Augmentation of natural killer cell activity in vivo against tumour cells by some wild plants from Jordan.

Twelve aqueous extracts prepared from Jordanian plants that are currently used in traditional medicine to treat various types of cancer were tested in mice for their augmentation of natural killer cells in vivo in generating cytotoxicity against YAC tumour targets. After 1 week of oral administration of aqueous extracts of fresh Nigella sativum (N.s.) seeds and Allium sativum (A.s.) bulbs significant augmentation of splenic natural killer (NK) cells (62.3% +/- 6.4% and 52.6% +/-5.4% cytotoxicity, respectively), was obtained in comparison with spontaneous activity (24.5% +/- 1.6%) at 200:1 effector:target ratio. Onopordum acanthium (O.a.) stem and leaves and Allium cepa (A.c.) bulbs showed intermediate augmentation (38.6% +/- 3.8% and 30.6% +/- 3.4% cytotoxicity, respectively) while the rest showed insignificant augmentation activity. The fresh aqueous extract of a mixture of the plants with high and intermediate activity showed little insignificant augmentation activity (72.7% +/- 6.7% cytotoxicity) of NK cells compared with that obtained with each plant alone.

Interferon gamma and interleukin-2 secretion in whole blood cell cultures from small-cell lung cancer patients.

Secretion of interleukin-2 (IL-2) and interferon-gamma (IFNgamma) in whole blood cell cultures after phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation was evaluated in 42 small-cell lung cancer patients before treatment. Blood cultures from patients with extensive and limited disease have lower IFNgamma secretions after PWM stimulation and lower IL-2 secretions after PWM and PHA stimulation, but this was not statistically significant. IFNgamma but not IL-2 secretion was significantly higher after PWM stimulation in cultures from patients with better clinical performance status (0 + 1, WHO scale). Blood cultures from patients with tumor regression after treatment had a higher secretion of IFNgamma after PWM stimulation. In contrast, blood cultures from patients with a poor prognosis had significantly lower IL-2 and IFNgamma secretions after stimulation with PWM, and a lower IL-2 secretion after stimulation with PHA.

Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan.

Thirteen aqueous extracts prepared from Jordanian plants, that are currently used in traditional medicine to treat various types of cancer, were tested in mice for their ability to augment natural killer (NK) cell function in vitro in generating cytotoxicity against YAC tumor targets. Lymphoid cells at a concentration of 5x10(6)/ml were incubated in medium alone or in medium containing different dilutions of either plant extract or purified interferon alpha for 20 h and tested for NK activity. Maximum NK activity (62. 3%) was obtained at 1:50 dilution of Nigella sativum fresh aqueous extract, 48.5% at 1:100 dilution for Allium sativum (and 38.3% at 1:50 dilution for Onopordum acanthium. Fresh aqueous plant extracts appeared to be more potent than old dried aqueous extract or ethanolic extracts. NK augmentation by plant extracts using nylon wool non-adherent spleen cells was slightly higher than the whole spleen cells.

Comparison of the effects of immunosuppressive factors from newly established colon carcinoma cell cultures on human lymphocyte proliferation and cytokine secretion.

Tumor cells may influence the host's immune reactivity by the production of immunosuppressive factors (ISFs). In this study, the effects of ISFs derived from nine polyclonal colorectal carcinoma (CRC) cell lines on PHA-induced lymphocyte proliferation and cytokine secretion was investigated. We found that most of the culture supernatants (8/9) from CRC cell lines contained ISFs, which inhibited T cell proliferation to a variable degree in a dose-dependent manner. Comparison of T cell proliferation in the presence or absence of monocytes showed that monocytes can modulate the effects of tumor-derived ISFs on lymphocyte function. In addition, exposure of activated PBMC to the tumor cell supernatants resulted in an altered secretion of cytokines by these cells, i.e. the secretion of IFN-gamma was generally reduced while the secretion of IL-1beta, IL-2 and TNF-alpha was little affected. We further investigated the supernatants' inhibitory effects on PBMC in respect to the production of prostaglandin E(2) (PGE(2)). It was found that PGE(2) was secreted by all tumor cell cultures. Therefore this substance is probably involved in the immunosuppression in vivo. However, the secreted PGE(2) was shown not to be solely responsible for the observed suppression of lymphocyte proliferation in vitro. Our results suggest that the secretion of ISF is a common property of CRCs as demonstrated with newly established CRC cell cultures, and therefore this may also be an important immune escape mechanism of colonic carcinomas in vivo.

