders were found among the patient population. Elevated numbers of circulating T lymphocytes and CD3-directed cytolysis correlated with the expression of oestrogen receptors independently of the clinical/histological parameters.

Expression of Fas and Fas ligand in Hodgkin's disease.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.

Altered release of tumor necrosis factor and its soluble receptor in non-Hodgkin's lymphoma patients.

Increased expression and elevated serum levels of tumor necrosis factor-alpha (TNF-alpha) have been shown to be associated with the presence of constitutional B symptoms and poor prognosis in non-Hodgkin's lymphoma (NHL) patients. Soluble TNF receptors (sTNF-R) are suggested to act as biological buffers in inflammatory conditions by binding and inactivating increased circulating TNF. Whereas studies have shown elevated TNF to be correlated with B symptoms, similar studies showing the status of soluble receptor release in these patients have not been conducted. Here, we show that there is increased soluble p75 TNF receptor release from peripheral blood mononuclear cells (PBMNC) in NHL patients in the early stages of the disease but it is severely depressed in patients with advanced disease. Decreased release is associated with presence of B symptoms in these patients. All NHL patients also show increased TNF secretion and a decreased rate of receptor release with time compared with healthy controls. These findings imply that decreased sTNF-R receptor release, in addition to increased TNF secretion, is also important in predisposing the patients to B symptoms. This opens up the possibility of the use of sTNF-Rs as a therapeutic tool to counter increased TNF and alleviate systemic symptoms in these patients and also as a marker for the progression of the disease.

Characterization of a murine monoclonal antibody against human esophageal squamous cell carcinoma-associated antigen.

A murine monoclonal antibody (MAb) 2G3 of the IgG1 type was raised using the human esophageal squamous cell carcinoma (SCC) cell line TE-2. Immunoblotting with 2G3 indicated that the antigen recognized by 2G3 has a molecular weight of 34 kD. Its activity was evaluated by immunoperoxidase and immunofluorescence on frozen and paraffin sections of various normal tissues, normal and benign tumors as well as various established cell lines. The pattern of reactivity revealed that the antigen recognized by 2G3 was expressed mainly by esophageal SCC. The only exception was represented by malignant breast tumors, where it reacted weakly. Scatchard analysis using 125I-labelled 2G3 showed that TE-2 has approximately 7.5 times more binding sites than the MCF-7 breast cancer cell line. The use of this new MAb is therefore proposed for the histopathological diagnosis of esophageal SCC.

Natural killer cell function and genetic instability in unaffected individuals from breast cancer families.

Several recent reports highlight the importance of modifying factors in determining the risk for cancer of a person carrying a mutant allele of a tumour susceptibility gene. The study of two such risk modifying factors namely, natural killer (NK) cell function and constitutional cytogenetic anomalies in members of families with familial breast cancer is presented in this paper. We observed that, compared to healthy controls, a significant proportion of unaffected persons from breast cancer families not only display lower NK cell function or genetic instability alone, but also in conjunction. The significance of these observations is discussed. We propose that amongst the unaffected members, persons with lower NK cell function as well as constitutive cytogenetic anomalies may be at a higher risk for cancer. The need for a set of suitable biomarkers to identify individuals at high risk from familial breast cancer families has been recognized for many years. Constitutional cytogenetic anomalies, otherwise seen in breast tumours, have also been observed in lymphocyte cultures from unaffected persons from such families. Lowered NK cell function has previously been demonstrated in first degree relatives of cancer patients. Both these parameters have been implicated in determining the risk of developing malignancy. In the present study these aspects have been investigated simultaneously in order to assess their utility as potential biomarkers.

CD40-ligation-mediated protection from apoptosis of a Fas-sensitive Hodgkin's-disease-derived cell line.

