Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.

The 27-kD diphtheria toxin receptor-associated protein (DRAP27) from vero cells is the monkey homologue of human CD9 antigen: expression of DRAP27 elevates the number of diphtheria toxin receptors on toxin-sensitive cells.

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence revealed that DRAP27 is the monkey homologue of human CD9 antigen. DRAP27 is recognized by CD9 antibodies. A human-mouse hybrid cell line (3279-10) possessing human chromosome 5, sensitive to DT, but not expressing CD9 antigen, was used for transfection experiments with DRAP27. When the cloned cDNA encoding DRAP27 was transiently expressed in 3279-10 cells, the total DT binding capacity was three to four times higher than that of untransfected controls. Transfectants stably expressing DRAP27 have an increased number of DT binding sites on the cell surface. Furthermore, the transfectants are 3-25 times more sensitive to DT than untransfected cells, and the sensitivity of these cells to DT is correlated with the number of DRAP27 molecules on the surface. However, when the cloned cDNA was introduced into mouse cell lines that do not express DT receptors, neither an increased DT binding nor enhancement of DT sensitivity was observed. Hence, we conclude that DRAP27 itself does not bind DT, but serves to increase DT binding and consequently enhances DT sensitivity of cells that have DT receptors. 12 proteins related to DRAP27/CD9 antigen were found through homology search analysis. These proteins appear to belong to a new family of transmembrane proteins.

Weekly paclitaxel/5-fluorouracil followed by platinum retreatment for patients with recurrent ovarian cancer: a single institution experience.

PURPOSE: Since the prognosis of recurrent ovarian cancer patients is still poor, we need to establish a useful treatment strategy to achieve their long-term survival. We treated recurrent ovarian cancer patients with weekly paclitaxel (PTX)/5-fluorouracil (5-FU) followed by platinum retreatment to investigate its clinical efficacy in a preliminary manner. METHODS: Sixteen patients with recurrent ovarian cancer, pretreated with taxane and platinum, were treated with weekly paclitaxel (PTX)/5-fluorouracil (FU). PTX (80 mg/m2) on day 1, 8, and 15 was combined with a bolus injection of 5-FU (500 mg/m2) on day 2, 9, and 16. Chemotherapy was given every four weeks. Patients with stable disease or progressive disease were subsequently retreated with a platinum-containing regimen. Response was evaluated by RECIST criteria or CA125 criteria. Toxicities were evaluated according to the National Cancer Institute-common toxicity criteria (NCI-CTC) version 3. RESULTS: Among five patients with sensitive disease, one of four patients with measurable tumor and one without measurable tumor responded to weekly PTX/5-FU. Among 11 patients with resistant disease, none of five patients with measurable tumor and three of six patients without measurable tumor responded to weekly PTX/5-FU. Overall objective response rate by weekly PTX/5-FU was 31.3% (5/16). Among 16 patients, 13 patients who showed no response or progressive disease (three with sensitive disease, ten with resistant disease) received platinum retreatment after weekly PTX/5FU. All three patients with sensitive disease and three of ten patients with resistant disease revealed response to platinum retreatment. Overall objective response rate by platinum retreatment after weekly PTX/5-FU was 46.2% (6/13). CONCLUSIONS: Weekly PTX/5FU followed by platinum retreatment could be a useful treatment strategy for recurrent ovarian cancer patients. We need to establish the standard treatment strategy for recurrent ovarian cancer patients with a poor prognosis.

Induction of anti-VEGF therapy resistance by upregulated expression of microseminoprotein (MSMP).

Anti-vascular endothelial growth factor (VEGF) therapy has demonstrated efficacy in treating human metastatic cancers, but therapeutic resistance is a practical limitation and most tumors eventually become unresponsive. To identify microenvironmental factors underlying the resistance of cancer to antiangiogenesis therapy, we conducted genomic analyses of intraperitoneal ovarian tumors in which adaptive resistance to anti-VEGF therapy (B20 antibody) developed. We found that expression of the microseminoprotein, prostate-associated (MSMP) gene was substantially upregulated in resistant compared with control tumors. MSMP secretion from cancer cells was induced by hypoxia, triggering MAPK signaling in endothelial cells to promote tube formation in vitro. Recruitment of the transcriptional repressor CCCTC-binding factor (CTCF) to the MSMP enhancer region was decreased by histone acetylation under hypoxic conditions in cancer cells. MSMP siRNA, delivered in vivo using the DOPC nanoliposomes, restored tumor sensitivity to anti-VEGF therapy. In ovarian cancer patients treated with bevacizumab, serum MSMP concentration increased significantly only in non-responders. These findings imply that MSMP inhibition combined with the use of antiangiogenesis drugs may be a new strategy to overcome resistance to antiangiogenesis therapy.