Influence of cytokines on the expression of fas ligand and CD44 splice variants in colon carcinoma cells.

The expression of Fas ligand (FasL) by malignant cells might be a mechanism for tumor immune escape. We investigated FasL expression by LS 174T colon carcinoma cells. Furthermore, the effects of in vitro stimulation with rIL-2, rIFN-gamma and rTNF-alpha were investigated with regard to a possible regulation of the FasL expression by cytokines. FasL expression was detected by flow cytometry and RT-PCR. We observed a spontaneous expression of FasL by LS 174T cells. Incubation with high-dose rTNF-alpha induced an upregulation of FasL of 23%. rIL-2 and rIFN-gamma did not significantly affect FasL expression. To control whether our cytokine stimulation experiments were suitable to prove an upregulation of membrane proteins by tumor cells, we investigated the expression of ICAM-1, N-CAM, CD44s, CD44v6 and CD44v10. These adhesion molecules were spontaneously expressed by LS 174T cells. Only ICAM-1 and CD44v10 were significantly upregulated by rIFN-gamma and rTNF-alpha, respectively. These results could indicate that cytokines, released by tumor-infiltrating leukocytes, may induce the FasL-dependent apoptotic signal by which tumors downregulate an immunological host response.

[Secretion of interleukin-2 (IL-2) and interferon (IFN gamma)in whole blood cell culture stimulated with mitogens in patients with lung neoplasms]. / Wydzielanie interleukiny-2 (IL-2) i interferonu (IFN gamma) w hodowli z pelnej krwi stymulowanej mitogenami u chorych na raka pluca.

UNLABELLED: The aim of this study was to compare the ability of blood lymphocytes from lung cancer patients to secrete interleukin-2 (IL-2) and interferon gamma (IFNg) upon stimulation with mitogens with that of healthy donors. 42 patients with small cell lung cancer (SCLC), 30 patients with non small cell lung cancer (NSCLC) and 30 healthy donors were studied. The test was done in lung cancer patients before treatment. IL-2 and IFNg levels were measured with Elisa ready kits (Genzyme) in the supernatants of whole blood culture after stimulation with Pokeweed (PWM) and Phytohemagglutinin (PHA) mitogens. The results of the cytokine levels after stimulation were not normally distributed and thus were transformed to logarthms for statistical evaluation. The t-test for transformed results were used to asses the difference between groups. The median level of IFNg in the supernatant of whole blood cultures was significantly lower in lung cancer patients than in healthy blood donors both after PWM as well as after PHA stimulation. When patients with NSCLC and SCLC were regarded separately the lower level of IFNg in comparison with healthy donors was found in the supernatant of the blood cultures only after stimulation with PWM. The median level of IL-2 in the supernatant of whole blood culture in lung cancer patients was lower than in healthy blood donors only after PWM stimulation. The same was true for SCLC patients. In NSCLC IL-2 levels were significantly lower after stimulation with PWM as well after PHA stimulation. IN CONCLUSION: secretion of IL-2 and IFNg in whole blood culture after mitogen stimulation in lung cancer patients is significantly lower than in healthy donors. No significant differences between SCLC and NSCLC were found.

Caring About Women and Cancer (CAWAC): a European survey of the perspectives and experiences of women with female cancers.

