Functional expression of Fas and Fas ligand on human colonic intraepithelial T lymphocytes.

The expression of Fas, a cell surface receptor directly responsible for triggering cell death by apoptosis, and its ligand (FasL) was investigated on both human colonic intraepithelial T lymphocytes (IELs) and peripheral blood mononuclear lymphocytes (PBMLs). FACS analysis indicated that IELs have increased expression of Fas compared with PBMLs, together with the progress activation marker, CD45RO. A discrete fraction of freshly isolated IELs also constitutively expressed FasL, perhaps as a result of recent in vivo activation. Using monoclonal antibody APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of anti-Fas monoclonal antibody (CH11) on both IELs and PBMLs. FACS analysis revealed that CH11 increased the percentage of apoptotic cells, in IELs but not in PBMLs. Culture with anti-FasL monoclonal antibody (4H9) significantly recovered cell viability in IELs, but not in PBMLs. These results indicate that IELs constitutively express both Fas and FasL and that Fas crosslinking generates signals resulting in apoptosis, outlining a potential mechanism involved in intestinal tolerance.

Circulating levels of soluble CD30 and other markers in colorectal cancer patients.

In a search for new biologic serum tumor markers with prognostic value we evaluated the soluble form of the CD30 (sCD30), a marker of cells producing T helper 2 (Th2)-type cytokines, interleukin-1 receptor antagonist (IL-1ra), soluble interleukin-2 receptor (sIL-2R), soluble tumor necrosis factor -type I, -type II (sTNF-R55, -75) and immunosuppressive acidic protein (IAP) in patients, with advanced colorectal cancer. The data showed that abnormal levels of sCD30 were detected in eight (80.0%) out of ten patients. In contrast, sCD30 levels were not detected in healthy volunteers. The relationship between sCD30, sIL-2R and IAP were positively correlated. In contrast, sCD30 and IL-1ra were negatively correlated. These results suggested that IL-1ra may play a role, at least in part, to inhibit CD30 release, and sCD30 appears to be a new biologic serum tumor marker of possible use in the clinical setting of cancer patients.

[A case of local recurrence of rectal cancer responding to local intraarterial infusion therapy].

A 42-year-old male developed pain in the right gluteal region due to local recurrence after curative resection of advanced lower rectal cancer. Radiotherapy (60 Gy) was performed, but satisfactory results were not obtained. Therefore, a reservoir was placed lowing cannulation of the internal iliac artery. The chemotherapy, in addition to intravenous administration of low dose CDDP (20 mg), included local intraarterial infusion therapy with 5-FU (1,500 mg/5 hour) once per week. After 10 courses of this chemotherapy (total dose: CDDP, 200 mg; 5-FU, 15,000 mg), the pain decreased, and the tumor size was reduced without side effects, improving the patient's QOL. At present, multidisciplinary treatments including such chemotherapy and radiotherapy is performed for local recurrence of rectal cancer, but adequate results are often not obtained. Local intraarterial infusion chemotherapy via the internal iliac artery accompanied by changes in blood flow can be safely performed on an outpatient basis, and appears to be effective for local recurrence of rectal cancer.

Recombinant adenovirus expressing wild-type p53 is antiangiogenic: a proposed mechanism for bystander effect.

