Oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD.

The pathogenicity of influenza virus infection in the mice involves, at least in part, overreaction of the immune responses of the host rather than a direct effect of virus multiplication. Xanthine oxidase, which is responsible for the generation of oxygen free radicals, was elevated in serum and lung tissue of mice infected with influenza virus. To test the theory that oxygen-free radicals are involved in pathogenesis, free radicals were removed by injecting superoxide dismutase (SOD), a specific superoxide radical scavenger, which was conjugated with a pyran copolymer. The conjugate protected mice against a potentially lethal influenza virus infection if administered 5 to 8 days after infection. These findings indicate that oxygen radicals are important in the pathogenesis of influenza virus infection, and that a polymer-conjugated SOD has therapeutic potential for this virus infection and other diseases associated with free radicals.

Establishment of an enzyme immunoassay system for gliostatin/platelet-derived endothelial cell growth factor (PD-ECGF).

A two-site enzyme immunoassay for gliostatin (GLS)/platelet-derived endothelial cell growth factor (PD-ECGF) has been developed. The detection limit of gliostatin/PD-ECGF was 30 pg/well, and the optimal assay range was 0.1 to ng/well. This assay system enabled us to confirm the immunochemical identity of both factors and to detect immunoreactive gliostatin/PD-ECGF (IR-GLS/PD-ECGF) in human biological body fluids. The age-related analysis from newborn to 69 years revealed that the serum IR-GLS/PD-ECGF level was high in infants younger than 1 year old (1.8 ng/ml) and in the 20-year-old age group (1.8 ng/ml), and highest in the umbilical cord blood (2.1 ng/ml). Curiously high concentrations were detected in saliva with a significant sex difference (11.3 ng/ml for males and 48.7 ng/ml for females), and in synovial fluids (3.7 ng/ml). A number of human tumor cells, gastric cancer cells, MKN-74, neuroblastoma cells, GOTO, as well as epidermoid carcinoma cells, A431, were found to produce a significant amount of IR-GLS/PD-ECGF (0.2 to 21.8 ng/mg protein), and some of them secreted the IR-GLS/PD-ECGF in the conditioned medium (approximately 0.5 ng/ml). The enzyme immunoassay system is sufficiently sensitive for the basic and clinical study of gliostatin/PD-ECGF in human body fluids, tissues and organs.

Safety evaluation of hemagglutinating virus of Japan--artificial viral envelope liposomes in nonhuman primates.

We tested, in cynomolgus monkeys, the safety and effectiveness of a hybrid liposome vector, hemagglutinating virus of Japan (HVJ)--artificial viral envelope (AVE) liposomes, for human therapeutic gene transfer in a series of experiments. In a repetitive intramuscular administration study, vehicle control macaques (n = 2), which were treated with HVJ--AVE liposome suspension, received repetitive intramuscular injections of 2 ml of test substance. Human hepatocyte growth factor (HGF) cDNA-inserted expression vector (pUC-SR alpha/HGF) injection animals (n = 2), which were treated with HVJ--AVE liposome suspension containing pUC-SR alpha/HGF, received repetitive intramuscular injection of 2 ml of test substance. General body condition, hematology, blood chemistry, and serum HGF were determined sequentially before treatment and 7, 21, 28, and 29 days after treatment. Elevations in HGF were detected in monkeys injected with pUC-SR alpha/HGF. After this observation period, macaques were killed for autopsy and histological examination. pUC-SR alpha/HGF was detected by polymerase chain reaction (PCR) analysis in the liver, spleen, and at the injection site. In single intravenous administration study, control macaques (n = 4) received a single intravenous injection of 10 ml of physiological saline. Vehicle control animals (n = 5) received a single intravenous injection of 10 ml of HVJ--AVE liposome suspension. DNA-treated animals (n = 7) received a single intravenous injection of 10 ml of HVJ--AVE liposome suspension containing plasmid DNA [pcDNA 3.1(+)]. General body condition, body weight, hematology, blood chemistry, and urine composition were determined sequentially before treatment and 1, 14, 21, and 28 days after treatment. After this observation period, macaques were killed for autopsy and histological examination. pcDNA 3.1(+) was detected by PCR analysis on day 1 in lung, liver, and spleen of all monkeys, in kidney of one of two monkeys, and in heart of one of two monkeys. However, no DNA was detected in any of the tissues examined on days 14, 21, and 28. No virus genomic RNA was detected by reverse transcription (RT)-PCR analysis with HVJ-specific primers. In this series of safety evaluations, the animals tolerated the safety study with no change in body weight or general condition. No hematological changes or alterations in blood chemistry or urine composition was detected. Moreover, no histological changes were observed. This safety evaluation study demonstrates the safety, feasibility, and therapeutic potential of the novel transfection vehicle, HVJ--AVE liposomes, in humans.