This paper reports on the findings of the largest ever European survey of female patients' perceptions of their cancer treatment. It has provided clarification of what women consider important in relation to their management and has identified several areas where more research is needed. It has shown that women's knowledge about cancer before diagnosis is poor and the number undergoing regular screening could be improved. Women are not being adequately prepared and educated about what to expect from treatment and steps should be taken as a matter of urgency to redress this shortcoming. It was revealed that whilst families were the primary source of support to female cancer patients, women also derive considerable support from healthcare professionals, particularly senior doctors; more attention should be paid by specialists and nurses to developing psychological skills to cope with this. In this context, further research is needed into how support groups may best meet patient needs.

Biliary glycoprotein (CD66a), a cell adhesion molecule of the immunoglobulin superfamily, on human lymphocytes: structure, expression and involvement in T cell activation.

The biliary glycoproteins (BGP or CD66a), a group of different splice variants of a single gene, are members of the carcinoembryonic antigen family and the immunoglobulin superfamily. Recently, we detected CD66a on IL-2 activated lymphocytes. In this study we characterized the structure and the expression pattern of BGP on human lymphocytes and investigated its role in T cell activation. Lymphocytes express 2 of the 13 known splice variants, i.e. BGPa and BGPb, which are glycosylated in a lymphocyte-specific manner. Both BGPa and BGPb have the long cytoplasmic tail, which contains two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs, but differ in their extracellular region containing 4 and 3 immunoglobulin-like domains, respectively. On PBL BGP is expressed in small amounts only on B cells and Th cells. Stimulation with IL-2 leads to a strong up-regulation of BGP by these cells, and induces de novo BGP expression on gammabeta T cells, CD8+ and CD56+ cells, but not on CD16+ lymphocytes. This up-regulation of BGP seems to be part of the physiological process of T cell activation, since stimulation with anti-CD3 mAb is sufficient to induce BGP up-regulation. Based on the presence of the two ITIM-like motifs, one may expect that BGP inhibits T cell activation, but surprisingly, engagement of BGP enhances the proliferation of anti-CD3-stimulated T cells.

Th1 and Th2 cytokine response patterns in leukocyte cultures of patients with urinary bladder, renal cell and prostate carcinomas.

As a decreased production of Th1 cytokines by stimulated peripheral blood leukocytes has recently been shown in patients with various carcinomas, the present study was performed to determine whether these patients also exhibit a Th1/Th2 imbalance compared to healthy controls. We measured the production of the Th1 cytokines IL-2 and IFN-gamma as well as the Th2 cytokines IL-4, IL-6 and IL-10 in mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures of patients with urinary bladder carcinomas (n = 47), prostate carcinomas (n = 111) and renal cell carcinomas (n = 67) as compared to 40 age-matched healthy controls. In the PBMC cultures of the tumor patients, the levels of the Th1 cytokines IL-2 and IFN-gamma were lower as compared to the controls. For IFN-gamma, the differences were highly significant and in the patients with renal cell carcinomas it could be shown that the values decreased with increasing tumor mass. In contrast, the levels of the Th2 cytokines IL-4, IL-6 and IL-10 were comparable in the PBMC cultures of tumor patients and controls. From these results, it is concluded that there is only a malfunction in Th1 cells but no switch from a Th1 type to a Th2 type cytokine profile in the PBMCs of cancer patients.

A distinct distribution of natural killer cell subgroups in human tissues and blood.

