Small bowel intussusception due to malignant melanoma: primary lesion or metastases? A case report.

Intussusception is a rare condition in the adult population: it is responsible for 1% of all bowel obstructions. In most of intussusceptions a malignant tumor is involved; a lot of studies show that approximately 50% of malignant metastases causing small bowel intussusception are metastatic melanomas. In present paper a case of a small bowel intussusception probably due to metastases of an occult melanoma, in a 69-year-old patient, is presented. Surgery resection, careful research of possible primitive neoplasms and an accurate follow-up program has been the treatment of choice. All the investigations carried out did not allow to identify a possible primitive neoplasm. The last whole body PET carried out 44 months after surgery resulted disease-free.

In vitro effect of reduced glutathione on platelet function.

BACKGROUND: Generation of reactive oxygen species has been suggested to represent an important regulatory mechanism of platelet reactivity in both physiological and pathological conditions, and free-radical scavengers may inhibit platelet activation. The purpose of the present study was to investigate the effect of reduced glutathione (GSH) on different platelet functions stimulated by ADP, collagen or PAF. METHODS: Platelet aggregation was investigated by Born's method. TxB2 and PDGF levels were measured by radioimmunoassay. RESULTS: GSH at the lowest dose (1 mM) did not significantly modify aggregation, TxB2 production or PDGF release induced by ADP or PAF, while at higher concentrations (3 mM or 10 mM) it significantly inhibited all parameters. Collagen-induced platelet activation was remarkably less sensitive to GSH, since aggregation was not significantly reduced, while TxB2 production was reduced by GSH when employed at concentrations of 3 mM or 10 mM, and PDGF release was inhibited only by the highest dose (10 mM). IC50 for inhibition of platelet aggregation, TxB2 production and PDGF release were between 1.43 and 2.36 mM when platelets were stimulated with ADP, between 2.23 and 8.90 mM when PAF was used and between 8.00 and 16.30 mM when collagen was the agonist. CONCLUSIONS: Our data suggest that GSH may act as a physiological inhibitor of platelet activation and may therefore contribute to the regulation of platelet reactivity.

In vitro Effect of Verapamil on Platelet Activation Induced by ADP, Collagen or Thrombin.

We studied the effects in Vitro of the calcium channel blocker verapamil (0.1, 0.2 or 0.3 mM) on platelet aggregation, on cytoplasmic Ca(+ +) levels and on TxB(2) production after activation of platelets with adenosine diphosphate (ADP) (100 µM), collagen (20 µg/ml) or thrombin (1 U/ml). A Platelet Ionized Calcium Aggregometer was used and washed, aequorin loaded platelets were employed. The drug was able to inhibit similarly and always significantly aggregation, Ca(+ +) fluxes and TxB(2) production when collagen was the agonist. Furthermore, inhibition of aggregation and TxB(2) production was significant at all the concentrations tested when platelets were activated by ADP or thrombin, but in this case inhibition of Ca (+ +) fluxes was observed only with the higher concentrations of the drug (0.2 or 0.3 mM). Hence, with these two last agonists inhibition of Ca(+ +) movements was less pronounced than inhibition of aggregation or TxB(2) production. These data suggest that platelet activation by collagen depends directly and almost exclusively on Ca(+ +) fluxes through biological membranes, while activation by ADP or thrombin is less strictly related to Ca(+ +) movements. Indeed, with these last two agonists verapamil may inhibit platelet activation also by calcium-independent mechanism(s).

Endothelin and aneurysmal subarachnoid haemorrhage: a study of subarachnoid cisternal cerebrospinal fluid.

Endothelin (ET) is considered one of the most potent vasoconstrictor polypeptides; several experimental studies have suggested its possible role in the pathogenesis of arterial vasospasm after subarachnoid haemorrhage (SAH). Previously reported data on plasma and CSF levels of endothelin in patients with a diagnosis of SAH have been controversial. Cisternal endothelin CSF levels and the possibility that they could be related to vasospasm and other clinical patterns of SAH were investigated. CSF samples were obtained from 55 patients admitted after angiographic diagnosis of intracranial aneurysm. Levels of ET-1 and ET-3 were measured through radio-immunoassay technique. Twelve patients who had operations for unruptured aneurysms were considered control cases; 43 patients with SAH were classified according to: Hunt and Hess grading at admission, vasospasm grading, CT classification and timing of surgery. In all 55 patients ET-1 was measured, while positive levels of ET-3 were found only in 17 cases of 48. No linear correlation was found between cisternal CSF ET-1 levels when considering time of surgery, CT classification, Hunt and Hess grading at admission, and vasospasm grading. The results of ET-3 assay should be considered with great caution because of the low percentage of positive cases. Cisternal CSF levels of ET-1 and ET-3 are not directly related to the occurrence of arterial vasospasm after the aneurysm rupture, or to other major clinical patterns of SAH; however, ET-1 expression occurs either in paraphysiological (unruptured aneurysm) or in pathological conditions (SAH). It is suggested that ET may potentiate, or may be potentiated by, other factors playing a consistent pathophysiological role in the development of vasospasm.