Reduced bone marrow toxicity of neocarzinostatin by conjugation with divinyl ether-maleic acid copolymer.

Neocarzinostatin (NCS) was conjugated with divinyl ether-maleic acid anhydride copolymer (pyran copolymer), and its therapeutic effect was compared with that of NCS. The conjugated NCS (pyran-NCS) with a molecular weight of about 23,000, exhibited in vitro cytotoxic activity against eight cell lines and bone marrow cells that was similar to the cytotoxic activity of NCS on a molar basis. Furthermore, both drugs had similar effects against a multidrug-resistant Chinese hamster ovary cell line (CHR C5) and its parent cell line (AUXB1) in vitro. However, pharmacological analysis showed that pyran-NCS had reduced accumulation in the spleen, and most important was three times less hematotoxic in vivo compared with NCS. Also, pyran-NCS had a 1.7-fold higher 50% lethal dose (LD50). Antitumor activity of pyran-NCS and NCS was tested against two different forms of Meth A tumor. In a solid tumor model, pyran-NCS and NCS suppressed tumor growth at three-fourths of the LD50 to 12.8 and 19.0% of the control tumor as evaluated on day 28, respectively (P less than 0.025). In an ascitic tumor model, the percentage increase in the median life span caused by pyran-NCS and NCS was more than 400 and 150% on day 60, respectively. Pyran-NCS is more effective than NCS because the reduced acute toxicity permits an increased drug dosage.

Degradation-resistant character of synthetic hydroxyapatite blocks filled in bone defects.

The long-term changes of both the implanted hydroxyapatite (HA) blocks and the bone around them were observed radiologically to investigate the clinical usefulness of HA. HA blocks were used as a space filler in surgically created bone defects of seven cases due to curettage of bone tumours or removal for bone grafts, and they were followed up for 78 to 109 months. Bone formation around HA blocks peaked within 1 year after implantation, and then it faded gradually. However, shapes of HA blocks were rarely changed in all the cases. A clear zone around HA blocks was never observed . HA blocks were biocompatible and demonstrated a character resistant to degradation.

Molecular design of polyvinylpyrrolidone-conjugated interleukin-6 for enhancement of in vivo thrombopoietic activity in mice.

Functional polyvinylpyrrolidone (PVP) was synthesized as a novel polymeric modifier for polymer-conjugated cytokines, and its efficiency and applicability as a drug delivery system (DDS) were evaluated. PVP with a carboxyl group at one end of the main chain was prepared by radical polymerization (M(n): 6000, M(w)/M(n): 1.14) with the aid of 4,4'-azobis(4-cyanovaleric acid) as a radical initiator and 3-mercaptopropionic acid as a transfer agent. Interleukin-6 (IL-6) was covalently conjugated via the formation of amino bonds between the lysine amino groups of IL-6 and PVP. PVP-conjugated IL-6, in which 60% of the fourteen lysine amino groups of IL-6 were estimated to be coupled with PVP (M-PVP-IL-6), showed more than 50-fold greater thrombopoietic potency in vivo than native IL-6. No side effects, such as body weight loss, were observed in the M-PVP-IL-6 treated mice. These results indicate that PVP as a polymeric modifier is a promising DDS for clinical application of cytokines and other therapeutic agents.

Effect of long-term administration of probucol on triglyceride turnover in rats.