Natural killer (NK) cells have been subdivided according to their CD16/CD56 expression into at least 2 subgroups. We examined the distribution of these NK subgroups in humans. In the blood of normal individuals, CD16++/CD56+/CD3- NK cells predominate, constituting more than 90% of all NK cells. In contrast, decidua is infiltrated almost exclusively by CD16(+/-)/CD56+/CD3- NK cells (>90%), a fact so far seen in context with decidua being an immunoprivileged tissue. However, this NK subgroup can also be detected in the blood, where it comprises about 10% of NK cells. We have found that normal (colon) as well as neoplastic (ovarian and urothelial carcinoma) tissues are also predominantly infiltrated by this CD16+/- NK subgroup. Lymphatic fluid draining solid tissues contains CD16+/- NK cells exclusively, with absolute numbers of NK cells being very low. No predominating NK subgroup was seen in ascites. CD16+/- NK cells, when tested against the target cell lines K562 and JAR, revealed a cytotoxic spectrum different from CD16++ NK cells and from T cells. A change in the CD16/CD56/CD3 phenotype was not seen in either subgroup in long-term cultures containing IL-2 (1,000 U/ml). Our data indicate that the decidua is not the only solid tissue infiltrated by CD16+/- NK cells. Other normal and malignant tissues were also infiltrated predominantly by this NK cell subgroup. We suggest that CD16+/- NK cells represent a functionally distinct NK subgroup involved in the surveillance of solid tissues.

Analysis of the T cell receptor variability of tumor-infiltrating lymphocytes in colorectal carcinomas.

While there is good clinical and experimental evidence for immunological tumor control in some tumors--malignant melanomas, for instance--the immunogenicity of colorectal carcinoma (CRC) still remains unsettled. We examined surgical specimens from 4 CRC patients for T cell clones among tumor-infiltrating lymphocytes (TIL). The growth of specific lymphocyte clones in a tumor indicates an immunological response in vivo. We used a T cell receptor V beta family-specific semiquantitative PCR with additional sequencing to examine TIL for clonal expansion. In CRC, specific T cell clones could not be demonstrated. However, we observed a predominance of V beta 9 in 3 of 4 tumors.

Isolated extracellular matrix-based three-dimensional in vitro models to study orthotopically cancer cell infiltration and invasion.

An initial event in colon cancer progression is the migration of epithelial cells through the basement membrane (BM) and the invasion of the colon submucosa, where tumour cells enter blood and lymph vessels to spread throughout the body. To interrupt this process would mean the prevention of metastasis. In order to investigate tumour cell invasion orthotopically in the human system, we established novel in vitro models which mimic normal human colon tissue (colon reproductions, CoRes) and primary colon carcinomas (artificial tumours, ArTs). These models are based on the isolated extracellular matrix (iECM) of the respective human tissues. Two isolation methods were established, the Digestion Method and the Lysis Method neither of which destroyed the characteristic architecture of the ECM found in the original tissues. BM components, i.e. laminin, fibronectin and collagen IV, were detectable in the iECM isolated with the Lysis Method but not those isolated with the Digestion Method. Scanning electron microscopic analysis of the normal colon iECM demonstrated that even if the BM was missing, the luminal surface consisted of densely packed ECM filaments which do not allow cell infiltration without degradation of the iECM. Furthermore, we demonstrated that iECM can be separately supplemented with different cell types, i.e. colorectal carcinoma cells, normal fibroblasts and immune cells at any desired concentration, combination and localisation. Therefore, these models could be used to determine the role of the BM and of the tumour cell/normal cell crosstalk in the infiltration process of human colorectal carcinoma cells.

Adhesion and migration are differentially regulated in hematopoietic progenitor cells by cytokines and extracellular matrix.

The conditions that control the migratory status of hematopoietic progenitor cells on extracellular matrix (ECM) and that decide whether a cell migrates or adheres are incompletely understood. We analyzed the migratory behavior of murine hematopoietic progenitor cells factor-dependent-cell-paterson (FDCP)-mix and purified lin-Sca1+ bone marrow cells on ECM. We found that migration on fibronectin (Fn) or laminin (Lam) becomes dependent on beta1-integrins if a surface restraint force is introduced by tilting the ECM-coated culture vessels. Under these conditions, migration specifically occured on Fn and Lam, and was not detected on collagen IV-, hyaluronate-, or bovine serum albumin- coated surfaces. Migration depended on the continuous presence of hematopoietic cytokines interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), or stem cell factor (SCF), whereas other cytokines, such as IL-8, macrophage inflammatory protein-1alpha, macrophage-chemotactic and activating factor, and erythropoietin resulted in very little or no migratory response. IL-3 induced migration was synergistically enhanced by other CSFs, but was completely inhibited by addition of transforming growth factor-beta1. In contrast to firm local adhesion of previously cytokine depleted progenitors that was rapidly inducible within 1 hour after exposure to cytokines, preincubation on Fn matrix for 4 to 6 hours was required before cytokines could induce migration. A sudden increase of cytokine concentration reversibly inhibited migration and induced a fully adhesive state; this effect could be prolonged by consecutive stimulation with heterologous cytokines. Whereas cytokines activated resting progenitor cells to migrate on ECM, cell migration speed was regulated by Fn concentration. These results indicate that beta1-integrin-mediated progenitor cell adhesion and migration are differentially regulated by external stimuli and suggest that this regulation corresponds to different activation states of beta1-integrins in hematopoietic progenitor cells.

