Cell-free Ascitic Fluid Research Articles

Reinfusion of autogenous ascitic fluid concentrated by freezing in patients with intractable ascites.

Eleven patients with intractable ascites were treated by intravenous reinfusion of concentrated autogenous ascitic fluid. The ascitic fluid was drained and centrifuged for removal of cells by a cell separator. Cell-free ascitic fluid was frozen in a medical freezer at -15 degrees C and thawed at room temperature. The fraction at the initial stage of melting contains a high concentration of protein and was thus collected and reinfused to the patient. In this initial fraction, the recovery rate of albumin was 42.0 +/- 9.0% and the concentration of protein was 7.1 +/- 2.4 g/dl. This frozen method is inexpensive and time saving, because it does not use a filter. Ascites was ameliorated in all patients with 1 to 19 times of reinfusion. Therefore, when renal function is not disturbed, the frozen method may be indicated for management of intractable ascites.

Generation of lymphokine-activated killer cells in human ovarian carcinoma ascitic fluid: identification of transforming growth factor-beta as a suppressive factor.

The effect of cell-free ascitic fluid from patients with epithelial ovarian carcinoma on the generation of lymphokine-activated killer cells (LAK) was compared to the activity generated in control medium containing 10% fetal bovine serum, using Daudi target cells. Samples of ascitic fluid from nine different patients tested inhibited LAK generation. Suppressive activity was evident as early as 24 h of incubation in the presence of ascitic fluid and increasing suppression developed with prolonged exposure. Suppression was concentration-dependent, present at 10%-20% and increasing with concentrations up to 80%. The suppressive effect of ascitic fluid was only partially reversed on increasing the concentration of interleukin-2 (IL-2) from 10 units to 1000 units/ml. Activated LAK appeared to maintain the majority of their activity on further culture in ascitic fluid in the presence of IL-2 but further enhancement of lytic activity was prevented. Fractionation of a suppressive sample by HPLC, using 0.1 M KCl/acetic acid buffer pH 2.6, revealed that the dominant peak of suppressive activity eluted at 25 kDa; with pH 7.0 TRIS-buffered saline, most of the activity was lost on the column. Antibody neutralization studies of the 25-kDa suppressive peak as well as on whole ascitic fluid have revealed that transforming growth factor beta (TGF beta) is the major suppressive factor present in ascitic fluid. Factors that suppress LAK generation in vitro were present in all samples tested. The effect on the lytic activity of activated LAK cells was minimal. This suggests that, in the clinical setting, the greatest impact would be achieved by activating LAK cells ex vivo and subsequently transferring them to the peritoneal cavity in the presence of IL-2 rather than by attempting to generate them in situ by injecting IL-2 into the peritoneal cavity. However, reversal of TGF beta-mediated suppression in situ may be necessary to allow local proliferation of LAK cells to achieve an effective killer-to-target ratio.

Phospholipase A2 in human ascitic fluid. Purification, characterization and immunochemical detection

A phospholipase A2 (PLA2, EC 3.1.1.4) was purified from human cell-free ascitic fluid (a-PLA2) by ion-exchange chromatography and h.p.l.c. on a reverse-phase column to apparent homogeneity. The enzyme had an Mr of approx. 10,000 as determined by SDS/PAGE. Polyclonal antibodies raised in a rabbit were specific to a-PLA2, as judged by immunoblotting. A time-resolved fluoroimmunoassay (TR-FIA) for measuring the concentration of a-PLA2 in various body fluids was developed. The detection limit of the assay was about 6 ng/ml. The antiserum did not cross-react with pancreatic secretory phospholipase A2 as measured by TR-FIA. The enzyme content was studied in various samples, including normal human serum, buffy-coat leucocytes, synovial fluid, and pancreas and spleen homogenates.

Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumor-bearing state.