Modulation of Fas expression and function by CD40 ligation was investigated in the Fas-sensitive human Hodgkin's disease cell line HDLM2. The recombinant human trimeric soluble CD40L (sCD40L) protected this cell line from apoptosis induced by an agonistic Fas antibody at all concentrations tested. sCD40L also protected HDLM2 when added up to 2 h after Fas ligation. Apoptosis induced by a cell-permeable synthetic ceramide could not be prevented by sCD40L. Thus, CD40 ligation is likely to intervene in the early phases of the Fas signal transduction pathway. When CD40 ligation preceded Fas ligation, it rendered the cells refractory to Fas-induced apoptosis. sCD40L-mediated protection could not be attributed to reduction in surface Fas expression, increase in Bcl-2 levels or to increase in the levels of soluble Fas isoforms.

Studies on functional status of circulating lymphocytes in unaffected members from cancer families.

That the inheritance of mutations in tumor susceptibility genes alone cannot determine risk for developing cancer is now well accepted. Immune functions have long been recognized as one of the important risk modifying factors in this regard. In an attempt to develop a multiparametric approach to identify high risk individuals from cancer families, we have examined NK cell function in unaffected members from familial breast cancer families. We have also carried out a parallel study of T lymphocyte functions in these individuals. Our studies demonstrate a significantly lower NK cell activity in members from cancer families. T lymphocyte activity also showed a similar trend, with the unaffected members demonstrating a notably lowered T lymphocyte function. In addition the data from patients reveals differential sensitivity of NK and T lymphocyte function to the disease phenotype. Implications of these observations are discussed.

Impaired T lymphocyte function and differential cytokine response pattern in members from cancer families.

In an attempt to understand the basis of lowered natural killer (NK) and T cell functions in unaffected members from cancer families, we investigated cytotoxic T lymphocyte function (CD3-directed lysis) and the ability of the lymphocytes to respond to cytokines such as IL-2, IFN-alpha and IL-12. We observed lower CD3-mediated cytotoxic activity in these individuals supported by significantly lower numbers of circulating CD3+ lymphocytes. The cytokine treatment studies revealed impaired response to IFN-alpha and IL-12 in unaffected members and breast cancer patients. The observations presented herein not only reinforce our earlier finding that lower NK and T lymphocyte function may be a feature of cancer families, but also suggest that such impaired responses may be one of the factors contributing to lower cytotoxic potential of the circulating lymphocytes.

Expression of adhesion molecules in B-cell chronic lymphocytic leukaemia: an analysis in lymphoid compartments--peripheral blood, bone marrow and lymph node.

The trafficking or homing of different lymphoid subsets to particular microenvironment is mediated by specific cell adhesion molecules (CAMs) expressed on lymphocytes and endothelial cells. B-cell chronic lymphocytic leukaemia (B-CLL) or Non-Hodgkin's lymphoma of small lymphocytic, B-cell type are monoclonal expansions of mature lymphocytes. The relative distribution of the tumor lymphocytes among various lymphoid compartments vary from patient to patient. Very few studies underlying this issue are available. To this effect, we have analysed the expression of LFA-1; VLA-4, ICAM-1; CD44H and CD44v6 (haematopoietic and variant form respectively) on freshly isolated lymphocytes obtained from bone marrow (BM), peripheral blood (PB) and lymph node (LN) by flow cytometry. Overall, we find strong expression of CD44H, low to moderate expression of LFA-1, negative to low expression of VLA-4 and lack of expression of CD44v6. ICAM-1 expression was observed only in patients with prominent lymphadenopathy. Higher expression of CD44H in PB lymphoid cells relative to that of BM lymphoid cells correlated with higher PB lymphocytosis (p < 0.001). Proliferating cell nuclear antigen expression in LN sections correlated inversely with VLA-4 expression on BM and PB lymphoid cells (p < 0.05). There was no significant correlation between expression of CAMs and bcl-2 protein.

Augmentation of cisplatin cytotoxicity using cytokines on cervical carcinoma cell lines.

