Effect of Pfaffia paniculata (Brazilian ginseng) on the Ehrlich tumor in its ascitic form

The roots of Pfaffia paniculata (Brazilian ginseng) have been indicated for the treatment of several diseases, among which the cancer. The purpose of this study was to investigate experimentally the possible antineoplastic effect of this root. Firstly, a toxicity study was performed in which the doses of 400 and 200 mg/Kg of the powdered root were administered by gavage for 10 days to BALB/cICB mice. The mice did not lose weight during the treatment. No increase in serum alanine-aminotransferase neither histopathological alteration (liver, kidney and spleen) was observed in mice treated with P. paniculata. The effect of this root on the ascitic Ehrlich tumor in BALB/cICB mice was then investigated. Male mice received, by gavage, once a day, 200 mg/Kg of the powdered root of P. paniculata or distilled water, as control, for 20 days. This protocol started 10 days before tumor inoculation with 5 x 10(6) cells i.p., and lasted until 10 days after. The ascitic tumor was evaluated by the quantification of the volume of the ascitic fluid, relative number of tumor cells and total number of tumor cells. A decrease in the total ascitic volume was observed in P. paniculata treated mice, that was followed by a numerical decrease in the total number of Ehrlich tumor cells. These results may indicate that P. paniculata anti-inflammatory effects were responsible by the decrease in the total ascitic fluid. In addition, the presence of tumor-cell inhibitory factors in P. paniculata roots is in agreement with other in vitro studies. The mechanisms of such tumor inhibition should be further investigated.     

A comparison of galactosyltransferase activity in mouse ascites lymphoma cells (Ly/Ya), in cultured lymphoma cells, in ascitic fluid and culture medium

Comparative studies were carried out on the galactosyltransferase activity in ascites lymphoma cells isolated from mouse with ascitic lymphoma Ly/Ya, in these cells grown in vitro (24 hrs culture), in ascitic fluid and culture medium. The effect of varying amounts of UDP-galactose on transfer rate of galactose to ovomucoid by the cell enzyme (ascitic and cultured lymphoma cells) and by the soluble enzyme (ascitic fluid and culture medium) was studied. The activity of the enzyme in the cell culture medium was 2.5-fold higher than that in ascitic fluid. The apparent Km values for UDP-galactose of the enzyme from both kinds of cells and from the two fluids was 7.14 x 10(-7) M. At saturating concentrations of donor substrate, V values for the cells and culture medium was 765 pmoles/10(6) cells/h and 180 pmoles/10(6) cells/h for the ascitic fluid.     

Presence of the atrial natriuretic factor (ANF) in human ascitic fluid

Presence of atrial natriuretic factor (ANF)-like material was demonstrated by radioimmunoassay in ascitic fluid of 14 patients with cirrhosis of the liver. Immunoreactive ANF concentrations (M +/- SEM) were 2.4 +/- 0.5 fmol/ml in ascites, significantly lower (p less than 0.001) than the corresponding plasma concentrations of 15.5 +/- 2.6 fmol/ml. High performance gel permeation chromatography and reverse phase high performance chromatography of the ascitic ANF immunoreactivity showed correspondence to the alpha human ANF (99-126). ANF levels in ascites were significantly (p less than 0.01) correlated to levels in plasma (r = 0.66).     

Passage of hybridomas in male BALB/c mice

For cultivating hybridomas in the ascitic form there are usually used female mice BALB/c and not male ones. Efficiency of production of monoclonal antibodies with cultivation of the hybridomas in male and female mice BALB/c was studied comparatively. The animals were stimulated to form ascite by administration of the incomplete Freund's adjuvant or 3 per cent peptone with petrolatum oil. Some parameters of the ascite formation were studied: viability of the hybridoma cells, ascitic fluid formation period and volume, hybridoma cell concentration and titers of monoclonal antibodies in the ascitic fluid. In regard to all the parameters studied the male animals were not inferior to the female ones and in case of one of the hybridomas even surpassed them twofold by the volume of the ascitic fluid formed. This is evident of possible using male mice for mass cultivation of hybridoma cells with a purpose of obtaining preparative amounts of monoclonal antibodies in production of immunodiagnostic agents on their basis.     