Glucocorticoid-induced elevation of serum high-density lipoprotein-cholesterol and its reversal by adrenocorticotropin in the rat.

The effect of administration of a high dose of glucocorticoid (triamcinolone) on serum lipids and lipoproteins was studied in rats. Changes in serum lipids, especially cholesterol, were most marked when 5 mg/kg body weight of triamcinolone was injected daily for 5 days. Serum lipoproteins were separated by ultracentrifugation followed by gel-filtration chromatography. Cholesterol distribution between apolipoprotein B-containing lipoproteins (very-low-density and low-density lipoproteins), high-density lipoprotein1 (HDL1), and HDL2 was determined after administration of triamcinolone with or without additional treatment with adrenocorticotropin (ACTH; Cortrosyn, 6 IU/rat). When triamcinolone was administered, cholesterol concentrations in HDL1 and HDL2 were elevated in a dose-dependent manner, but there was no significant change in apolipoprotein B-containing lipoprotein cholesterol levels. When ACTH was administered in combination with triamcinolone, the concentrations of all serum lipids except triacylglycerol were significantly lowered compared with rats treated with triamcinolone alone. HDL1-cholesterol concentration in serum was significantly (P less than 0.001) lowered from 69 +/- 13 mg/dl (mean +/- S.D.) in triamcinolone-treated rats to 36 +/- 4 mg/dl by the administration of ACTH plus triamcinolone. The additional administration of ACTH in triamcinolone-treated rats caused a slight, but significant, decrease in cholesterol concentration in apolipoprotein B-containing lipoproteins; however, HDL2-cholesterol level was not significantly affected, although there was a tendency for it to be lowered.

Alterations of high density lipoproteins in experimental cholestasis in the rat.

High density lipoproteins (HDL) in the plasma from cholestatic rats were studied by gel filtration, polyacrylamide gel electrophoresis and electron microscopy. HDL from cholestatic rats separated into two subfractions, abnormally enlarged particles and smaller ones, on gel filtration through Bio-Gel A 1.5 m. Polyacrylamide gel electrophoresis in 8 M urea revealed that the relative proportion of C-III-3 peptide in C-apoproteins was higher in the two HDL subfractions from cholestatic rats than in normal HDL. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that the abnormally enlarged HDL contained the 'arginine-rich' apoprotein, whereas a band corresponding to 'arginine-rich' apoprotein was only faintly visible in the smaller HDL subfraction. Normal HDL contained much smaller amount 'arginine-rich' apoprotein than the enlarged HDL. In electron microscopy, both of the two subfractions of cholestatic HDL appeared spherical. The abnormally enlarged HDL were consisted of particles with a diameter of 17 +/- 4.5 nm (mean +/- S.D.). The other subfraction contained smaller particles with a diameter (about 10 nm) similar to normal HDL.

Situs ambiguous with polysplenia complicated by renal adenocarcinoma.

Two men developed renal adenocarcinoma in association with situs ambiguous off with polysplenia (SAP) (also known as the polysplenia syndrome). Features of their diseases included (1) no normal spleen--just splenuli, (2) interruption of the inferior vena cava with azygos or hemiazygos continuation, (3) bilateral hyparterial bronchi, (4) cardiac malformations, (5) renal adenocarcinomas originating from the kidneys, ipsilateral to the anomalous spleens. The association of renal adenocarcinomas and SAP has not been previously reported, to our knowledge. We suggest that renal adenocarcinoma and SAP may share a common pathogenetic link.

Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials.

We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.

Preventive effects of a herbal medicine on bone loss in rats treated with a GnRH agonist.

