Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells.

Beta-hydroxy-beta-methylglutaryl coA reductase inhibitors (HRIs) inhibit isoprenylation of several members of the Ras superfamily of proteins and therefore have important cellular effects, including the reduction of proliferation and increasing apoptosis. Significant toxicity at high doses has precluded the use of HRIs as a monotherapy for cancers. We therefore studied whether combinations of the HRI lovastatin with standard chemotherapeutic agents would augment apoptosis in colon cancer cells. In the colon cancer cell lines SW480, HCT116, LoVo, and HT29, lovastatin induced apoptosis with differing sensitivity. Pretreatment with lovastatin significantly increased apoptosis induced by 5-fluorouracil (5-FU) or cisplatin in all four cell lines. Lovastatin treatment resulted in decreased expression of the antiapoptotic protein bcl-2 and increased the expression of the proapoptotic protein bax. The addition of geranylgeranylpyrophospate (10 microM) prevented lovastatin-induced augmentation of 5-FU and cisplatin-induced apoptosis; mevalonate (100 microM) was partially effective, whereas cotreatment with farnesyl pyrophosphate (100 microM) had no effect. These data imply that lovastatin acts by inhibiting geranylgeranylation and not farnesylation of target protein(s). Our data suggest that lovastatin may potentially be combined with 5-FU or cisplatin as chemotherapy for colon cancers.

Apoptosis in gastric epithelial cells is induced by Helicobacter pylori and accompanied by increased expression of BAK.

Carriage of the bacterium H. pylori in the human stomach is associated with evidence of increased epithelial cell apoptosis. This may be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. The ability of H. pylori to directly induce epithelial apoptosis was examined in vitro by fluorescence and electron microscopy, flow cytometry, and DNA fragmentation ELISA. The induction of apoptosis by H. pylori was time and concentration-dependent and inhibited by preventing direct bacterial-epithelial cell contact. Apoptosis was accompanied by increased expression of Bak, with little change in expression of other Bcl-2 family proteins. The expression of Bak was also increased in gastric biopsies from patients colonized by H. pylori. Thus, H. pylori induces gastric epithelial cell apoptosis, by a Bak-dependent pathway.

The nitrite/elastin reaction: implications for in vivo degenerative effects.

Nitrite ion is a by-product of nitrogen oxides (nitric oxide and nitrogen dioxide) from cigarette smoke and is used as a preservative for curing meats. Therefore, study of the reaction of nitrite with elastin in vitro was undertaken. By colorimetric assay, reactivity of nitrite with insoluble elastin at neutral pH, 37 degrees C, and physiologic concentration was confirmed. In histochemical studies on in situ human aortic elastin, nitrite-treated sections displayed marked structural disruptions. Determinations of fluorescence and absorbance on nitrite-treated soluble bovine elastin revealed marked alterations of fluorescence, and increased UV and visible absorbance. Amino acid analysis confirmed that it reacted with tyrosine. The findings indicate that non-enzymatic nitration by nitrite may have deleterious effects on elastin in vivo and may provide insights into the pathogenesis of chronic elastin degenerative processes, including aortic aneurysms, pulmonary emphysema, and premature skin wrinkling, all of which have been well known to have associations with cigarette smoking.

Cytokines that activate proteolysis are increased in abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) disease is characterized by an increase in proteolysis and loss of matrix components. The cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), products of activated macrophages and T cells, are known to increase the production of matrix-degrading enzymes in some pathological states. METHODS AND RESULTS: Seven AAA and five control aortic tissue extracts were assayed for TNF-alpha and IL-1 beta with ELISA. TNF-alpha was elevated significantly in AAA extracts compared with controls (86 +/- 34 pg/mg of total protein versus 1 +/- 1 pg/mg of total protein; P < .001). IL-1 beta concentration also was significantly increased in the AAA specimens (48 +/- 14 pg/mg of total protein versus 12 +/- 5 pg/mg of total protein; P < .05). Immunoblotting demonstrated secreted forms of TNF-alpha in the AAA extracts, and possible membrane-bound forms were observed when the tissues were detergent-extracted. Known forms of IL-1 beta also were observed on immunoblots of AAA tissue extracts. CONCLUSIONS: The presence of TNF-alpha and IL-1 beta in AAA tissue underscores the importance of the infiltrating inflammatory cells present in the media and adventitia of aneurysmal aortic wall and further implicates an inflammatory process in the pathogenesis of AAA.

The elastase infusion model of experimental aortic aneurysms: synchrony of induction of endogenous proteinases with matrix destruction and inflammatory cell response.