BACKGROUND: Our previous observation revealed that probucol, a well known cholesterol-lowering drug, can improve impaired catabolism of plasma triglyceride in streptozotocin-diabetic rats without reducing plasma glucose. The present study was conducted using Triton WR1339 and endogenously radiolabeled lipoproteins in order to examine the mechanisms whereby the drug can stimulate triglyceride removal from the circulation in rats independent of glucose metabolism. METHODS: The short- and long-term effects of probucol on triglyceride turnover were examined in rats. Male Wistar rats received a diet containing probucol (1%) for 2 weeks and 4 months. Triglyceride turnover was estimated in rats using Triton WR1339. RESULTS: After 2 weeks of probucol administration, triglyceride and cholesterol concentrations in plasma and in the very-low-density lipoprotein (VLDL) fraction were suppressed slightly but significantly in the probucol-administered rats compared with standard chow-fed control rats. However, triglyceride secretion rates were similar in the two groups. Probucol-administered rats showed markedly suppressed triglyceride concentration in plasma and the VLDL fraction compared with chow-fed control rats after 4 months of administration. However, triglyceride secretion rate was significantly increased in rats given probucol. The radioisotope experiment revealed that probucol-administered rats can remove VLDLs (lipoproteins either from probucol-administered or control rats) from their system faster than normal rats and that probucol-containing VLDL can be removed from the circulation of both groups of rats more rapidly than normal VLDL. Therefore, probucol-administered rats as well as probucol-containing VLDL (lipoprotein from probucol-administered rats) are responsible for accelerated catabolism of VLDL-triglyceride. CONCLUSION: No significant differences in the lipid composition of newly-secreted VLDL particles between control rats and those given probucol were noted after Triton injection. The precise mechanism behind increased triglyceride secretion rate in rats after long-term administration of probucol is not known. However, we concluded from the present data that long-term administration of probucol results in markedly accelerated VLDL-triglyceride turnover without affecting lipid composition of newly secreted VLDL particles in normal rats.

Comparison of the isotopical tracer and the Triton WR 1339 methods for triglyceride kinetics in carbohydrate-fed rats.

Radiolabeled tracer [3H] very low density lipoprotein (VLDL)-triglyceride (TG) and non-tracer (Triton WR 1339) methods were used to determine VLDL-TG kinetics in normal rats and in rats given either a 10% glucose or a 10% fructose drinking solution for 16 h ad libitum. The carbohydrate-fed rats were hypertriglyceridemic compared to control animals. VLDL-TG was endogenously prelabeled with [3H] glycerol in control, glucose- and fructose-fed rats, and injected into identically treated recipients. Triton WR 1339, a potent inhibitor of VLDL-TG catabolism, was injected into the same animal 30 min after the end of the tracer method to measure TG secretion rate (TGSR). The tracer and non-tracer methods showed that fructose-fed rats had a significantly lower fractional catabolic rate (FCR) than either control or glucose-fed rats. In contrast, glucose-fed rats had a TGSR greater than control without a reduction in FCR. For all treatments, TG concentration correlated with FCR for the tracer method and with TGSR for the non-tracer method. There was good correlation of TGSR determined by the tracer and non-tracer method for control rats despite the substantial difference in the absolute values. No such relationship was observed in carbohydrate-fed rats. Control rats were made hypertriglyceridemic by Intralipid infusion. A lower FCR was observed when determined by the tracer method, but this was not observed by the triton method. These results suggest that TG kinetics determined by the tracer and the triton methods cannot be readily correlated, especially in hypertriglyceridemic state. However, the two kinetic studies both suggested that overproduction of VLDL-TG in glucose-fed rats and impaired VLDL-TG catabolism in fructose-fed rats were the primary cause for their hypertriglyceridemia, respectively.

[An adult case of Chédiak-Higashi syndrome with parkinsonism and marked atrophy of the central nervous system].