Effect of interferon alpha, interferon gamma and tumor necrosis factor on the procoagulant activity of human cancer cells.

BACKGROUND: It is not known whether the different cytokines may influence the procoagulant activity of cancer cells; the purpose of this study was to investigate the effect of interferon alpha, interferon gamma and tumor necrosis factor on the procoagulation capacity of human cancer cells cultured "in vitro" or isolated from tumor tissues. METHODS: "In vitro" cultured tumor cell lines were derived from a patient with malignant mesothelioma and a patient with lung adenocarcinoma. Cells isolated from 6 carcinomas of different origin were also investigated. The procoagulant activity of the cells before and after treatment with the cytokines was expressed as RBT U/10(5) cells or RVV U/10(5) cells. RESULTS: Short-term incubation of tumor cells cultured "in vitro" with cytokines did not modify their procoagulant activity; after longer incubation however, interferon alpha induced a significant increase in the procoagulant activity of mesothelioma cells, while interferon gamma induced and increase in the procoagulant activity of lung adenocarcinoma cells. Furthermore, short-term incubation of cells isolated from tumor tissues with interferon gamma or tumor necrosis factor resulted in a significant increase of procoagulant activity, while interferon alpha had no effect. CONCLUSIONS: Altogether, these data demonstrate that the cytokines may influence the expression of the different procoagulant activities of tumor cells.

Different expression of procoagulant activity in human cancer cells cultured "in vitro" or in cells isolated from human tumor tissues.

We studied in a homologous system the procoagulant activity of human tumor cells cultured "in vitro" (1402 primary melanoma, Me 7110/2 metastatic melanoma, Hep G2 hepatoma and GLC1 small cell lung carcinoma) or of cells freshly isolated from different human tumor tissues. Tumor cells cultured "in vitro" possessed and released a factor VII dependent procoagulant activity, which was inhibited by concanavalin A and unaffected by iodoacetamide or HgCl2. The activity released by the cells of metastatic melanoma was higher than that released by the cells of the primary tumor. On the contrary, cancer cells isolated from tumor tissues possessed and released a factor VII independent activity which was inhibited by iodoacetamide of HgCl2 and was not modified by concanavalin A. Therefore, different methods for the preparation of tumor cell suspensions have to be used for the study of tumor procoagulants, since their expression depends very largely on the source of tumor cells. Furthermore, cultured human tumor cells are not an appropriate model for the "in vivo" procoagulant effect of tumor cells.

Tumor cells induce platelet aggregation and intraplatelet calcium ion movements.

We studied platelet aggregation and changes in cytosolic Ca(++) concentrations induced by cells isolated from 5 human tumor tissues (2 hepatocellular carcinomas, 1 colon carcinoma, 1 gastric carcinoma and 1 pancreatic carcinoma). A Platelet Ionized Calcium Aggregometer was used and washed, aequorin loaded platelets were employed. Tumor cells were able to induce aggregation and an increase in cytoplasmic Ca(++) concentrations in the presence of trace amounts (10 µl) of PPP, while no aggregating response was found after addition of fibrinogen alone to washed platelets. The platelet aggregating activity of tumor cells was maintained in the presence of factor VII deficient plasma or of factor VIII deficient plasma, and disappeared completely when factor X deficient plasma was added to washed platelets. Furthermore, tumor cell induced platelet aggregation and Ca (++) movements were inhibited by hirudin (100 U/ml), a specific thrombin inhibitor, while concanavalin A (100 µg/ml), a tissue factor inhibitor, had no effect. Finally, preincubation of neoplastic cells with HgCl(2) (0.5 mM), a cysteine protease inhibitor, markedly decreased their ability to induce aggregation and Ca(++) movements; on the contrary, incubation of cells with soybean trypsin inhibitor (10 µg/ml), a serine protease inhibitor, or with concanavalin A (100 µg/ml) had no effect. These data suggest that cells isolated from human tumor tissues activate platelet function through the generation of thrombin, due to a cysteine protease which directly activates factor X.

Platelet activation by cells isolated from human tumor tissues: effect of cyclooxygenase blockade.