The present study investigates the nature of humoral component(s) generated in tumor-bearing hosts to induce immune dysfunction of T cells. Cell-free ascitic fluid and culture supernatant (SN) were obtained from the ascites and cultures allowing MH134 hepatoma cells to grow. These ascites and SN samples were tested for their abilities to influence the generation of CTL responses to TNP and alloantigens. The generation of the anti-TNP CTL responses that require self H-2-restricted CD4+ Th cells was markedly suppressed by addition of the ascites or SN under conditions in which these samples did not inhibit anti-allo CTL responses capable of using alternate pathways of allo-restricted CD4+ and CD8+ Th. The activation of CD8+ CTL precursors and CTL activity were also resistant to the ascites or SN. The ascites- or SN-induced suppressive effect to which CD4+ Th were most susceptible was found to be mediated by transforming growth factor-beta (TGF-beta) activity, because: 1) the TGF-beta activity was detected in the MH134 ascites and culture SN; 2) the suppression of CD4+ Th function required for anti-TNP CTL responses was almost completely prevented by addition of anti-TGF-beta antibody to cultures and; 3) rTGF-beta also induced similar patterns of immunosuppression to those observed by ascites or SN. These results indicate that TGF-beta produced by tumor cells induces deleterious effects on T cell, especially on the CD4+ Th subset, and provide an explanation for the molecular mechanism underlying the previously observed CD4+ Th-selective suppression in the tumor-bearing state.

Antigen shedding vs. development of natural suppressor cells as mechanism of tumor escape in mice bearing ehrlich tumor

C57BL/6J mice immunized with devitalized Ehrlich tumor (ET) cells produce high serum levels of IgM antibodies to ET cell-surface carbohydrates that are critical in the observed resistance against this tumor. However, this response is not found in ET-bearing mice at any stage of tumor development. Since previous studies had shown splenic natural suppressor (NS) cells in ET-bearers, their role in such IgM impairment was assessed. Here we show that tumor-bearers' spleen cells (TBSC) are unable to produce IgM in vitro in response to LPS, due to the presence of NS cells. Nevertheless, TBSC do produce IgM antibodies to ET cell-surface carbohydrates in increasing amounts as the tumor progresses. Yet these antibodies are not detected in sera of ET-bearers and are greatly decreased in immunized mice with a growing tumor. Moreover, increasing amounts of circulating carbohydrates, able to absorb most specific IgM, are found in ET-bearing sera associated with a large molecular size structure(s). These carbohydrates are also found in ET cell-culture supernatants and cell-free ascites fluid derived from this tumor, indicating their tumor origin. Taken together, our results indicate that lack of specific IgM antibodies in ET-bearing mice is not due to faulty production, but to in vivo absorption by carbohydrates shed from ET cells in increasing amounts as the tumor progresses. Thus, NS cells are unable to suppress this IgM production in vivo, despite the strong suppressor activity they show for many responses in vitro.

Hydrocortisone protects cycloheximide-challenged mice pretreated with Ehrlich ascites tumour cells or with cell-free Ehrlich ascites tumour fluid

The invariably fatal outcome following cycloheximide challenge of mice pretreated 24 h earlier with either 1 x 10(7) washed Ehrlich ascites tumour cells i.v. or with 0.2 ml of cell-free Ehrlich ascites tumour fluid i.v. was prevented by hydrocortisone treatment. However, glucocorticosteroids failed to prevent the fatal outcome of cycloheximide challenge in mice bearing 5-day-old Ehrlich ascites tumours. The results are interpreted as indicating that there is a steroid demand which must be met to ensure survival of cycloheximide-challenged mice pretreated with tumour cells or with cell-free tumour ascites fluid and that an additional and essential requirement for uninterrupted protein synthesis exists in the case of mice with established ascites tumours and challenged with cycloheximide.

The inhibition of endotoxin-induced local inflammation by LDH virus or LDH virus-infected tumors is mediated by interferon.

The footpad swelling reaction induced by local injection of S. marcescens lipopolysaccharide was found to be inhibited in mice given a transplantable tumor (TA3) or cell-free ascitic fluid from tumor-bearing mice. The tumor was shown to contain LDH virus, which is known to cause inapparent persistent infections in mice. Monoclonal antibodies directed against protein VP3 of the LDH virus could partially abrogate the anti-inflammatory effect of the TA3-ascitic fluid, and, conversely, the anti-inflammatory effect could be obtained by LDH virus isolated from the tumor and reproduced by serial passage of cell-free fluids. Inhibition of the footpad reaction was seen in the acute but not in the chronic phase of LDH virus infection, suggesting that the anti-inflammatory effect might be due to endogenous interferon (IFN) which, similarly, was only detectable in the acute phase. Newcastle disease virus, another potent interferon inducer, had a similar inhibitory effect on the footpad reactivity. Moreover, the inhibitory effect of LDH virus infection could partially be abrogated by administration of a polyclonal antibody directed against murine IFN-alpha,beta. Finally, passively administered natural murine IFN-alpha,beta or recombinant murine IFN-alpha 1 (but not recombinant murine IFN-beta) was found to cause inhibition of the footpad reaction. Since Gram-negative bacteria and their lipopolysaccharides have the ability to induce a systemic interferon response, our findings suggest that this interferon may play a modulatory role in local inflammation caused by these bacteria. Our findings also open a new perspective for interferon therapy of certain inflammatory reactions to bacterial infections.