We investigated whether the biological response modifiers like IFN-alpha, IFN-gamma and TNF-alpha could enhance the cytotoxic action of cisplatin on cervical carcinoma cell lines in vitro. The sensitivity of three cell lines SiHa, ME 180 and C33A to these agents was tested using colorimetric MTT assay as well as tritiated thymidine uptake. All the three cell lines demonstrated range of sensitivity to cisplatin and cytokines. Interferons and TNF when used in combination with lower dose of cisplatin showed a significant enhancement of cytotoxic action of the drug in all the cell lines. Thus these data indicate that cytokines in concert with the drug may have a potential to improve the 'in vivo' therapy in these patients.

Characterization of a pan-lymphocyte monoclonal antibody useful in differentiating leukemic from normal myeloid progenitors and monocytes from macrophages.

A monoclonal antibody (McAb) designated 3A2 that recognizes a 51 kDa epitope having surface density of 37 x 10(8) per MOLT-4 cells is described. This epitope appears to be expressed on (i) lymphocytes at all stages of differentiation; (ii) leukemic myeloid progenitors; (iii) peripheral blood monocytes (MO). The epitope is specifically absent from normal myeloid progenitors and macrophages. The McAb may, therefore, be useful in studying myeloid lineage leukemias and, as a marker for monocyte to macrophage (MO + MAC) differentiation.

Tumor cytotoxicity and production of interleukin-1 and tumor necrosis factor from blood monocytes of cervical carcinoma patients.

To assess the functional integrity of monocytes in patients with cervical carcinoma, cytotoxic potential of peripheral blood monocytes and their ability to secrete interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) after activation with different stimulating agents were studied. Comparable levels of monocyte-mediated cytotoxicity were observed in healthy donors and patients. The production of IL-1 and TNF-alpha by monocytes of healthy donors and patients was similar indicating no defect in monocyte functions in these patients.

A specific and rapid receptor assay for squamous-cell carcinoma of human esophagus.

A specific radioreceptor assay, for squamous-cell carcinoma of the human esophagus, using 2G3 monoclonal antibody, has been developed for the first time. In this assay a new mathematical parameter of the Scatchard plot has been introduced for the correct measure of binding capacity (BCcm = x cos alpha). This is the perpendicular distance from the origin to the Scatchard plot line. It is always a positive quantity and is directly proportional to both the x and y intercepts for expressing the binding capacity. The assay is highly sensitive and can be used to differentiate various types of esophageal tumors such as squamous-cell carcinoma, adenocarcinoma, ulcerative growth and also a benign growth. BCcm significantly varies in the case of squamous-cell carcinoma tumors compared to adenocarcinoma and other types of tumor of the human esophagus. The assay can be completed in about 4 h and it may be used as a clinical diagnostic test.

Characterization of lymphokine-activated killer cells from peripheral blood and lymph nodes of non-Hodgkin's lymphoma patients.

Peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) from non-Hodgkin's lymphoma patients were tested for LAK cell cytotoxicity using appropriate targets in a short-term 51chromium-release assay. The results showed a significant depression in LNL-LAK activity suggesting the reduced capacity of LNL to generate LAK cells. LNL-LAK cells demonstrated significantly low percentages of cells expressing CD16, CD56 and CD25 as compared to PBL-LAK of patients and healthy donors. The reduced capacity to generate LAK cells in lymph nodes could be due to the presence of low numbers of NK cells which are thought to be the main precursors of LAK cells. The IL-2 producing ability of lymph node mononuclear cells was found to be significantly higher than that of peripheral blood mononuclear cells from both healthy donors and NHL patients.

Lymphokine activated killer cells and interleukin-2 production in patients with cervical carcinoma.

Lymphokine activated killer cells were generated from peripheral blood lymphocytes (PBL) in patients with cervical carcinoma. The cytotoxic potential of these cells was determined against three tumor cell lines (K-562, RAJI and MCF-7) using a short-term 51Cr-release assay. Both normals and patients showed comparable levels of LAK activity. Phenotypic analysis of cells having LAK activity showed a heterogeneous population. A significant increase in the expression of CD25 marker (IL-2R, Tac) of PBL population was observed after IL-2 culture. In addition, the indigenous ability of peripheral blood mononuclear cells to produce IL-2 in response to mitogen PHA was almost the same as observed in case of normal individuals.