PURPOSE: Perfusion of the isolated aorta of the rat with a saline solution containing pancreatic elastase induces an abdominal aortic aneurysm (AAA). An interesting feature of this model is the phenomenon of latency, suggesting that additional steps beyond the initial injury are required for AAA formation. This study was performed to determine whether the latency period for aortic dilation to aneurysmal proportions is correlated with the appearance of proteinases of endogenous origin and the interval for infiltration of inflammatory cells. METHODS: Twenty Wistar rat aortas were perfused with the test solution, and 20 with normal saline solution. Laparotomy was performed on days 1, 2, 3, and 6 for measurement and harvest of the aorta. Histochemical studies were performed to analyze changes in matrix proteins, and substrate gel enzymography was used to determine the appearance of endogenous proteinases. Immunohistochemical studies were performed with monoclonal antibodies to T cells (CD-4, -5, and -8), monocytes/macrophages (ED-2), B cells (LC-A), immunoglobulin G, and immunoglobulin M. RESULTS: The exogenously administered elastase was not detectable beyond day 2, but the aortic diameter did not progress to aneurysmal dimensions until the interval between days 3 and 6. During the period from day 3 to day 6, multiple endogenous matrix proteinases became detectable in the aortic tissue preparations. Immunohistochemical study revealed progressive infiltration of the aorta with various subsets of inflammatory cells. CONCLUSION: The results suggest that the latency in AAA formation in this model corresponds with a complex sequence of biochemical and cellular events. The model provides an "early window" into these interesting early phases leading to aneurysm formation.

Matrix metalloproteinases in abdominal aortic aneurysm: characterization, purification, and their possible sources.

One of the most consistent observations in abdominal aortic aneurysm (AAA) disease is the disorganization and disruption of elastin and other matrix components of the aortic wall. The enzymatic basis for the biochemical features of AAA has been investigated beginning with the demonstration on substrate gel enzymography of a typical "profile" of proteinase activities in AAA tissue extracts which degrade gelatin, casein and elastin. A recombinant TIMP-1 affinity column was developed and three of the elastolytic/caseinolytic activities with approximate molecular weights of approximately 80 kDa, approximately 50 kDa and approximately 32 kDa were partially purified from these extracts. Affinity for rTIMP-1 suggests that these enzymes are members of the matrix metalloproteinase (MMP) family. High molecular weight forms of two MMPs, collagenase (MMP-1) and stromelysin-1 (MMP-3), were also isolated from the AAA tissue on this column; active forms of MMP-1 could be demonstrated by immunoblotting techniques in this preparation under reducing conditions. Infiltrating inflammatory cells are known sources of these proteolytic activities; analysis of these cell populations in the aneurysmal aortic wall using fluorescence-activated cell counting revealed a fifty-fold increase in macrophages (a well-known source of matrix-degrading enzymes) as well as a significant increase in lymphocytes.

Monoclonal antibody-defined phenotypes of regional lymph node and peripheral blood lymphocyte subpopulations in early breast cancer.

Lymphocyte subset phenotypes in peripheral blood and axillary lymph node cell isolated from 28 patients undergoing surgery for breast cancer were determined by two-color immunofluorescence with monoclonal antibodies and flow cytometric analysis. Lymphocyte subpopulation proportions were determined with combinations of monoclonal antibodies directed against the Leu 2, Leu 3, Leu 7, Leu 8, Leu 11, Leu 12, Leu 15, Leu M3, and HLA-DR surface markers. Patients were staged according to the postsurgical-pathological modification of the Tumor-Node-Metastases staging system, for analysis of tissue source (lymph node versus peripheral blood) and stage of disease as factors influencing lymphocyte subset size. Activated Leu 2+DR+ and Leu 3+DR+T-cells were elevated in stage 2 carcinoma compared to Stage 1. Elevation of Leu 2+8+ circulating T-cells and a reciprocal depression of Leu 2+8- T-cells were also seen in Stage 2 patients when compared to Stage 1. Total T-cells, B-cells, Leu 2+, and Leu 3+ T-cell subsets and natural killer phenotypes defined by Leu 7 and Leu 11 were unchanged in the peripheral blood of Stages 1 and 2 breast cancer. Regional lymph nodes from Stage 1 were found to contain a high frequency of Leu 3+ cells which dropped significantly in Stage 2 patients; this was found to be numerically due to a sharp decrease in the Leu 3+8- subpopulation in Stage 2 patients. Elevated B-cells (Leu 12+), activated T-cells (Leu 2+DR+ and Leu 3+DR+), total Leu 2+ cells, and Leu 7-11+ natural killer cells were demonstrated in Stage 2 lymph nodes when compared to Stage 1. Generally, no differences in subpopulations were seen when level 1 (low axillary) lymph node cells were compared to level 3 (high axillary) lymph node cells at each stage of the disease. These findings demonstrate substantial differences in the profile of lymphocyte phenotypes between Stage 1 and Stage 2 breast carcinoma, especially in the ipsilateral regional nodes. The findings presented in this study suggest that changes in local-regional immunocompetent cell subsets may be related to metastasis of tumor to the regional nodes and progression of disease without being fully reflected in the systemic circulation.