The study was designed to evaluate the effects of a traditional Chinese herbal medicine Hochu-ekki-to (Bu-zong-yi-qi-tang), which was composed of 10 herbal medicines and had been used for the treatment of oligospermia and as a postoperative medication in Japan, on bone loss in rats treated with a gonadotropin-releasing hormone (GnRH) agonist. Female rats at 40 weeks of age were divided into 4 groups of 8 rats each. In the three experimental groups, each animal received subcutaneous injections of the long-acting GnRH agonist, buserelin acetate, once every four weeks throughout the experiment. Beginning at 48 weeks of age, the experimental groups were given diets containing conjugated estrogens or Hochu-ekki-to for 8 weeks. The administration of the GnRH agonist reduced the bone mineral density in the whole femur to 91.0% of that in the control group. However, administration of conjugated estrogens and Hochu-ekki-to increased the serum concentrations of estradiol 16.8- and 5.3-fold respectively compared with concentrations in the GnRH agonist-treated group, resulting in the augmentation of the bone mineral density to 110.3% and 106.2% respectively. These findings indicate that Hochu-ekki-to enhances the reduced bone mineral density and causes a slight elevation of the serum estradiol levels in the chemically castrated rats.

Alterations of high density lipoproteins in experimental intrahepatic cholestasis in the rat induced by administration of alpha-naphthylisothiocyanate.

It has previously been reported that abnormally enlarged high density lipoproteins (HDL) appear in rats with extrahepatic cholestasis induced by ligation of the common bile duct. To see whether similar changes in HDL occur in intrahepatic cholestasis in rats, we studied HDL alterations in rats treated with alpha-naphthylisothiocyanate (ANIT), which is known to produce a cholestatic response in rats similar to intrahepatic cholestasis in man. Findings were obtained which indicated changes in HDL similar to those in bile duct-ligated rat serum: HDL from ANIT-treated rats were separated into two subfractions, enlarged particles and smaller ones, on Bio-Gel A5m column chromatography. In electron micrographs, the two subfractions appeared spherical and the diameters of the enlarged particles and the other ones were 15.0 +/- 2.6 nm and 11.5 +/- 2.2 nm, respectively. Both subfractions showed slow alpha-mobility in agarose gel electrophoresis. The enlarged HDL had apoE as their major apoprotein, while apoA-I was the major apoprotein in the other HDL subfraction. The enlarged HDL contained less protein and more cholesterol than the other HDL subfraction. The two HDL subfractions were also separated by heparin-Sepharose affinity chromatography.

Separation of cholesterol-induced high density lipoproteins (HDLc) by concanavalin A-Sepharose affinity chromatography.

Cholesterol-induced density lipoprotein(s), termed HDLc, is one of the abnormal lipoproteins which occur in experimental hypercholesterolemia in several animal species, including rats. Thus far, HDLc has been exclusively isolated by sequential ultracentrifugation followed by Geon-Pevikon block electrophoresis, which is time-consuming and requires some specialized knowledge. In this report, a faster and more convenient alternative method for the isolation of HDLc is described. A combination of a single ultracentrifugation and agarose gel chromatography followed by concanavalin A-Sepharose affinity chromatography was employed. HDLc thus obtained was similar to and probably identical with the HDLc isolated by Geon-Pevikon electrophoresis, with respect to chemical composition, electrophoretic properties in agarose gel, apoprotein patterns in SDS polyacrylamide gel electrophoresis, and electron micrographic appearance.

Apolipoproteins in rat cerebrospinal fluid: a comparison with plasma lipoprotein metabolism and effect of aging.

Cerebrospinal fluid (CSF) apo E concentrations, determined by a sensitive sandwich ELISA, were 411.3 +/- 76.0 and 454.3 +/- 51.8 micrograms/dl (mean +/- S.D.) for young rats (8-12 weeks old, n = 7) and old rats (36-40 weeks old, n = 10), respectively. Age-related increase, which was conspicuous in serum apo E (21.2 +/- 2.4 vs 60.9 +/- 14.1 mg/dl for young and old rats, respectively), was not observed in CSF apo E. CSF apo A-I concentrations, determined by ELISA, were extremely low in the both groups (less than 10 micrograms/dl). Neither CSF apo A-I nor CSF apo E correlated to any of the plasma lipoprotein components, indicating the presence of largely independent lipoprotein metabolism in the rat central nervous system. Apo E is present in CSF in the form of apo E-rich HDL1 with particle sizes similar to those of plasma E-rich HDL1.