烟草花叶病毒单克隆抗体杂交瘤细胞株的建立

用TMV免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,获得10株能稳定传代並分泌抗烟草花叶病毒(TMV)单克隆抗体(McAb)的杂交瘤细胞株,其中T73McAb滴度最高,ELISA滴度高达1/655,360,能被TMV兔免疫血清所阻断,能中和TMV的感染性。但与其它植物病毒无交叉反应,与TMV不同毒株均有反应。     

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions

Objective: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. Methods: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. Results: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. Conclusions: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.     

Gastrointestinal torsion in a channel catfish (Ictalurus punctatus)

A case of gastrointestinal torsion with dilatation in a farm-raised channel catfish (Ictalurus punctatus) was examined at the Thad Cochran National Warmwater Aquaculture Center (Stoneville, Mississippi, USA). The affected fish was a gravid female broodfish, which displayed pale gills and a markedly distended abdomen. Internal examination revealed that the gastrointestinal tract and ovaries were rotated around each other four times in a counterclockwise direction as viewed in right lateral recumbency. The catfish had a markedly distended gastrointestinal tract, pale liver, hypoplastic spleen, hypoplastic swim bladder, and high volume of ascitic fluid. Blood analysis indicated multiple abnormalities, including severe anemia and metabolic acidosis. The etiology of the torsion was uncertain; however, the presence of a hypoplastic swim bladder most likely allowed for increased movement of the gastrointestinal tract and ovaries. When examining cases of abdominal distention in fish, gastrointestinal torsion can be considered among the differential diagnoses.     

Purification process monitoring in monoclonal antibody preparation: contamination with viruses, DNA and peptide growth factors.

Administration in vivo of monoclonal antibodies to humans is challenged by considerations regarding their safety. Contamination with viruses, potentially oncogenic nucleic acids and biologically active components like growth factors and hormones forms a serious point of concern in this respect. We have investigated the potential risk of viral contamination by measuring the reduction of 12 different viruses (after spiking) in the standard downstream purification process of ascitic fluid. Depending on the type of virus added and the purification step employed, the reduction of infectious virus particles varies considerably. The overall reduction ranges from about 10(3), observed for a member of the family of Papovaviridae, to more than 10(12) for members of the families of Herpesviridae and Orthomyxoviridae. Using hybridization analysis with a mouse (genomic) DNA probe, we show that the amount of residual DNA in ascitic fluids may also vary considerably, ranging from 75 ng/ml to 1 microgram/ml. In crude preparations produced in cell culture, much lower DNA concentrations are found (0.3 ng/ml). When standard downstream purification procedures are applied to ascitic fluid, a significant reduction of residual DNA levels is observed in the purified monoclonal antibody preparations and in intermediate fractions. The overall reduction factors vary from about 10(3) to 10(4), which is also confirmed by spiking experiments with either purified DNA or crude chromatin-like DNA. Using in-vitro cellular assays, we further show that peptide growth factors like PDGF and TGF beta are present in considerable amounts in ascitic fluids. The observed biological activities, however, are completely eliminated during the purification steps applied.     

In vitro recognition of alloantigens: nature of responding and stimulating cells.

Immunosuppressive and immunostimulatory activity of human cancer ascitic fluids has been examined using the in vitro primary plaque-forming cell response (PFR) to sheep erythrocytes (Mishell-Dutton assay). We have prepared three fractions of ascitic fluid by precipitation with ammonium sulfate. Suppressive activity occurred in the fraction which was insoluble at 50% of saturation but not those fractions which precipitated at 30 or 80% of saturation. The fractions which were precipitated at 30 and 80% were stimulatory in the assay system. Normal human serum also had suppressive activity in the fraction precipitated at 50% of saturation but not as much as was found in ascitic fluid. Serum did not yield any fractions with stimulatory activity.     

Comparison of the action of pristan and Freund's incomplete adjuvant on the development of ascites in mice--the recipients of hybridoma cells

Production of immunodiagnostic preparations based on monoclonal antibodies requires large amounts of such antibodies. Most frequently preparative quantities of monoclonal antibodies are provided by ascitic fluid from hybridoma-carrying mice. Prior to intraperitoneal administration of hybridoma cells mice are subjected to stimulation with pristan, a mineral oil component. The authors showed that the Freund's incomplete adjuvant (FIA) may be as well used for this purpose. The effect of pristan and the FIA on ascites development in mice with hybridomas producing monoclonal antibodies to the Ia-like antigens of man was studied. The stimulants were administered in amounts of 0.5 ml per a mouse. It was shown that irrespective of the stimulant injection time and the number of the administered hybridoma cells the amount of the ascitic fluid formed in female mice BALB/c stimulated with the FIA was 1.5-3 times higher as that in the animals stimulated with Pristane, the antibody titer in the ascitic fluid being unchanged.     