Angiogenesis is required for the growth and progression of malignancies. Recent studies have demonstrated that genetic alterations may accompany acquisition of the angiogenic phenotype. The tumor suppressor gene p53 is most frequently mutated in human cancers and is also known to be a transcriptional regulator of a variety of genes. Here, we investigated the antiangiogenic effect of the wild-type p53 (wt-p53) gene transfer on a human non-small cell lung cancer cell line. Mutant p53-expressing H226Br non-small cell lung cancer cells were transduced with the wt-p53 gene using a recombinant adenoviral vector (Ad5CMVp53) and applied to semiquantitative reverse transcription-PCRs for the detection of altered mRNA expression of angiogenic and/or antiangiogenic factors. In vivo neovascularization assay of Ad5CMVp53-infected cells was then performed using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice. We also evaluated the effect of Ad5CMVp53-infected H226Br cells on nontransduced tumor cells in vivo by s.c. inoculating mixture of cells into nu/nu mice. Ad5CMVp53 infection markedly inhibited the expression of an angiogenic factor, vascular endothelial growth factor, and increased the expression of a novel antiangiogenic factor, brain-specific angiogenesis inhibitor 1, resulting in reduced neovascularization in vivo. Mixing experiments showed that tumor cells transduced with the wt-p53 gene inhibited the in vivo tumor growth of adjacent nontransduced cells. Our data suggest that a recombinant adenovirus expressing the wt-p53 gene is antiangiogenic, which may explain, in part, the mechanism of the bystander effect induced by the wt-p53 gene transfer on adjacent tumor cells.

Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells.

The CD95 (Fas/APO-1) system regulates a number of physiological and pathological processes of cell death. The ligand for CD95 induces apoptosis in sensitive target cells by interacting with a transmembrane cell surface CD95 receptor. We previously reported that the recombinant adenovirus-mediated transfer of the wild-type p53 gene caused apoptotic cell death in a variety of human cancer cells. To better understand the mechanism responsible for this cell death signaling, we have investigated the potential involvement of the CD95 receptor/ligand system in p53-mediated apoptosis. The transient expression of the wild-type p53 gene upregulated the CD95 ligand mRNA as well as protein expression in H1299 human lung cancer cells deficient for p53 and in DLD-1 and SW620 human colon cancer cells with mutated p53, all of which constitutively expressed CD95 receptor as shown by a flow cytometric analysis, and induced rapid apoptotic cell death as early as 24 h after gene transfer. However, the sensitivity to the cytolytic effect of agonistic anti-CD95 antibody (CH11) varied among these cell lines: CH11 induced apoptosis in H1299 cells, but not in DLD-1 and SW620 cells despite their abundant CD95 receptor expression, suggesting that the CD95 receptors on DLD-1 and SW620 cells might be inactivated. In addition, an antagonistic anti-CD95 ligand antibody (4H9) that interfered with the CD95-receptor-ligand interaction partially reduced the apoptosis induced by the wild-type p53 gene transfer in H1299 cells, whereas apoptosis of DLD-1 and SW620 cells occurred in the presence of 4H9. Taken together, these findings led us to conclude that the CD95 receptor/ligand system is differentially involved in p53-mediated apoptosis, suggesting that the restoration of the wild-type p53 function may mediate apoptosis through CD95 receptor/ligand interactions as well as an alternative pathway.

Functional expression of Fas and Fas ligand on human intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) constitute the first lymphoid compartment to encounter dietary antigens and intestinal pathogens. IEL are proposed to be involved in the defence against bacterial and viral invasion and to play an important role in mucosal immunity. Fas (CD95/APO-1) is a surface receptor that induces apoptotic cell death upon ligation with Fas ligand (FasL). The aim of this study was to examine the expression and function of Fas and FasL on freshly isolated normal human colonic IEL. The expression and function of Fas and FasL on IEL isolated from 40 normal colonic specimens were examined by flow cytometry, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and DNA-release cytotoxicity assay. Virtually all CD3+ IEL (95.2 +/- 4.3%) expressed Fas and were sensitive to agonistic anti-Fas antibody, whereas only 56.6 +/- 8.4% of peripheral T lymphocytes expressed Fas and were resistant to the antibody. We also detected FasL mRNA and protein (40.1 +/- 4.2%) on IEL, and found that IEL exerted FasL-mediated cytotoxicity against Fas-expressing target cells. These findings suggest that human IEL are activated in situ but are tightly regulated by the constitutive expression of functional Fas and FasL to maintain homeostasis of the mucosal immune system.