Chédiak-Higashi syndrome (CHS) is often a fatal disease of childhood characterized by oculocutaneous forms of albinism with congenital gigantism of peroxidase granules, granulation anomaly of leukocytes, hereditary gigantism of cytoplasmic organelles and a marked susceptibility to infections. A few patients have survived to age 20 years. A 39-year-old woman developed tremor and gait disturbance at age 22 years. At age 25 years, she was admitted to the Hospital for evaluation. Mental impairment, horizontal nystagmus, bradyphrenia, cogwheel rigidity, tremor at tongue, mandible and hands, bradykinesia, and unsteady gait were found and a juvenile parkinsonism was diagnosed. However, there was no favorable response by levodopa therapy. She became unable to walk at age 33 years. On admission, Oct. 27, 1988, at age 39 years, she was bedridden with a posture of decorticate rigidity. She was found to have partial depigmentation of the retina and choroid, pale and atrophic optic disc and subluxation of the mandible, Onuaguluchi's finger deformities and pes cavus. Neurological examination disclosed that she was alert but had marked difficulty in speaking. The communication was only possible by giving a sign of grasping of the left hand. The patient also showed oculogyric crisis, dystonic rigidity of the neck, diffuse muscular atrophy, complete paraplegia and decreased deep tendon reflexes with Babinski sign. On laboratory studies, at age 39, the white cells count was decreased (2,510/mm3), the hemoglobin level was 10.3 g/dl, the serum iron was 12 micrograms/dl, IgG 2,828 mg/dl, IgA 1,002 mg/dl, and the activity of natural killer cell was profoundly decreased (2%, normal; 18-40). Hematological examination revealed peroxidase positive giant granules in leukocytes. Chest X-ray film disclosed marked abnormal colon gas which located right subdiaphragma (Chilaiditi syndrome). Cerebrospinal fluid contained 12 cells/mm3, 99% lymphocytes; protein, 58.8 mg/dl; IgG, 6.8 mg/dl; HVA, 4.5 ng/ml (normal 41.8-44.6); 5-HIAA, 1.3 ng/ml (11.3-29.2); MHPG, 5.8 ng/ml (13.2-22.2).(ABSTRACT TRUNCATED AT 250 WORDS)

[Antitumor activity of polyanion and its application for drug delivery system of antitumor drugs].

Polyanionid copolymer of divinyl ether and maleic anhydride (DIVEMA) with narrow molecular weight distribution was synthesized and tested of its antitumor activity. DIVEMA showed a significant antitumor activity against colon 26 adenocarcinoma and FSaI fibrosarcoma transplanted in syngenic mice. Furthermore, DIVEMA was used as a polymeric drug carrier of antitumor drugs to reduce side effects and enhance the antitumor activity of the drugs. Adriamycin and neocarzinostatin were attached covalently to DIVEMA and the polymeric conjugates showed higher antitumor activity than the corresponding mother drugs against P 388 leukemic mice.

Regulon controlled by the GppX hybrid two component system in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis experiences a number of environmental conditions in the oral cavity, and must monitor and respond to a variety of environmental cues. However, the organism possesses only five full two-component systems, one of which is the hybrid system GppX. To investigate the regulon controlled by GppX we performed RNA-Seq on a ΔGppX mutant. Fifty-three genes were upregulated and 37 genes were downregulated in the ΔGppX mutant. Pathway analyses revealed no systemic function for GppX under nutrient-replete conditions; however, over 40% of the differentially abundant genes were annotated as encoding hypothetical proteins indicating a novel role for GppX. Abundance of small RNA was, in general, not affected by the absence of GppX. To further define the role of GppX with respect to regulation of a hypothetical protein observed with the greatest significant relative abundance change relative to a wild-type control, PGN_0151, we constructed a series of strains in which the ΔgppX mutation was complemented with a GppX protein containing specific domain and phosphotransfer mutations. The transmembrane domains, the DNA-binding domain and the phosphotransfer residues were all required for regulation of PGN_0151. In addition, binding of GppX to the PGN_0151 promoter regions was confirmed by an electrophoretic mobility shift assay. Both the ΔGppX mutant and a ΔPGN_0151 mutant were deficient in monospecies biofilm formation, suggesting a role for the GppX-PGN_0151 regulon in colonization and survival of the organism.

Human trophoblast responses to Porphyromonas gingivalis infection.