Isolation of intestinal mononuclear cells from colonoscopic biopsies for immunofluorescence analysis by flow cytometry.

A method for isolating and characterizing intestinal lymphoid cells from colonoscopic biopsies is presented. Intraepithelial lymphocytes were separated from the lamina propria by incubation in edetic acid (EDTA) and lamina propria lymphoid cells isolated by incubation in collagenase followed by Ficoll-Hypaque density flotation. Quantitation of T lymphocyte helper (OKT4) and suppressor (OKT8) cells was performed using monoclonal antibodies to cell surface markers and analyzed on a flow cytometer. The isolation procedure yielded approximately 400,000 lamina propria cells and 100,000 intraepithelial cells per sample, with better than 90% viability. Surface marker analysis demonstrated significant differences in the ratios of helper to suppressor cells between the intraepithelial lymphocytes and the lamina propria lymphocytes. These demonstrate the feasibility of lymphoid cell isolation from colonoscopic biopsy specimens for surface marker analysis by flow cytofluorimetry. These techniques could prove important in the study of immune mechanisms in inflammatory bowel diseases.

Carcinosarcoma and carcinoma of the gallbladder.

Carcinosarcoma is a tumor-containing malignant epithelial and connective tissue elements. The 18th case of carcinosarcoma of the gallbladder is reported in the context of 17 prior cases from the world literature. The 18-year experience of St. Luke's Hospital with extrahepatic and primary gallbladder cancer is presented to provide perspective on this unusual tumor. The diagnosis of malignant disease of the gallbladder is reviewed with special emphasis on the roles of sonography, computerized tomography, and arteriography. The importance of staging as a guideline to prognosis and therapy of gallbladder cancer is discussed. Carcinosarcoma of the gallbladder behaves clinically like carcinoma and guidelines for surgical and palliative therapy of the more common malignancies of the gallbladder should be followed for carcinosarcoma as well.

Diagnosis, stage, and prognosis of lung carcinoma by preoperative assay of lung tumor antigen-sensitive T lymphocytes.

Levels of circulating T lymphocytes sensitized to human lung tumor--associated antigens (LTA) were determined by the antigen-stimulated active rosette-forming T cell (AgARFC) assay. These levels were correlated with detection, pathological tumor stage, and postassay survival of patients with lung carcinoma. Peripheral blood lymphocytes (PBLs), from patients found to have lung cancer, were incubated with LTA and produced increased AgARFC compared to PBLs incubated without LTA. Significant levels of LTA-sensitive T cells were found in preoperative PBLs of 80% of patients with Stage I disease (8/10, p < 0.0005), 60% of those with Stage II disease (3/5, p < 0.025), and 46% of those with Stage III primary lung cancer (12/26, p < 0.01), compared with 11% of patients with either benign lung lesions (2/12) or lung metastases (0/6) of nonpulmonary malignant tumors (by chi square analysis). Postoperative survival correlated significantly with preoperative levels of LTA-sensitive T cells by AgARFC assay within Stage I lung cancer (r = 0.807, p < 0.0005). Stage I + II (r = 0.689, p < 0.001), and Stage III (r = 0.657, p < 0.001, not treated with chemotherapy). Preoperative PBL from patients with Stage I + II lung cancer were more frequently sensitized to LTA in the AgARFC assay than from patients with nonpulmonary carcinomas (0/22) or cigarette smokers (1/7) without pulmonary lesions (p < 0.0005). These findings demonstrate a high rate of detection of early, resectable lung carcinomas by preoperative AgARFC assay of PBL sensitized to LTA, and a significant correlation of LTA-sensitive T cell levels with tumor stage and patient survival. The AgARFC assay may be of prognostic as well as diagnostic value in the evaluation of patients with lung carcinoma.

The kinetics of T lymphocyte subpopulations in guinea-pigs sensitized with allogeneic transplantation antigens.