Mechanisms of malignant ascites production

The accumulation of malignant ascites is determined primarily by the obstruction of diaphragmatic lymphatics with tumor inhibiting the outflow of peritoneal fluid. An abnormal increase in peritoneal fluid production has been shown to contribute to ascites formation by a marked neovascularization of the parietal peritoneum. Cell-free malignant ascitic fluid obtained from rats with intra-abdominal Walker 256 carcinoma when infused into the peritoneal cavities of normal animals causes an increase in edema formation and an increase in the permeability of protein from normal omental vessels. Protamine sulfate, a known inhibitor of angiogenesis when infused into the peritoneal cavity along with cell free malignant ascitic fluid, significantly reduces the leak of protein from the intravascular space when compared to ascites alone. Persistent permeability changes continue to exist even after the inhibition of vessel proliferation. These results indicate that angiogenesis is responsible for a major portion of the increase in permeability caused by malignant ascitic fluids. Other tumor-induced factors may be present which alter vascular permeability by other mechanisms which remain to be elucidated.

Increased Tϵ cells in BALB/c mice with an IgE-secreting hybridoma

Since it has been proposed that T cells with receptors for the Fc portion of IgE (Tϵ) in rats, mice and humans are IgE-specific regulatory cells, it was investigated whether mice with an IgE-secreting hybridoma might be a source of large numbers of Tϵ cells. BALB/c mice with ascitic tumors of the IgE hybridoma B53 (ϵ, k, anti-DNP) developed high serum concns of monoclonal IgE which was followed by the appearance of large numbers of Lyt1 −2 + L3T4 − Tϵ cells. In contrast, mice bearing a non-IgE producing variant of B53 failed to develop Tϵ cells. Mice infused with cell-free ascites fluid (containing high levels of monoclonal IgE) from the IgE-secreting B53 hybridoma developed high serum IgE levels and also developed high numbers of Tϵ cells. Mice infused with cell-free ascites obtained from the non-IgE-secreting variant ENP-1 (containing very low amounts of IgE) did not develop either high serum IgE levels or Tϵ cells. These findings suggest that high serum IgE concns induce large numbers of T cells that express phenotypic markers of suppressor cells and have surface IgE-Fc receptors. These studies extend to IgE the principle that hybridomas and plasmacytomas induce large numbers of immunoregulatory T cells that express Fc receptors specific for the heavy chain class of the secreted monoclonal immunoglobulin. Since Tϵ cells are normally present only in small numbers, their marked increase in mice with IgE-secreting hybridomas identifies a ready source of large numbers of Tϵ cells that can be used to investigate their regulatory properties.

Malignant ascites. Clinical and experimental observations.

Malignant ascites formation is a grave prognostic sign, but palliative efforts seem justified in some patients. Lack of knowledge concerning the natural history of this process hinders the choice of therapeutic options. Over 5 years, 107 patients with untreated malignant ascites were reviewed to define their survival. Pancreas (20), ovary (18), and colon (18) were the most frequent tumors, with 52% of patients presenting with ascites at the time of the initial cancer diagnosis. Cytology evaluation of the ascitic fluid was positive for tumor cells in 57% of cases and a high protein content was noted in 65%. Mean survival of the entire series was only 20 weeks from the time of diagnosis of ascites, with tumors of ovarian and lymphatic origin having better mean survivals of 32 and 58 weeks, respectively. Patients with high ascitic protein levels fared better than those with low levels. In an effort to explain this correlation of elevated protein levels and a favorable survival rate, a hypothesis was proposed that certain tumors secrete a factor, which alters vascular permeability and causes fluid accumulation in the absence of lymphatic obstruction. In an experimental rat model of malignant ascites, the intraperitoneal infusion of cell-free malignant ascitic fluid caused an increase in edema formation and a significant increase in capillary permeability to protein in the omentum. This demonstrated change in the leak of protein explains the formation of ascites by some tumors in the absence of tumor obstruction of the draining lymphatics of the peritoneal cavity and suggests another important mechanism in the genesis of malignant ascites.

