Peracute Bacterial Meningitis due to Infection with Klebsiella pneumoniae in Captive-bred Ruffed Lemurs (Varecia variegate).

We describe the development of neurological signs in four juvenile black-and-white ruffed lemurs (Varecia variegate), housed at a petting zoo in Japan. The clinical course was severe, with three lemurs dying within 1 day of the appearance of clinical signs. The other lemur was treated and survived. Pathological analyses demonstrated meningitis and the presence of gram-negative bacilli in the cerebrum, cerebellum, palatine tonsil and liver. Klebsiella pneumoniae was isolated from the brain of all of the dead lemurs. Multilocus sequence typing analysis showed that all the isolates were sequence type 86 (ST86). To our knowledge, this is the first determination of K. pneumoniae infection in ruffed lemurs of this genus. K. pneumoniae infection may represent a risk to lemurs and people who come into contact with infected animals.

Prevalence of Baylisascaris Roundworm in Captive Kinkajous in Japan.

Baylisascaris potosis causes larva migrans in animals. The present study evaluated the prevalence of B. potosis in captive kinkajous ( Potos flavus ) and the ability of milbemycin to treat natural infections of B. potosis in 2 female wild-caught kinkajous. In 2012, fecal samples were collected from 16 kinkajous in 6 zoological gardens and 29 imported captive kinkajous from 4 pet traders in Japan. Although all samples from zoological gardens were negative, 8 kinkajous from traders were positive for Baylisascaris eggs, at least 4 of which were wild caught in the Republic of Guyana. No associated human illness was reported from any of the facilities. The 2 infected kinkajous received a single oral administration of Milbemycin® A Tablets, which delivers 0.69-0.89 mg/kg milbemycin oxime. Fecal examinations on days 14 and 30 were negative for Baylisascaris eggs. These results demonstrated that milbemycin oxime has possible anthelmintic efficacy against Baylisascaris roundworms in captive kinkajous. We conclude that Baylisascaris infections are highly prevalent in wild-caught kinkajous in Japan and that most of the infected kinkajous were imported from the Republic of Guyana.

Experimental infection of Mongolian gerbils with Baylisascaris potosis.

The present study evaluated the pathogenicity of Baylisascaris potosis, a newly described ascarid nematode, in Mongolian gerbils. Gerbils were infected with varying doses of either B. potosis or Baylisascaris transfuga embryonated eggs (100, 1,000, and 4,000) for 30 days postinfection (pi). Baylisascaris potosis-infected gerbils showed no clinical signs of disease; however, gerbils exposed to 1,000 and 4,000 B. transfuga eggs showed severe neurologic signs at 22-29 days and 14-15 days pi, respectively. Histopathologic examination revealed larvae and lesions in the intestine, lung, liver, and muscles of B. potosis-infected gerbils, but not in the brain, whereas B. transfuga larvae were found only in the brain and muscle. These results indicate that B. potosis larvae migrate through numerous organs and are associated with visceral larva migrans in gerbils, but less frequently migrate to the nervous system in gerbils than does B. transfuga .

(+)-Syringaresinol-di-O-beta-D-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-kappaB activities.

(+)-Syringaresinol-di-O-beta-D-glucoside (SR), syringin, and isofraxidin isolated from the stem bark of Acanthopanax senticosus Harms are its major constituents. The present work was undertaken to analyze effects of these compounds on inflammatory functions in SW982 human synovial sarcoma cell system. When cells were exposed to SR, syringin, or isofraxidin, only isofraxidin had significant inhibitory effects on cell growth, although a slight inhibition was observed at the highest concentration of SR. SR suppressed the production of IL-6 at lower concentrations than syringin and isofraxidin. SR and syringin significantly suppressed the production of prostaglandin E(2), while isofraxidin suppressed only slightly. SR was more potent than syringin and isofraxidin at inhibiting the expression of IL-1beta, IL-6, cyclooxygenase (COX)-2 and matrix metalloproteinases (MMP)-1 mRNA, but was less potent than syringin at inhibiting the expression of MMP-2. We further demonstrated that SR significantly reduced MMP-1 promoter luciferase activity and DNA-binding activity of transcriptional factors AP-1 and NF-kappaB. Taken together, these results suggest that SR, an active component of the stem bark of A. senticosus, modulates the inflammatory process involved in arthritis by suppressing various gene expression through inhibiting AP-1 and/or NF-kappaB activities.

Differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line as liver progenitor cells.

We report the differentiation potential of an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b. Under basic culture conditions THLE-5b showed undifferentiated phenotypes. When grown as cell aggregates, THLE-5b exhibited a hepatocyte-like ultrastructure, ammonia metabolic activity and several other indicators that suggest hepatocytic maturation, including up-regulation or induction of liver-specific genes such as albumin and tryptophane 2,3-dioxygenase, and down-regulation of biliary cell markers such as gamma-glutamyl transpeptidase (GGT). Under these conditions, transcriptional factors such as HNF-1 and HNF-4alpha were also up-regulated or induced. In Matrigel culture, expression of GGT was up-regulated. THLE-5b expressed both albumin and alpha 1-antitrypsin, but lost expression of CK19 in severe combined immunodeficient mice. Thus, THLE-5b can be aligned with progenitor cells, which are committed to the hepatocytic or biliary epithelial cell lineage. These results imply that bipotent progenitor cell populations similar to THLE-5b cells may exist in adult human liver.