We have studied in a homologous system the effect on different platelet functions of cells isolated from 26 human tumor tissues (11 breast carcinomas, 11 colon carcinomas, 2 pancreatic carcinomas, 1 gastric carcinoma and 1 esophageal carcinoma). Tumor cells (10(5)/ml) significantly increased platelet adhesion to glass beads; they were also found to possess a potent platelet aggregating activity and aggregation was accompanied by significant release of ATP and platelet derived growth factor (PDGF) and by production of TXB(2). Preincubation of platelets with a low concentration (1 µM) of indobufen, a cyclooxygenase inhibitor, significantly reduced tumor cell induced TXB(2) production and ATP release, while the other platelet functions were not modified. Higher concentrations of the drug (10 or 100 µM) were also able to inhibit tumor cell-induced platelet aggregation and PDGF release, while platelet adhesion to glass beads was unchanged even at these doses. Finally, preincubation of neoplastic cells with indobufen (400µM) had no effect on their ability to induce platelet aggregation, TXB(2) production and ATP release. These data demonstrate that cyclooxygenase blockade in platelets has different effects on several platelet functions activated by the tumor cells that were investigated.

Proaggregating and procoagulant activities of human mesothelioma tumor cells at different stages of "in vitro" culture.

BACKGROUND: The mechanisms of the interactions between tumor cells and the hemostatic system are not completely understood; the purpose of this study was to elucidate whether tumor cells grown "in vitro" express the same proaggregating and procoagulant activities as cells isolated from tumor tissues, and whether the activities of such cultures are constant and consistent over time. METHODS: Tumor cells were collected and cultured from the pleural fluid of a 71-year-old patient with a sarcomatous malignant mesothelioma. Platelet aggregating activity was studied by adding tumor cells to platelet rich plasma or to washed, aequorin-loaded platelets. The procoagulant activity of the tumor cells was measured by the one-stage recalcification time of different humans plasma substrates. RESULTS: Cells harvested after 4 culture passages possessed low, ADP-dependent platelet aggregating activity, while those studied after 16 or 40 passages activated platelets through the production of thrombin. In the washed platelet system and in the presence of trace amounts of platelet poor plasma, the difference in the aggregating activity of various tumor cell populations was more evident. Normal mesothelial cells did not induce platelet aggregation. Procoagulant activity (tissue factor-like) was low in normal mesothelial cells and in tumor cells after 4 passages, and it was about 10 times higher in tumor cells after 16 or 40 passages. CONCLUSIONS: Results obtained with tumor cells cultured "in vitro" should be considered with caution because their effects are different from those of freshly isolated cells and may not be constant in the different culture passages.

Thromboxane production by platelets during tumor cell-induced platelet activation.

We have evaluated in a homologous system the mechanisms of platelet activation by cells isolated from fresh human tumor tissues and the role of thromboxane B2 (TxB2) generation in this process. Thirty-eight of the 46 tumor tissues considered showed a high platelet-aggregating activity, with no particular distribution in any specific tumor type. Apyrase caused a nonsignificant reduction in the aggregation response, hirudin did not change it, while iodoacetic acid or p-hydroxymercuriphenylsulfonate, specific cysteine proteinase inhibitors, significantly reduced the platelet-aggregating capacity of these tumor cells. In 9 colon carcinomas and in 8 breast carcinomas the levels of TxB2 produced by platelets after addition of tumor cells were measured: tumor cell-induced platelet aggregation was accompanied by a significant production of the metabolite; indobufen, a cyclooxygenase inhibitor, significantly reduced aggregation and particularly TxB2 production, while the drug had no effect on both parameters if preincubated with tumor cells only. These data suggest that cells isolated from different human tumor tissues activate platelets through the activity of tumor-associated cysteine proteinase(s); platelet aggregation by tumor cells is largely dependent on arachidonic acid metabolism in platelets, while such metabolism in tumor cells does not play a significant role.

Human tumor cells cultured "in vitro" activate platelet function by producing ADP or thrombin.

We studied the effects on platelet function of different human tumour cells cultured "in vitro": Mo T lymphocyte cell line, NCI-N592 small cell lung carcinoma cell line, and 5637 bladder carcinoma cell line. Mo and NCI-N592 cells possessed a slight, dose-dependent platelet aggregating activity, which was completely abolished by apyrase and unaffected by hirudin. The cell-free supernatant also induced an aggregation response, which was very similar to that obtained with tumour cell suspensions. The presence of ADP in the cell-free supernatants of cell suspensions was confirmed by HPLC analysis. On the contrary, aggregation induced by 5637 cells was preceded by a significant lag phase; it was not affected by apyrase but it was abolished by hirudin, and the cell-free supernatant had no effect. These data suggest that Mo and NCI-N592 cells activate platelets by producing ADP, while 5637 cells stimulate platelet function by generating thrombin. The amount of ADP produced by the first two tumour cell lines was measured by bioassay: the extent of such production was similar for both cell lines and the maximum was reached after 60 minutes and maintained for up to 3 hours. These results suggest that neoplastic cells can activate platelets by different mechanisms: such investigations should be performed in homologous systems and in well-defined experimental conditions.

Mechanisms of platelet activation by cultured human cancer cells and cells freshly isolated from tumor tissues.