Isolation of MHC class I-restricted tumor antigen peptide and its precursors associated with heat shock proteins hsp70, hsp90, and gp96.

We have previously demonstrated that vaccination with heat shock proteins hsp70, hsp90, and gp96 elicits specific immunity against the tumor from which the hsps were purified. Although the association of tumor Ag peptides with these hsps have been suggested, the identification of the peptides or their precursors stripped from the hsps remained to be resolved. We show in this report that an Ld-restricted cytotoxic T lymphocyte epitope of a mouse leukemia RLmale symbol1 and its precursors are associated with the chaperones hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic reticulum. Hsp70 was associated with only final sized octamer, while hsp90 was found to associate with the octamer and two distinct precursor peptides. The gp96 was associated with the octamer and one of the two precursors. Thus, each of the hsps bound a distinct set of peptides. Our results have demonstrated for the first time that the hsps associate not only with final sized tumor Ag peptide but also with its precursors. The implication of this evidence is also discussed in terms of the roles of hsps in MHC class I Ag processing/presentation.

Involvement of apoptosis in activation-induced cell death of bacteria-reactive human CD45RO+ T cells.

Although the identity of the T cells that protect against bacteria in humans remains unknown, it is clear that patients with bacterial infection have reduced numbers of T cells in their blood. Here we have determined whether this T cell loss is a consequence of bacterial antigen-mediated activation-induced cell death (AICD). By flowcytometric analysis, less than 0.3% of freshly isolated T cells from healthy volunteers and patients with severe pneumonia were identified as apoptotic. However, during culture the rate of apoptosis in peripheral blood T cells from patients was 3.0 +/- 0.9%; and increased further in the presence of anti-CD3 (7.4 +/- 2.1%) and decreased when IL-2 was added (4.4 +/- 1.3%). In contrast, no changes were observed in healthy volunteers on addition of anti-CD3. Further, anti-CD3 significantly increased the susceptibility to apoptosis of CD45RO+ T cells, but not CD45RA+ T cells from patients, and the percentage of CD45RO+ T cells in patients was significantly higher than that in healthy volunteers. Flowcytometric analysis revealed the expression level of Fas to be higher in the patients than healthy volunteers. Collectively, these findings demonstrated that bacteria-reactive T cells were more susceptible to AICD and that Fas-FasL pathways of apoptosis were involved. AICD of CD45RO+ T cells, therefore, provides an explanation for the loss of bacteria-reactive T cells during bacterial infection.

Effect of streptococcal lyzate OK-432 on peripheral blood mononuclear cells in gastric cancer patients.

OK-432, a killed preparation of Streptococcus pyogenes, as well as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum are all known biological response modifiers. To examine the immunomodulatory effects of OK-432, natural killer cell activity and cytokine production by peripheral blood mononuclear cells (PBMCs) were assessed in 32 patients with gastric cancer. Skin tests for Streptococcus pyogenes A-3Su (Su-PS) and BCG were performed in all patients. Other nutritional and immunological parameters were also determined. OK-432-treated PBMCs showed a significant increase of cytotoxicity against K562 cells (p < 0.01). Increased levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2) were found in the supernatants of cultures treated with OK-432 in 29 (90.6%), 20 (62.5%), and 8 (25.0%) out of 32 patients, respectively. Natural killer cell activity, IFN-gamma production, and the Su-PS skin test were positively correlated (p < 0.01). In contrast, the BCG test and other markers were not correlated with natural killer cell activity and IFN-gamma production. These results suggest that the Su-PS skin test could predict OK-432-induced natural killer cell activity and IFN-gamma production in patients with gastric cancer, and was therefore useful to determine whether patients were responders to OK-432.

Penile metastasis secondary to cecum carcinoma: a case report.