REINFUSION OF CELL-FREE AND CONCENTRATED AUTOGENOUS ASCITIC FLUID FOR INTRACTABLE ASCITES IN THE TERMINAL CANCER PATIENT

Intravenous reinfusion of autogenous ascitic fluid was primarily applied for intractable ascites in the cirrhotic patient. Recently, this procedure was modified using a technique of cell removal and fluid concentration and applied for intractable ascites in the terminal cancer patient. In the present study, the effect of the reinfusion of cell-free and concentrated autogenous ascitic fluid for in-tractable ascites in the terminal cancer patient was evaluated. Fourteen cases of terminal cancer with intractable ascites were treated with 21 reinfusions. The reinfusion was effective in 85.7% of the cases decreasing the complaints caused by ascitic retension. It caused neither hypoprotenemia nor imbalance of serum electrolytes. In addition, the repeated reinfusion led to a restraint of the re-retension of ascitic fluid. Although pyrexia occurred in 71.4% of the reinfusions, in most of the cases, this was subsided with the administration of the usual antipyretics. These results indicate that the reinfusion of cell-free and concentrated autogenous ascitic fluid is useful for the control of intractable ascites in the terminal cancer patient.

Tumour-induced suppression of delayed-type hypersensitivity in the mouse: independence of cyclophosphamide-sensitive suppressor cells.

Delayed-type hypersensitivity (DTH) responses to sheep red blood cells and ovalbumin were markedly reduced in mice receiving viable Landschütz ascites tumour cells at the time of immunization. Similar inhibitory effects were observed in control immunized animals when cell-free ascitic fluid or tumour bearer serum was administered together with antigen at the challenge site. Normal mouse serum exhibited much less pronounced inhibitory activity over the range of concentrations tested. High-dose cyclophosphamide (Cy) prior to immunization enhanced DTH responses to both antigens in normal mice. However, in animals also given tumour or soluble tumour-associated material, the immunosuppressive effect which had been observed in non-Cy-treated controls was preserved. It is concluded that the observed suppression of DTH is not dependent on Cy-sensitive T suppressor cells or on the presence of immunosuppressive factors during induction of the immune response.

Detection of a cell proliferation-inhibiting factor in the cell-free ascitic fluid of intraperitoneal mouse tumors.

Three cell-free ascitic fluids: MCG3AF, P815AF and SL2AF were obtained from mice injected with MCG3, P815 and SL2 tumor cells, respectively. These ascitic fluids were tested in culture with different normal and tumor mouse cells showing an inhibitory effect on 3H-thymidine and 3H-uridine uptake. The MCG3AF, selected for further study, was tested with a larger number of mouse tumor cell lines and also with normal mouse and human cells. This ascitic fluid inhibited 3H-thymidine and 3H-uridine uptake in all cases. The cell proliferation-inhibiting factor(s) (CPIF) detected in MCG3AF was found to be thermostable and nondialyzable. Further biochemical analysis showed that CPIF is a lipid but with no relationship with very low-density lipoproteins.

Enhanced concanavalin A-agglutinability of erythrocytes in rats bearing Yoshida ascites sarcoma: a study of the mechanism