Modulations of the effector function and cytokine production of human lymphocytes by secreted factors derived from colorectal-carcinoma cells.

We investigated the in vitro effects of factors secreted by 3 freshly explanted human colorectal-carcinoma (CRC) cell lines on lymphocyte proliferation, IL-2-receptor expression, LAK-cell generation and cytokine secretion. We found that the supernatants of all 3 CRC cell lines inhibited T-cell proliferation in a dose-dependent manner, due to the secretion of immunosuppressive factors (ISFs). In addition, the supernatants of 2 cell lines were able to inhibit LAK-cell generation and to depress IL-2R, but not HLA-DR expression, on PHA-activated T cells. Furthermore, the secretion of cytokines, i.e., IFN-gamma, IL-1beta, IL-2 and TNF-alpha, by peripheral-blood mononuclear cells (PBMC) was differently modulated by the tumor-cell supernatants, e.g., the production of IFN-gamma was reduced in normal PBMC stimulated with PHA. However, the effects induced by the supernatants were not identical: for example, factors from one CRC cell line (w25) influenced early and late events of T-cell activation and division, while 2 others (w19 and te6) contributed only to the inhibition of early events. Some biochemical properties of the ISFs were characterized. Our results suggest that colon-tumor cells can secrete ISFs, which may lead to the in vivo immunosuppression often observed in patients with these tumors.

Expression of CD44, ICAM-1 and N-CAM in colorectal cancer. Correlation with the tumor stage and the phenotypical characteristics of tumor-infiltrating lymphocytes.

The cellular adhesion molecules (CAMs) CD44s, CD44v6, CD44v10, ICAM-1 and N-CAM were immunohistologically detected in colorectal cancers using the APAAP method. The expression of CD44s and CD44v6 was associated with the presence of lymph node metastases in the examined tumors. The pattern of ICAM-1 expression was inversely related to that of CD44, i.e. lower numbers of ICAM-1 positive cells were observed in metastasizing tumors. An intense focal staining of N-CAM was observed in the majority of the metastasizing tumors. The expression of CD44v, ICAM-1 or N-CAM on tumor cells did not correlate with the density of the tumor-infiltrating lymphocytes (TIL) within the tumors. The flowcytometric analysis of TIL showed a significant accumulation of CD25+ and HLA-DR+ cells and a reduced number of CD45RA+ cells as compared to autologous peripheral blood lymphocytes (PBL) or intraepithelial lymphocytes of the colon mucosa (IEL). These phenotypic characteristics of TIL did not correlate with the CAMexpression on tumor cells.

The carcinoembryonic antigen (CEA) modulates effector-target cell interaction by binding to activated lymphocytes.