Porphyromonas gingivalis is a periodontal pathogen that is also associated with preterm low-birthweight delivery. We investigated the transcriptional responses of human extravillous trophoblasts (HTR-8) to infection with P. gingivalis. Over 2000 genes were differentially regulated in HTR-8 cells by P. gingivalis. In ontology analyses of regulated genes, overpopulated biological pathways included mitogen-activated protein (MAP) kinase signaling and cytokine production. Immunoblots confirmed overexpression of the MAP kinase pathway components MEK3, p38 and Max. Furthermore, P. gingivalis infection induced phosphorylation and activation of MEK3 and p38. Increased production of interleukin (IL)-1beta and IL-8 by HTR-8 cells was demonstrated phenotypically by enzyme-linked immunosorbent assay of HTR-8 cell lysates and culture supernatants. Hence, infection of trophoblasts by P. gingivalis can impact signal transduction pathways and modulate cytokine expression, outcomes that could disrupt the maintenance of pregnancy.

Studies on immunity in hybridoma-bearing mice. A. Immune response to antigens. II. inhibition of production of anti-dinitrophenyl antibodies in mice which have rejected the B 53 anti-dinitrophenyl-producing IgE hybridoma.

When BALB/c mice, which had rejected the anti-dinitrophenyl (DNP) IgE-producing hybridoma B 53, were immunized with DNP proteins, they produced much less anti-DNP antibodies than control (normal) mice. The anti-DNP plaque-forming cell (PFC) number was much less when spleen cells from mice immunized with DNP proteins were treated with sera of mice which had rejected the hybridoma B 53 than the PFC number from the same spleen cells not treated by the sera. The sera of mice which had rejected the hybridoma B 53 contained an inhibitor which was adsorbed and eluted from an anti-mouse immunoglobulin column and also a mouse anti-DNP IgG2a column. The inhibition of PFC was hapten-reversible. In Western blotting the eluates from the anti-DNP IgG2a column reacted as well with the blotted anti-DNP IgE B 53 as an anti-idiotypic antibody to anti-DNP IgE B 53. These criteria establish that the inhibitor in the sera of the mice which had rejected the B 53 tumor was an anti-idiotypic antibody of the type which mimics the epitope (DNP) of the immunizing antigen.

Degradation of bisphenol A by the lignin-degrading enzyme, manganese peroxidase, produced by the white-rot basidiomycete, Pleurotus ostreatus.

Degradation of 2,2-Bis(4-hydroxyphenyl)propane (bisphenol A, BPA), an endocrine-disturbing chemical, by the growing mycelia of the white-rot basidiomycete, Pleurotus ostreatus, was examined. About 80% of BPA initially present decreased in 12 days of culture with this fungus. By in vitro experiments using the lignin-degrading enzyme manganese peroxidase (MnP), BPA was metabolized to phenol, 4-isopropenylphenol, 4-isopropylphenol, and hexestrol. The degradation products of BPA were assumed to be formed by the one-electron oxidation of the substrate.

Wear of alumina ceramics prosthesis.

To investigate the process of wear in the total knee prosthesis consisting of alumina ceramics (Al-ceramics) and ultra-high molecular weight polyethylene (UHMWP) in vivo, we observed the fine structures of the articular surface of the prostheses used for four patients with osteosarcoma of the distal femur. We also examined the wear debris in the surrounding soft tissues. The prostheses were extracted at autopsy between 13 and 48 months after surgery. An increase of pores, indicating where Al-ceramics grains fell off, and many streaks on the UHMWP running parallel to the gliding direction of the joint were observed on the articular surface. The streaks had similar widths to the Al-ceramics grains. Al-ceramics debris was detected in the soft tissues around the joint mixed with UHMWP debris. We concluded that the wear of UHMWP in Al-ceramics prosthesis was promoted by interposition of Al-ceramics debris.

A fragile X female with Down syndrome.

Clinical and cytogenetic aspects of a female infant with trisomy 21 and the fragile X [fra(X)] chromosome are reported. Most of the facial characteristics of the patient are those observed in Down syndrome, but some features such as long face with prominent forehead and lower jaw, and large ears are related to the fra(X) syndrome. The origin of an additional chromosome 21 may be ascribed to maternal first meiotic nondisjunction in our case. It has been suspected that female carriers of the fra(X) chromosome may be predisposed to meiotic nondisjunctional events. However, there is probably no relationship between the two chromosomal abnormalities in our case because of the maternal age at the delivery.