To date, only around 300 cases of metastasis to the penis have been reported and the primary cancer has been generally found in genitourinary structures. However, exceptional cases in which the primary site has been situated in organs like esophagus, pancreas, stomach have been published. We report an uncommon case in which the primary tumor was located in the cecum. To our knowledge, this is the first case of penile metastasis in which the responsible tumor was located in the cecum and in which the mode of metastasis was not direct invasion. As the tumor was restricted to the colon wall without invasion to neighbouring structures, and because colon cancer follows mainly the lymph-vascular route to its dissemination, this route is the most likely mode of the spread. By magnetic resonance imaging (MRI) on penile shaft, multiple nodules were clearly visualized. Nevertheless, as in most cases, in spite of the availability of advanced and precise diagnostic methods, the information was of little value for the patient. His survival was short.

Human colon cancer cells express the functional Fas ligand.

Fas ligand (FasL) belongs to the TNF superfamily. It is induced in activated lymphocytes and eliminates Fas-positive lymphocytes, resulting in the down-regulation of immune responses. FasL has also been detected in tissues other than lymphoid cells. We investigated the expression and function of FasL on human colon cancer cells. FasL mRNA was detected by RT-PCR in all six colon cancer cell lines tested and was not found on fibroblasts. FasL protein was detected in DLD-1, LoVo, HCT-116 and RPMI 4788 cells by immunohistochemical staining. DLD-1, LoVo and WiDr were cytotoxic against mouse T lymphoma cells which were transfected with human Fas receptor cDNA. The cytotoxicity was significantly enhanced by phorbol 12-myristate 13-acetate (PMA) and ionomycin. Our data suggest that the FasL expressed in human colon cancer cells may be regulated by endogenous factors in the microenvironment of the host and facilitates the escape from the host immune system.

Soluble intercellular adhesion molecule-1 and natural killer cell activity in gastric cancer patients.

Intercellular adhesion molecule-1 (ICAM-1), a molecule bound to the cell surface, is a ligand for leukocyte function antigen-1 (LFA-1), and the ICAM-1/LFA-1 system mediates various cell-cell interactions involved in immunity. Soluble ICAM-1 (sICAM-1) is a circulating substance and binds with LFA-1 of leukocytes, thus, making leukocytes less available for binding with cell surface ICAM-1 on target cells. The serum level of soluble ICAM-1 (sICAM-1) was found to be significantly elevated (p<0.01) in patients with early and advanced gastric cancer compared with healthy controls. Natural killer activity (NK activity) was assessed by measuring the cytotoxicity of peripheral blood mononuclear cells (PBMCs) for K562 cells. There was no significant difference in NK activity between gastric cancer patients and healthy controls when heat-inactivated fetal calf serum was used in assays. However, addition of patient serum significantly decreased (p<0.05) NK activity when the serum was from patients with advanced gastric cancer compared with healthy volunteers. Addition of anti-ICAM-1 monoclonal antibody 0 to 5.0 microg/ml caused little change in NK activity in healthy controls, but its addition at 10 microg/ml remarkably decreased NK-activity in gastric cancer patients, probably through antibody binding with ICAM-1 on target cells. In other experiments, liver metastasis was induced in mice by inoculation of colon 26 murine colon cancer cells. In vitro pretreatment of colon 26 cells with the anti-ICAM-1 monoclonal antibody significantly increased the number of metastatic nodules. These results suggest that both sICAM-1 and anti-ICAM-1 monoclonal antibody act as immunosuppressive factors by inhibiting the ICAM-1/LFA-1 system.

Can POSSUM, a scoring system for perioperative surgical risk, predict postoperative clinical course?