The effect of ascitic fluid globulins on the growth of leukemia P388/DOX and Ehrlich's carcinoma in mice]

The study was performed to investigate the effect of ascitic fluid globulins of tumor on tumor growth and life span of mice. The globulins are shown to shorten the life span of Ehrlich tumor mice from 86.8 to 61.8 days, to increase 3-5-fold the growth rate of Ehrlich carcinoma and P388/DOX tumor. It was found that globulins of ascitic fluids and serum globulins of tumor have equal effects of tumor growth. It is proposed to use globulins of ascitic fluid to study the globulin role in tumor growth.     

Intraperitoneal amyloid formation by amyloid enhancing factor--rich macrophages in ascitic fluid.

Although resident peritoneal cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing the precursor protein of secondary amyloidosis, serum amyloid A (SAA) to amyloid fibrils, the peritoneum is a rare site for amyloid deposition. This is considered to be due to a deficiency of SAA in the peritoneum. To increase the supply of SAA to the peritoneum, ascitic fluid containing about the same protein constituents as in the serum was induced in mice. Amyloidotic peritoneal cells were packed in a microchamber which was shielded with filter membranes, and cultured in ascitic fluid supplemented with additional inflammatory factors. On the 7th day, Congo red-positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or -SAA immunostaining, amyloid deposits and the cell surfaces of macrophages were positive. Immunologic depletion of T- and B-lymphocytes from the amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers. These results suggest that either ascitic fluid containing sufficient amounts of SAA, or peritoneal macrophages with a high amyloid enhancing factor (AEF) activity are indispensable for AA amyloid fibrillogenesis in the peritoneum.     

Immunogenic gangliosides in human ovarian carcinoma

Ganglioside signatures of four poorly and three moderately differentiated ovarian epithelial cancer (OEC) cell lines reveal the presence of GM3, GM2, GD2, O-AcGD2, GD1a and GM1b. The expression of GM3, presence of GD1a and GM1b in the ascitic fluid and plasma, together with a positive correlation in the total-gangliosides levels between ascitic fluid and plasma of OEC patients support the earlier contention that the tumor-gangliosides may be released (or shed) into the tumor-microenvironment. The immunogenicity of OEC-gangliosides is determined by comparing anti-ganglioside-IgM titers in ascitic fluid (n = 14) and plasma (n = 23) of OEC-patients and age-matched healthy (n = 14). The titers were measured by ELISA. Strikingly, the level of anti-GD1a-IgM is significantly higher in ascitic fluid and plasma of patients than in the plasma of healthy volunteers. Paired sample analysis of ascitic fluid and plasma from the same patients confirmed the significant expression of anti-GD1a IgM in OEC patients, while no such difference was observed with other anti-ganglioside IgMs among different groups. The significance of the endogenous IgM response to GD1a may be to eliminate this immunosuppressive-ganglioside from the tumor-microenvironment.     

The bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents

The present study investigated the effects of a new bradykinin B₁ receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2 mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B₁ antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE₂ production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84-94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B₁ receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats.     

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

Summary 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 (TGF) 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-mediated suppression in situ may be necessary to allow local proliferation of LAK cells to achieve an effective killer-totarget ratio.Supported by a grant from the National Cancer Institute of Canada D.A.C. is a recipient of a Scientist Award from the Medical Research Council (Canada)     

Inhibition of lymphocyte mitogenesis by factor(s) released from macrophages isolated from ascitic fluid of advanced ovarian cancer patients

Summary Ascitic fluid from women with advanced ovarian carcinomas was shown to contain factor(s) which inhibit(s) T lymphocyte mitogenesis. The factor(s) was (were) demonstrated to be associated with the infiltrating macrophages. The inhibition was reversible and inhibited mitogenesis at some late event in the cell cycle. The inhibitory substance(s) was (were) noncytotoxic, dialyzable, heat-stable at 70° C for 10 min (but unstable at 100° C for 15 min), and partially resistant to protease treatment (55%–70%). Further experiments demonstrated that macrophages isolated from the ascitic fluid of patients with cirrhosis of the liver also released factor(s) which inhibit(s) T lymphocyte mitogenesis. On the basis of our data and data from other investigators, we propose that in advanced human ovarian cancer of epithelial origin, macrophages which infiltrate the ascitic fluid elaborate nonspecific inhibitors of T lymphocyte blastogenesis within the proximal environment, resulting in localized immunosuppression and the subsequent enhancement of metastasis within the peritoneal cavity, the tumor cells themselves being resistant to the cytocidal action of the macrophages due to genetic selection and/or their inherent biochemical ability to circumvent normal immunosurveillance mechanisms. This may account, at least in part, for the rapid metastasis and poor prognosis of human ovarian adenocarcinomas.     