We and others have shown that the carcinoembryonic antigen (CEA) modulates the susceptibility of human colorectal carcinoma cells to cytotoxic lymphocytes. We now demonstrate that the density of the CEA molecules on the tumor cell surface has a determining influence on its protective function. In contrast, CEA released by tumor cells has no protective effect for CEA-negative cells. To elucidate the responsible mechanism, we analyzed the binding properties of CEA to lymphokine activated killer (LAK) cells. In agreement with our observation that only membrane-bound CEA provides protection, we found that intercellular contact between LAK cells and CEA-expressing tumor cells is required for binding of CEA to LAK cells. Using FACScan analysis, we demonstrate the presence of CEA on lymphocytes co-cultured with CEA-transfected cells but not after co-culture with their parental cells and after incubation with soluble CEA. Interestingly, following overnight co-culture the amount of bound CEA on LAK cells was identical regardless of adherence on tumor cells or loss of contact with tumor cells. This indicates that CEA is released from the tumor cells after binding to LAK cells. Our results suggest that tumor cells can modulate effector cell adhesion by regulating the turnover rate of CEA on the tumor cell membrane.

Phenotypical and functional differences of tumor-infiltrating lymphocytes from human colorectal cancers induced by costimulation with rIL-2 and rIL-4 in vitro.

The influence of recombinant human interleukin-4 (rIL-4) on the proliferation and cytotoxic activity of tumor-infiltrating lymphocytes (TIL) from human colorectal cancers was investigated TIL and peripheral blood lymphocytes (PBL) were cultured for 3-5 weeks. Under simultaneous stimulation with rIL-2 and rIL-4 the proliferation of TIL was less pronounced compared to stimulation with rIL-2 alone. In rIL-2 expanded TIL an outgrowth of CD56+ cells was observed. Concordantly, the expression of CD3+ cells was low. The number of CD56+ cells could be reduced significantly in TIL and PBL by stimulation with rIL-2 and rIL-4 simultaneously while the number of CD3+ cells increased. In TIL and PBL co-stimulation with rIL2 and rIL-4 resulted in higher CD4/CD8 ratios as compared to expansion with rIL-2 alone. In a 72 hour cytotoxicity assay the rIL-2/rIL-4 expanded TIL and PBL showed a lower lytic activity against autologous tumor targets in comparison to the rIL-2 expanded lymphocytes. In contrast, the cytotoxic activity of rIL-2/rIL-4 cultured TIL against allogeneic tumor cells was higher than in rIL-2 stimulated TIL.

Prognostic factors in breast cancer: theoretical and clinical aspects (review).

To estimate the prognosis of a patient is extremely important, because it strongly influences the therapeutic strategy. For this purpose a battery of prognostic factors has been described and evaluated, some of which are discussed in the present paper. They are divided into four categories (epidemiologic/demographic, anatomic/morphologic, hormonal/cellular and genetic/molecular factors). Although there are still many difficulties in the evaluation of prognostic factors, that must be overcome and although the ideal prognostic factor(s) are not yet defined, there are some that have been proved useful and should be employed for the benefit of the patients.

Comparative evaluation of four tumor markers, CA 242, CA 19/9, TPA and CEA in carcinomas of the colon.

At present there is at least one optimal tumor marker and/or optimal marker combination available for the most frequent carcinomas. For colorectal carcinomas (CRC) the general consensus is that CEA is the best single marker, however, other markers, like CA 19/9, TPA and more recently CA 242 were reported to be just as useful. Since CA 242 is a relatively new marker we decided to assess comparatively the value of the four markers in CRCs. Although 308 sera from patients with CRCs, benign digestive diseases (n = 128) and healthy controls (n = 45) were analyzed using commercially available testkits. None of the mean values of the four markers were elevated above their respective cut-off levels in the controls. CEA was the most sensitive marker in early stage cancer, while CA 19/9 was the least sensitive marker, and hence should not be used for the study of this kind of malignancy. However, it remains usefull for carcinomas of the pancreas. As regards specificity, CA 242 was the most specific marker in hepatobiliary diseases. Used concomitantly, CEA and TPA and CEA plus CA 242 augmented the sensitivity markedly, hence these combinations can be recommended. CEA remains the most reliable marker for the follow-up of colon cancer patients, the novel marker CA 242 has a similar performance, the combination of the two markers ameliorates the specificity of CEA used as a single marker.