The kinetics of specifically sensitized T lymphocytes in the circulation and lymphoid tissues of guinea-pigs immunized with allogeneic transplantation antigens or with synthetic peptide sequence known to induce delayed-type hypersensitivity were documented by the antigen-stimulated active rosette-forming T cell (AgARFC) assay. The results show that immunologically functional cells sensitized to a particular antigen do not remain in the circulation when the antigenic source has been withdrawn. These cells become sequestered in lymphoid tissue and may be recalled into the circulation shortly after the de novo administration of sensitizing antigen. The detection of antigen-sensitive T cells in the circulation was indicative of the presence of and failure to detect these cells and their eventual appearance in lymphoid tissues was related to depletion of the antigenic source.

Circulating breast tumour antigen-sensitive T lymphocytes in early breast cancer and high risk benign breast disease.

Levels of circulating T lymphocytes sensitized to breast tumour associated antigens (BTA) were correlated with pathological tumour stage or benign histopathology in preoperative studies of 180 patients by the antigen-stimulated active rosette-forming T cell (AgARFC) assay. Incubation of lymphocytes with allogeneic BTA extracts produced increased AgARFC compared with incubation without BTA. Significant levels of BTA-sensitive T cells were found in 78 per cent of breast cancer patients compared with 23 per cent of patients with benign disease (P less than 0.0005, by X2). Over 93 per cent of stage I cancer patients responded to BTA, compared with 69 per cent of stage II patients (P less than 0.025) and 59 per cent of stages III-IV patients (P less than 0.005). Twenty-nine per cent of 42 patients with fibrocystic disease were positive to BTA in contrast to 8 per cent of 25 patients with fibroadenomas. This was a 3.6-fold higher incidence of BTA-sensitive T cells associated with fibrocystic disease than with fibroadenomas, which was in agreement with the increased breast cancer risk rate associated with fibrocystic disease. These findings suggest that the AgARFC assay may detect early malignant change in fibrocystic disease. The AgARFC assay was found to reliably detect early invasive carcinoma.

Cell-mediated immunity to donor antigens in renal allograft recipients.

The antigen-stimulated active rosette-forming T-cell (AgARFC) assay was adapted for the preoperative study of 21 consecutive kidney transplants (17 cadaver donors and four living related donors; five retransplants). Recipient peripheral blood lymphocytes were incubated for 15 minutes with donor histocompatibility antigens preparaed by sonication of donor peripheral blood or splenic lymphocytes. Recipient presensitization to donor antigens was expressed as the difference between active rosette formation in the presence (%AgARFC) and in the absence (%ARFC) of donor antigens. This antigen-induced difference is rosette formation (%AgARFC - %ARFC) for all patients ranged from - 7.0% to 24.2%. Of those patients with pretransplant sensitization greater than 6.3% (group I: mean, 13.2 +/- 3.0; n = 7), 71% had severe acute rejection requiring dialysis within the first 2 weeks of transplantation. In contrast, none of the patients with pretransplant values below 6.3% (group II: mean, -0.8 +/- 1.0; n = 14) had rejection requiring dialysis within the first 2 weeks. Group I patients had 43% graft survival at 1 month and 14% survival at 2 months, whereas group II had 86% graft survival at 1 month and 71% at 2 months. The AgARFC assay provided a rapid means of measuring recipient T-cell presensitization to donor alloantigens, which was correlated with the accelerated rejection of renal allografts.

A new assay for T-cell mediated immunity to transplantation antigens.

The kinetics of circulating antigen sensitive T-cells were studied in Hartley strain guinea pig recipients of Shorthair strain first- and second-set skin allografts. Peripheral blood donor antigen sensitive T-cells (AST) were quantitated by the antigen-stimulated active rosette-forming T-cell (AgARFC) assay by incubating lymphocytes in the presence and in the absence of soluble transplantation antigens. The number of circulating AST/cu mm rose to maximum levels (1,165 +/- 272 SEM) by day 3 and fell sharply before first-set graft rejection (453 +/- 117 SEM) on day 7 after transplant. In contrast, there were no detectable antigen-sensitive cells when lymphocytes from both recipient and control guinea pigs were stimulated with soluble recipient-strain antigen. Significant numbers (212 +/- 159 SEM) of circulating AST remained through day 68 after first-set grafts. Following placement of sencon-set allografts on day 73, the AST disappeared from the circulation for 2.5 days and then rose to peak levels (825 +/- 167 SEM) in circulating AST (579 +/- 327 SEM) preceded rejection of second-set skin allografts. When control guinea pigs were immunized with a single dose of soluble donor antigens, a progressive increase in circulating AST (579 +/- 327 SEM) was found through day 17 after sensitization without the fall associated with graft rejection. The antigen-stimulated, rosette-forming T-cell assay may prove useful in the detection of cellular presensitization and in the monitoring of graft rejection in clinical transplantation.