Conditions for forming ascitic tumors in hybridoma cultivation in vivo

The conditions of the formation of ascitic cells in BALB/c mice injected with hybridoma cells were studied. All the hybridomas under study, producing monoclonal antibodies to viral antigens, induced the formation of ascitic tumors when introduced into the abdominal cavity of BALB/c mice pretreated with sensitizing agents. In the mice pretreated with pristane hybridoma cells took at a rate of 43-80% and in the mice pretreated with Freund's complete adjuvant, 31-70%. Angara oil and perfume oil, as well as Bayol F, were less effective. The time of the formation of ascites was inversely proportional to the dose of the injected cells, while the volume of ascitic fluid depended rather on the type of hybridoma and not on the dose of the injected cells. The study showed that the use of physiological saline or culture medium without serum for washing the abdominal cavity of mice after withdrawing ascites permitted the additional collection of 2.6-13.7 million hybrid cells, as well as a considerable amount of immunoglobulins.     

Sulfated glycosaminoglycan and collagen patterns in parietal yolk sac carcinoma (PYSC)

Electrophoretic analyses of collagenous material have shown that the parietal yolk sac carcinoma (PYSC) ascitic tumour synthesizes polypeptide chains that migrate as type IV procollagen. Having molecular weights of 185,000 and 160,000, these polypeptides are sensitive to collagenase. When the PYSC cells are injected subcutaneously, they form a solid tumour, and type I collagen predominates. The electrophoretic analyses of sulfated glycosaminoglycans and enzymatic degradation have shown a predominance of heparan sulfate in the ascitic tumour, and of chondroitin sulfate B in the solid tumour. Cells cultured from ascitic tumours have maintained the same collagen and sulfated glycosaminoglycan patterns as the original cells, whereas in the solid tumour culture only chondroitin sulfate AC has been detected.     

Pancreatic ascites hemoglobin contributes to the systemic response in acute pancreatitis

Upon hemolysis extracellular hemoglobin causes oxidative stress and cytotoxicity due to its peroxidase activity. Extracellular hemoglobin may release free hemin, which increases vascular permeability, leukocyte recruitment, and adhesion molecule expression. Pancreatitis-associated ascitic fluid is reddish and may contain extracellular hemoglobin. Our aim has been to determine the role of extracellular hemoglobin in the local and systemic inflammatory response during severe acute pancreatitis in rats. To this end we studied taurocholate-induced necrotizing pancreatitis in rats. First, extracellular hemoglobin in ascites and plasma was quantified and the hemolytic action of ascitic fluid was tested. Second, we assessed whether peritoneal lavage prevented the increase in extracellular hemoglobin in plasma during pancreatitis. Third, hemoglobin was purified from rat erythrocytes and administered intraperitoneally to assess the local and systemic effects of ascitic-associated extracellular hemoglobin during acute pancreatitis. Extracellular hemoglobin and hemin levels markedly increased in ascitic fluid and plasma during necrotizing pancreatitis. Peroxidase activity was very high in ascites. The peritoneal lavage abrogated the increase in extracellular hemoglobin in plasma. The administration of extracellular hemoglobin enhanced ascites; dramatically increased abdominal fat necrosis; upregulated tumor necrosis factor-α, interleukin-1β, and interleukin-6 gene expression; and decreased expression of interleukin-10 in abdominal adipose tissue during pancreatitis. Extracellular hemoglobin enhanced the gene expression and protein levels of vascular endothelial growth factor (VEGF) and other hypoxia-inducible factor-related genes in the lung. Extracellular hemoglobin also increased myeloperoxidase activity in the lung. In conclusion, extracellular hemoglobin contributes to the inflammatory response in severe acute pancreatitis through abdominal fat necrosis and inflammation and by increasing VEGF and leukocyte infiltration into the lung.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.