Lipoprotein cholesterol concentrations in patients with Behçet's disease.

Serum cholesterol concentrations among very low, low, and high density lipoproteins (VLDL, LDL and HDL) in 12 male patients with Behçet's disease were compared with those of 12 normal male subjects. Serum lipoproteins were separated by a combination of ultracentrifugation and gel filtration chromatography. The patients had significantly (p less than 0.001) lower concentrations of HDL-cholesterol than the control subjects (356 +/- 62 mg/l vs. 573 +/- 108 mg/l, means +/- SD). The cholesterol concentrations in apolipoprotein B-containing lipoproteins (VLDL and LDL) from the patients tended to be reciprocally higher than those of the controls, though not statistically significant. There was no difference in serum total cholesterol concentrations. The chemical composition of HDL from the patient group was characterized by higher protein and lower cholesterol (both esterified and free) contents compared with the control HDL.

Protective effects of an antiulcer agent, ecabet sodium on colorectal carcinogenesis in rodents.

A new antiulcer agent, ecabet sodium is one of dehydroabietic acid derivatives prepared from pine resin. The effects of ecabet sodium on colorectal carcinogenesis were investigated in azoxymethane-pretreated mice with chronic ulcerative colitis induced by 3 repeated administration of 3% dextran sulfate sodium and in 1, 2-dimethylhydrazine-treated rats. Although daily treatment with ecabet sodium did not affect the colorectal DNA-synthesizing enzyme activities and bromodeoxyuridine-immunoreactive S-phase cells, high-grade dysplasia in ecabet sodium-treated mice was less frequent than in untreated mice. In rats, ecabet sodium administration reduced the elevated activity of thymidylate synthetase in colorectal tumors.

Pathological and enzymatic features of 1,2-dimethylhydrazine induced colorectal carcinomas in rats.

Well- and moderately well-differentiated colorectal adenocarcinomas accounted for 86% of all tumors induced by 1,2-dimethylhydrazine in rats, and were distributed throughout the colorectal tract. Poorly differentiated carcinomas, 14% of all tumors, were markedly restricted to the proximal half of colon, i.e., 94% of the poorly differentiated type was found in the proximal colon and caecum. Thymidylate synthetase and thymidine kinase, key enzymes in the de novo and salvage pathways, respectively, for pyrimidine nucleotide synthesis were found to be reduced and elevated, respectively, with increasing cellular differentiation. These results suggest that biochemical differences in colorectal tumors may be associated with differences in tumor frequency, distribution, and histological type.

Effects of a new antiulcer agent, ecabet sodium on a cutaneous tumorigenesis induced with 7,12-dimethylbenz(a)anthracene in mice.

A new antiulcer agent, ecabet sodium is one of dehydroabietic acid derivatives prepared from pine resin, and is known to have a potent protective effect on gastric hemorrhagic lesions induced with carcinogens by covering a gastric mucosa after oral administration. In the present study, we investigated the effects of synthetic ecabet sodium on cutaneous tumorigenesis and development of skin-tumors induced by 7,12-dimethylbenz(a)anthracene application on mouse back skin. It was tentatively concluded that ecabet sodium reduced the incidence of skin-tumors (squamous cell papillomas) by inhibition of DNA synthesis in de novo pathway, and suppressed the development of papillomas by decrease of DNA synthesis in a salvage pathway.

Effects of lentinan on colorectal carcinogenesis in mice with ulcerative colitis.

Lentinan; i.e., polysaccharides extracted from a kind of black mushroom shiitake, has been clinically applied as an antitumor and antimetastatic drug, and has been reported to prevent both chemical and viral carcinogenesis. It is known that lentinan affects the tumorous vascular system resulting in the induction of hemorrhagic necrosis which is dependent on T-cells in the tumor. Repeated mucosal necrosis-regeneration sequence in chronic ulcerative colitis induced with 3% dextran sulfate sodium led to colorectal carcinogenesis in azoxymethane-pretreated mice. In the present study, the additive treatment with lentinan in the azoxymethane-dextran sulfate sodium treated mice enhanced the colorectal high-grade dysplasia, though not significantly, and the splenic weight. This may show the proliferation of pathogenic splenic T cells resulting in a change for the worse of ulcerative colitis, anemia induced with hemorrhage and colorectal carcinogenesis; i.e., high-grade dysplasia of the mucosa and/or invasive adenocarcinomas of the colorectum. The present results may recommend chemoimmunotherapy while using lentinan, but not immunotherapy using lentinan alone, is indicated for the management of cancer patients.