POSSUM, a Physiological and Operative Severity Score for the enUmeration of Mortality and morbidity, is a scoring system which assesses perioperative surgical risks (Copeland GP et al.: Br J Surg, 1991, Vol 78, 356-360). The POSSUM scoring system consists of two categories of assessment to assess the risk of surgery. A 12-factor (age, cardiac status, pulse rate, systolic blood pressure, respiratory status, Glasgow Coma Score, serum concentration of urea, potassium and sodium, hemoglobin concentration, white cell count and findings on electrocardiography) and 4-grade physiological score (PS) were developed. This was combined with a 6-factor (type of surgical procedure, number of procedures, blood loss, peritoneal soiling, presence of malignancy and mode of surgery) and 4-grade operative severity score (OSS). The present paper attempts to validate it retrospectively. Postoperative hospitalization period and duration of antibiotics administration were both significantly correlated with OSS, but not with PS. These results suggest that the POSSUM scoring system is useful for predicting the postoperative clinical course.

p53 expression overcomes p21WAF1/CIP1-mediated G1 arrest and induces apoptosis in human cancer cells.

The p21WAF1/CIP1 gene, which encodes a cyclin-dependent kinase inhibitor, may be critical for tumor suppressor gene p53-induced cell cycle arrest. The p53 gene is known to regulate G1 checkpoint, which can either induce G1 arrest or initiate apoptosis. To directly examine the role of p21WAF1/CIP1 in the control of p53 function, we have introduced human p21WAF1/CIP1 gene into a p53-deficient human non-small cell lung cancer cell line H1299 using a p21WAF1/CIP1-expressing adenoviral vector (AdCMVp21). Infection with AdCMVp21 resulted in high levels of p21WAF1/CIP1 expression and significantly suppressed the growth of H1299 cells through the G1 arrest of the cell cycle. In contrast, transient expression of the wild-type p53 gene by a recombinant adenoviral vector (AdCMVp53) in H1299 cells induced apoptotic cell death and resulted in a rapid loss of cell viability. We then examined the effects of combined infection with AdCMVp21 and AdCMVp53 on H1299 cells to explore the dominant function of these molecules. Interestingly, introduction of exogenous p53 overcame p21WAF1/CIP1-mediated cell cycle arrest at G1 and induced apoptosis, although viral-transduced p21WAF1/CIP1 expression level was unaffected. These observations suggest that p53 expression converts a p21WAF1/CIP1-mediated growth arrest into apoptosis. The result was repeated with two additional human colon adenocarcinoma cell lines with the different p53 status, mutant p53-expressing DLD-1 and wild-type p53-expressing LoVo, suggesting that this phemonenon is a general event among human cancer cells. Thus, p53-mediated apoptotic pathway is dominant over the growth arrest pathway, indicating that p53 may be an essential upstream mediator of p21WAF1/CIP1 in the regulation of a cell process leading either to growth arrest or to apoptotic suicide.

Novel combination therapy for human colon cancer with adenovirus-mediated wild-type p53 gene transfer and DNA-damaging chemotherapeutic agent.

Alteration of the wild-type (wt) p53 gene by mutation, deletion or re-arrangement is a major factor in the development of human colon cancer. Recent studies have demonstrated that p53 might be an essential component of the apoptotic pathway triggered by DNA-damaging stimuli such as chemotherapeutic agents and ionizing radiation. We examined the anti-tumor effects of adenovirus-mediated wt-p53 gene transfer in combination with a chemotherapeutic drug on the human colon cancer cell line WiDr, which is homozygous for a mutation in the p53 gene. Treatment with the chemotherapeutic drug cisplatin following infection with a replication-deficient, recombinant adenoviral vector expressing wt-p53 (termed AdCMVp53) significantly suppressed the growth of WiDr cells compared to single treatments alone. To evaluate the in vivo efficacy of AdCMVp53 and cisplatin given sequentially, WiDr cells were inoculated s.c. in nu/nu mice. After 3 days, AdCMVp53 was injected s.c. into the area where tumor cells were implanted, followed by i.p. administration of cisplatin. Analysis of initial growth inhibition at 21 days demonstrated a profound therapeutic cooperativity, though administration of either AdCMVp53 or cisplatin alone was followed only by a slowing of growth. Our results suggest that gene therapy using wt-p53-expressing adenovirus in combination with a chemotherapeutic DNA-damaging drug could be a useful strategy for treating human colon cancer.