Erythrocytes of normal Wistar rats are maximally agglutinated at 250 μg/ml concanavalin A (Con A). In rats bearing Yoshida ascites tumor (i. p.), however, the red cells in circulation and those contaminating the tumor fluid agglutinate the most at 50 μg/ml of the lectin. The enhanced agglutinability of circulatory RBCs arises 3 days after tumor transplantation, but the erythrocytes in the ascites fluid show the alteration within a day. Incubation of normal erythrocytes in the cell-free ascites fluid or its soluble or membranous component modifies their agglutinability. Intraperitoneal injection of the cell-free ascites fluid in a normal rat also leads to modification of its erythrocytes in 24 hr. Thus the modification activity resides in the fluid, from which it can enter into circulation. The blood plasma of the animal, collected on the third day after tumor transplantation but not earlier, is also able to modify red cells in vitro. The erythrocyte modification activity of the fluid is insensitive to the temperature of incubation, and prior boiling enhances its activity. Comparison of the sugar content, and protein and glycoprotein profiles of the normal and modified circulatory erythrocytes shows no differences. Thus proteolytic enzymes are not responsible for the enhanced agglutinability of red cells from tumor-bearing animals. The membranes of erythrocytes from the ascites fluid, however, have somewhat reduced contents of sugars and diminished amounts of glycoproteins, indicating that they are probably acted upon by proteases in the ascites fluid. Approximately 5% of the protein in the ascites fluid binds to Con A-Sepharose. This glycoprotein fraction, consisting of 14 polypeptides, possesses in vitro modification activity, while the protein devoid of affinity for Con A is inactive. It is proposed that the tumor cells secrete or shed Con A-binding glycoproteins in the ascites fluid, which bind to the surface of erythrocytes present in the fluid and modify their agglutinability with the lectin. The glycoproteins enter circulation and, after reaching a sufficient concentration in plasma, alter the membranes of red blood cells.

Immunosuppression related to ascitic fluid in patients with ovarian carcinoma.

The immune response to SRBC (PFC assay) was suppressed in mice injected with cell-free ascitic fluid from patients with ovarian carcinoma. The immunosuppressive effect of ascitic fluid obtained from stage IV patients was stronger than that of stage III patients. These data were correlated with the patient's immune status (number of E and EAC rosettes, PHA reactivity of lymphocytes, skin reactivity in recall antigens) and with changes in protein fractions in the serum and ascitic fluid. A good correlation was found between the immunosuppressive effect in the mouse PEC assay and the increased quantity of alpha-1-globulins in ascites. Skin nonreactivity to PPD also correlated with the immunosuppressive effect of ascitic fluid. However, the lymphocyte response to PHA and the numbers of E and EAC rosettes did not correlate either with skin reactivity or recall antigens or with the suppression of PFC response in mice.

Heterogeneity in surface glycoproteins of mouse peritoneal macrophage populations

Murine peritoneal macrophages were activated in vitro by repeated addition to the culture medium of cell-free tumour ascites fluid from a syngeneic methylcholanthrene-induced sarcoma. Cell morphology was studied by phase-contrast microscopy and scanning electron microscopy (SEM). The ascites-activated cells were extensively spread out on the glass surface and showed extensive membrane ruffling. To investigate the protein metabolism of the cells, total protein content and incorporation and turnover of [ 14C]glucosamine and [ 35S]methionine into TCA-precipitable macromolecules were analysed. Activated cells had a total protein content 2–3 times higher than that found in control cells. [ 35S]Methionine and [ 14C]glucosamine were incorporated into ascites-treated cells at a rate 2.7–3 times higher than into control cells. Cell glycoproteins were lost with biphasic kinetics with a reduced half-life for both phases in the activated cells as compared to controls. Radiolabelled proteins were isolated from the plasma membrane by immunoprecipitation of trinitrobenzene sulphonic acid-derivated cells and analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Whereas most of the protein peaks were the same in both cultures, the control cells had a single broad protein peak in the range of 100–110 K, whereas the ascitesactivated cells had three peaks of 90, 100–110 and 135 K. The 135 K peak appeared to be a ‘new’ surface glycoprotein expressed in the activated but not in the control cells.

Non-bicarbonate buffering of ascites tumor cells in the rat as titrated by strong acids

Buffer equations originally derived for blood were applied to the in vitro system ‘ascites tumor cells and ascitic fluid’. Non-bicarbonate buffer values β(mmol·pH −1·1 −1) were determined experimentally by titration of ascites, cell free ascitic fluid and cell homogenates with NaOH in the absence of CO 2. β in ascitic fluid, in native ascites and in cell homogenate were β(F) = 5.9, β(A) = 16.4 and β(H) = 24.9, respectively. Intracellular β value β(C) calculated from buffer equations depended on the ratio ΔpH i/ ΔpH e. With ΔpH i/ ΔpH e = 1, β(C) was 38.8. The difference between β(C) and β(H) could be partly accounted for by differences in the cellular protein content. With ΔpH i/ ΔpH e = 0.62 as in the presence of CO 2, β(C) becomes 62.6. In the presence of CO 2 (see Albers et al., 1981), the extracellular buffering was higher and the intracellular buffering lower than the values titrated in the absence of CO 2. It is concluded that in the presence of CO 2 and/or HCO 3 − the apparent intracellular buffering of intact tumor cells results from non-bicarbonate buffering by cell proteins as well as from active or passive ionic exchanges with the extracellular fluid.