We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2). The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor. On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves. Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate. Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines.

Human breast and colon carcinomas express cysteine proteinase activities with pro-aggregating and pro-coagulant properties.

We have investigated concomitantly the pro-aggregating and pro-coagulant activities of 11 breast and 2 colon human carcinomas. Tumor tissues, obtained at surgery, were immediately processed to prepare tumor-cell suspensions for the study of aggregating activity and tissue extracts for the study of procoagulant capacity. Nine carcinomas (8 breast and 1 colon) possessed a high, dose-dependent platelet-aggregating activity, which was present in the cell-free supernatant and was inhibited by HgCl2 and iodoacetic acid, specific cysteine proteinase inhibitors, while apyrase and hirudin had no significant effect; in contrast, the other tumors did not aggregate platelets. All the tumor extracts tested from 12 carcinomas (11 breast and 1 colon) were able to activate blood coagulation in both the presence and the absence of F VII. The activity was inhibited by HgCl2 and iodoacetamide, while Con A was less effective. Therefore, these tumors do not aggregate platelets through the production of ADP or thrombin, nor promote blood coagulation through the production and release of tissue factor; a tumor-associated cysteine proteinase plays a major role in both pro-aggregating and pro-coagulant activities.

Severe platelet dysfunction in a patient with autoantibodies against membrane glycoproteins IIb-IIIa.

A young women affected by Hodgkin's disease developed chronic autoimmune thrombocytopenic purpura. Splenectomy induced normalization of her platelet count, but hemorrhagic symptoms did not disappear. The patient's platelets did not aggregate in response to collagen and ADP and the IgG fraction of the patient's plasma induced the same defect in normal platelets. The women's IgG recognized glycoproteins IIb and IIIa of normal platelet membranes. Prednisone therapy induced the disappearance of bleeding symptoms and the normalization of platelet aggregation.

Characterization of the platelet-aggregating activity of cancer cells with different metastatic potential.

We studied the mechanisms of platelet activation by sublines exhibiting different metastatic potential of two murine experimental tumors: sublines M4 and M9 of the benzopyrene-induced mFS6 sarcoma and sublines B77-AA6 and B77-3T3 of RSV-transformed BALB/c 3T3 fibroblasts. The neoplastic cells of both models induced platelet aggregation, secretion and prostaglandin biosynthesis. In the first model but not in the second, all these processes correlated with the in vivo malignancy of cells. Pretreatment of B77-AA6 and B77-3T3 cells with apyrase significantly decreased platelet aggregation, while pretreatment of M4 cells was ineffective. However, pretreatment with trypsin or neuraminidase was effective in reducing platelet aggregation induced by M4 cells, but not that induced by any of the others; furthermore, phospholipase A2 reduced the platelet response by all sublines. Finally, platelet-activating activity was also found in the pellets obtained following centrifugation of culture media. These results suggest that platelets are stimulated by cancer cells through different mechanisms; platelet activation by a sialo-lipo-protein complex of the cellular membrane was found to be characteristic of the model in which the platelet-aggregating activity of neoplastic cells correlated with their in vivo metastatic behavior.

Role of tumor proteinases in tumor cell-induced platelet aggregation.

In a previous work we found a correlation between in vivo metastatic potential of cancer cells and their platelet aggregating activity in sublines of the mFS6 murine fibrosarcoma. In the present study the effects of different proteinase inhibitors on platelet aggregation induced by these cells were investigated. When the platelets were incubated with the inhibitors, only those effective against cysteine proteinases strongly reduced platelet aggregation by cancer cells; serine protease inhibitors, including hirudin, had no effect on platelet response. Incubation of neoplastic cells with the same inhibitors gave similar, though less evident results. Addition of neoplastic cells to platelet-rich plasma also caused significant production of fibrinopeptide A, more by the less malignant cells. Thus, in this experimental model a cysteine proteinase of the neoplastic cells appears to play an important role in the platelet aggregation induced by them, and this property was detected in the M4 cells with high metastatic in vivo activity.

[Invagination of a gastric leiomyoma causing duodenal subocclusion and cholestasis]. / Invaginazione di leiomioma gastrico causante subocclusione duodenale e stasi biliare.

The case is reported of a 78 year old female admitted to our Department with symptoms compatible with intestinal obstruction and melena. Upper gastrointestinal x-ray examination and endoscopy did not lead to a precise diagnosis. On the contrary, double contrast x-ray examination revealed the presence of a probably benign gastric tumour with gastroduodenal intussusception. Cholestasis was also present, as suggested by elevated serum conjugated bilirubin levels and by intravenous cholangiography. These findings were confirmed during surgical operation; a partial gastrectomy with gastroentero anastomosis was performed; histologic examination showed that the tumour was a gastric leiomyoma. All the symptoms disappeared quickly and the patient is still in good health after 2 years.