Cancer cachexia and depressive states: a neuro-endocrine-immunological disease?

Plasma 5-hydroxytryptamine (serotonin), tryptophan, neopterin and cortisol levels were measured in patients with depressive cancer cachexia and in healthy controls during the same time period. Patients with advanced cancers had significantly raised neopterin, a marker of endogenous gamma-interferon (IFN-gamma) production, and cortisol values, but decreased serotonin and tryptophan levels. Much work has been done to elucidate the possible role of serotonin in depressive states. IFN-gamma induces a high level of indoleamine dioxygenase (IDO), a tryptophan degrading enzyme, and high cortisol levels induce high tryptophan oxygenase activity, which in turn increases metabolism along the tryptophan-nicotinic acid pathway. These results suggest that persistent immune activation and intense adrenal activity occur in patients with cancer cachexia, resulting in disorders involving tryptophan metabolism followed by depression in cancer cachexia.

Interleukin-1 receptor antagonists and other markers in colorectal cancer patients.

BACKGROUND: Although the interleukin-1 receptor antagonist (IL-1ra) has been suggested as a potentially valuable therapeutic agent and has been shown to improve outcome in various animal models of arthritis, septic shock, and inflammatory bowel disease, there is little information available about its level in the circulation in patients with cancer. METHODS: Serum levels of IL-1ra, soluble interleukin-2 receptor (sIL-2r), soluble intercellular adhesion molecule-1 (sICAM-1), and cortisol were measured in normal controls and patients with colorectal cancer. RESULTS: The data showed that serum IL-1ra levels in patients were significantly lower than those of healthy controls (P < 0.05). In contrast, serum sIL-2r and cortisol levels in patients were significantly higher than those of normal controls (P < 0.01). Serum sICAM-1 levels in patients were the same as in normal controls. CONCLUSIONS: These results suggested that a reduced level of IL-1ra exists in colorectal cancer patients relative to normal controls, indicating that cancer patients have an immunologic disorder and that exogenous IL-1ra administration might be a future alternative for cancer treatment.

Clinical value of cytokine antagonists in infectious complications.

Plasma levels of antiinflammatory compounds (which counteract inflammation, cortisol, IL-1 receptor antagonist, IL-1ra; soluble IL-2 receptor, sIL-2r, soluble intercellular adhesion molecule-1, sICAM-1; interleukin-10, IL-10) were synchronously determined in a consecutive series of 25 patients with severe bacterial infections. Serum levels of cortisol, IL-1ra, sIL-2r, sICAM-1 and IL-10 were significantly higher in patients with infection compared with healthy volunteers. Bacterial infection results in the production of inflammatory and proinflammatory cytokines from macrophage/monocyte, which are thought to be involved in the pathogenesis of systemic inflammatory response syndrome (SIRS). We found that counter-inflammatory compounds can also be released during infectious insults. These results suggested that the biological activity of inflammatory mediators is inhibited by natural antiinflammatory compounds, and the body itself might down-regulate excessive inflammatory cascades through counteracting the inflammatory responses and restore homeostasis.

Effect of low dose cyclophosphamide on the synthesis of acute phase protein and its significance for cancer chemotherapy.

Patients with far advanced colorectal cancers received chemotherapy consisting of low-dose cyclophosphamide (LDCY) 333 mg/m2 every four weeks intravenously and by oral administration of 5'-DFUR (a masked compound of 5-Fluorouracil). Serum levels of immunosuppressive acidic protein (IAP), an acute phase protein, were measured every four weeks for a total of thirty-one LDCY trials of ten patients. LDCY chemotherapy significantly decreased the IAP levels in cancer patients with high IAP levels. These results suggested that LDCY chemotherapy could counteract host responses against tumors and could have decreased immunosuppressive responses in cancer patients.