The effect of Ehrlich ascites tumour growth on lactate dehydrogenase activities in tissues and physiological fluids of tumour bearing mice

1. 1. Tumour growth kinetics and the activity of lactate dehydrogenase (LDH) in body fluids and host tissues were studied during the Ehrlich ascites tumour growth. The rate of tumour cell death was determined by measuring the loss of 125I from mice bearing [5- 125I]iodo-2′-deoxyuridine labelled tumour cells. 2. 2. The activity of LDH in serum increased progressively with increasing tumour mass during the first period of tumour growth, whereas that of cell free ascites fluid exhibited a peak level during early plateau phase growth. The analysis of LDH activity in cell free ascites fluid revealed that the increased LDH activity could only partly be attributed to a release from dead tumour cells. 3. 3. A marked increase in host liver LDH activity was observed. In addition, the growing tumour induced an increase in RNA synthesis activity and Kupffer cell proliferation in the liver. The LDH activity in skeletal muscle decreased significantly with increasing tumour mass. 4. 4. The observations show that the growing tumour exerts a marked effect on the metabolic activity of host tissues. It is suggested that an altered metabolism affects the cell membrane integrity of certain host tissues and causes a leakage of LDH molecules, which contributes to the increased extracellular LDH activity of tumour bearing animals.

Regulation of cholesterol synthesis in L1210 murine leukemia cells by dietary cholesterol

Studies were undertaken to measure cholesterol synthesis by L 1210 murine leukemia cells and to determine if there is modulation of the rate of cholesterol synthesis by the content of cholesterol in the diets of the host animals. When the animals were fed a cholesterol-free diet for 6 weeks, the L 1210 leukemia cells synthesized digitonin-precipitable sterols from [2- 14C] acetate at rates that were 2–3 fold greater than cells from animals fed a 5% cholesterol diet. This difference was also evident after only 2 weeks of feeding. The total and free cholesterol content of the cell-free ascites fluid of the animals fed the 5% cholesterol diet was higher than the ascites fluid of the animals fed the cholesterol-free diet. However, there were no differences in the cholesterol content of the L 1210 cells from the two diet groups. These results suggest that there is dietary regulation of the rate of cholesterol synthesis by L 1210 leukemia cells.

Leukokinin-H generated from human ascites fluid; its isolation and pharmacology.

Leukokinin-H, a pharmacologically active peptide, has been isolated from human ascites fluid following its generation in vitro at acid pH. The peptide has a molecular weight of approximately 2500. The partial sequence of Leu-Ala-Tyr-Thr — was obtained from the amino-terminal end. Arginine is the terminal amino acid at the carboxyl end. It was demonstrated that while cell-free ascites fluid generates leukokinin-H, the presence of neoplastic cells enhances the formation about 5 fold. A bradykinin-like peptide was formed in the fluid following storage at a low temperature of 4°C. The presence of neoplastic cells, however, did not enhance this peptide formation. Procedures are described for the isolation of leukokinin-H by chromatography. The peptide was clearly differentiated from bradykinin, Lys-bradykinin and Met-Lys-bradykinin by treatment of the leukokinin and other kinins with fluorescamine and separation of the fluorescamine peptides by gel electrophoresis. The pharmacology of leukokinin-H isolated from human ascites fluid is described and compared with bradykinin and leukokinins PMN and M. Like the other kinins, it is a potent agent in causing vascular permeability. Likewise it is a vasodepressor substance. Unlike bradykinin it has little spasmogenic activity on the guinea pig ileum. Its pharmacological properties as well as other evidence cited indicates that it may play an important role as a permeability factor in ascites fluid accumulation of neoplastic etiology.