Does macrophage-colony stimulating factor stimulate rat haematopoietic cells?

It is known that colony stimulating factors (CSFs) stimulate the myeloid cells of bone marrow and splenic cells in rodents. The effects of macrophage (M)-CSF on the activities of thymidylate synthase and thymidine kinase, involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively, in haematopoietic cells of bone marrow and spleen were investigated in rats. A single M-CSF injection did not elevate the mRNA expression levels of the enzymes in bone marrow cells 6 h after treatment, but it enhanced the splenic thymidylate synthase mRNA expression. M-CSF stimulated the splenic thymidylate synthase activity without an increase of the peripheral granulocytes. The effect of M-CSF on granulocytes is considered to be weak compared with that of granulocyte (G)-CSF, because of the indirect secretion of endogenous G-CSF from the cells with M-CSF receptors stimulated by exogenous M-CSF. Since M-CSF was able temporarily to lead progenitor cells from long G1-phase into S-phase, M-CSF might accelerate the anticancer effects when used together with anticancer agents.

Correlation between histological differentiation and DNA-synthesizing enzymes in rat colorectal tumors induced with 1, 2-dimethylhydrazine.

Thymidylate synthase and thymidine kinase are key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis. Colorectal carcinogenesis induced with 1,2-dimethylhydrazine in rats enhanced mRNA expression levels of both enzymes, resulting in the increase of both enzyme activities and bromodeoxyuridine-immunoreactive S-phase cells. Poorly and well differentiated adenocarcinomas of the colorectum showed the relative elevation of activities of thymidylate synthase and thymidine kinase, respectively. These results indicate that the relationship between de novo and salvage pathways for pyrimidine nucleotide synthesis may depend on the histopathological grades of cell differentiation.

Cataract development induced by repeated oral dosing with FK506 (tacrolimus) in adult rats.

FK506 (tacrolimus), a potent immunosuppressant, is used for inhibiting allograft rejection in the organ transplantation field. In a preclinical toxicity study in rats, FK506 induced various toxicities, including renal and pancreatic injuries. One of these toxic findings was cataract, and we have found that cataract appeared in rats dosed orally with FK506 for 13 weeks and more. Therefore, to better elucidate the onset mechanism of FK506-induced cataract, we measured biochemical parameters, such as sorbitol, Na,K-ATPase and glutathione in the lens of rats. Rats were dosed with FK506 in oral daily doses of 0.2, 1 or 5 mg/kg for 13 weeks, the lowest dose of which approximated the expected clinical dosage. Cataract developed in the 5-mg/kg/day group, with an incidence of 25%, whereas no cataract formation was observed in the 0.2- or 1-mg/kg/day groups. Five mg/kg/day led an increase of sorbitol and a decrease of reduced type glutathione, but did not affect Na,K-ATPase activity of the lens. FK506 is known to have diabetogenicity mediated through pancreatic injury, which appears as vacuolation of islet cell in rats. Five mg/kg/day of FK506 induced an elevation of blood glucose associated with glucose intolerance, and decrease of both basal insulin level and insulin content in the pancreas, and the changes were in parallel with the cataract development in the present study. On the other hand, diabetic parameters did not change in the 0.2- or 1-mg/kg/day groups. These observation suggest that diabetes developed in the rats dosed with 5 mg/kg/day of FK506. Coadministration of a novel aldose reductase inhibitor, Zenarestat, at an oral dose of 50 mg/kg/day resulted in a reduction of incidence of the FK506-induced cataract and a decrease of sorbitol levels in the lens when compared to that in the lens of rats dosed with 5 mg/kg/day of FK506. These results suggest that FK506-induced cataract in rats is due to an accumulation of sorbitol in the lens, secondary to the diabetogenic effect of FK506. FK506 treatment at the doses of 0.2 and 1 mg/kg/day neither affected parameters indicative of diabetes nor induced cataract in rats, suggesting that the cataract would not develop with FK506 if diabetic parameters were kept under control.