Isolation and cultivation of porcine hepatocytes for extracorporeal artificial liver support system.

OBJECTIVE: To develop procedures for the successful harvesting of large quantities of viable and functional pig liver cells from abattoir organs. METHODS: The procedure included partial liver lobe retrograde perfusion and mechanical/enzymatic digestion of the liver tissue, followed by separation of the hepatocytes, based on size and density, from contaminating cell types. RESULTS: Digestion of the partial liver lobe resulted in an average yield of 1.39 x 10(9) cells (9.9 x 10(8) cells/g liver) with an average viability of 92.5%. The yield and viability of cells were improved by dispase/collagenase resultant digestion. The emergence of blebby cells was blocked by supplying oxygen to the cell isolation buffers. Isolated hepatocytes seeded onto polystyrene surfaces remained viable and functional at a level comparable to that of rat hepatocytes, although their function decreased over time. CONCLUSIONS: Adult pig hepatocytes can be harvested with high yields and retain viability and differentiated function using this method. Abattoir pig livers can be an excellent source of hepatocytes for use as the biological component of artificial liver assist devices.

The in vitro differentiating capacity of nonparenchymal epithelial cells derived from adult porcine livers.

Specific nonparenchymal epithelial cell (NPEC) clusters derived from normal adult porcine livers demonstrate a characteristic developmental pattern in the presence of other types of nonparenchymal cells in vitro. This pattern includes scattering, colonial growth, and an emergence of duct-like structures (DLSs) in the colonies. It has been confirmed that 96% of the scattered cell clusters in these cultures develop into colonies containing DLSs. In this study, we examine the differentiation of NPEC clusters using the scattered formation as a marker of the DLS-emerged colonies. We report that the NPECs expressed albumin, alpha-fetoprotein, transferrin, cytokeratin (CK) 18, CK7, and c-met, but not alpha-1-antitrypsin (AAT), at the scattering stage. In addition, at the same stage, NPECs expressed oval-cell-related markers such as OV6, but not biliary epithelial cell (BEC) markers such as gamma-glutamyltransferase, CK19, and CK14. At the DLS emerging stage, hepatocyte markers, including AAT, were detectable in the cells either at the periphery of colonies or in the cells surrounded by the DLSs. On the other hand, the cells constituting DLSs expressed BEC markers, suggesting a bile duct nature of the DLSs. Furthermore, the cells in the colonies possessed an ultrastructural appearance of differentiated hepatocytes and BECs. These results suggest that certain NPECs are bipotent, and that, in culture, they mimic hepatoblast development in vivo.

A mild transient decrease of peripheral red blood cell counts induced by a suprapharmacological dose of pegylated human megakaryocyte growth and development factor in rats.

Previous studies have shown that pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) at suprapharmacological dose induces a mild transient decrease of red blood cell counts according to thrombopoiesis in normal mice. To unravel the mechanism underlying this mild transient decrease of red blood cells, we have studied the effect of PEG-rHuMGDF on the circulating plasma and blood volume, and the serum biochemical parameters of anaemia and splenectomy. Also, we have performed histological studies of the bone marrow and the spleen of PEG-rHuMGDF-treated rats. PEG-rHuMGDF (300 microg kg(-1)]) or vehicle was subcutaneously administered to rats once a day for up to five days. From day 6 after the start of PEG-rHuMGDF administration, the platelet counts and plateletcrit levels were significantly increased, reaching peak values on day 10, and recovering to normal by day 20. The red blood cell counts and the haematocrit levels were significantly decreased on day 6 to 13. The decreases in red blood cell levels and haematocrit produced by PEG-rHuMGDF treatment were mild and had recovered by day 15. The plasma and blood volumes were significantly increased on day 10 in PEG-rHuMGDF-treated rats. No alteration of the serum biochemical parameters for anaemia, iron or total bilirubin, were observed on day 10. The histological examination on day 10 revealed a marked increase in megakaryocytes and a slight decrease in erythropoiesis in the bone marrow of rats that received PEG-rHuMGDF (300 microg kg(-1)). There was also a slight increase in splenic megakaryocytes and erythropoiesis. The decrease of red blood cells by PEG-rHuMGDF was not affected by splenectomy. These results suggest that the mild transient decrease of red blood cells induced by PEG-rHuMGDF treatment for up to five days is based mainly on the increases in the plasma and blood volume. These events are secondary changes due to the regulation of the excess production of megakaryocytes in the marrow and the peripheral platelets.

Effects of KRN4884 (a novel K+ channel opener), levcromakalim, nilvadipine and propranolol on endothelin-1-induced heart disorders in anesthetized rats.

The effects of KRN4884 (5-amino-N-[2-(2-chrolophenyl)ethyl]-N'-cyano-3-pyridinecarboxa midine), a novel K+ channel opener, on the electrocardiogram changes caused by the intracoronary administration of endothelin-1 (ET-1) were studied in anesthetized rats and compared with the effects of levcromakalim, a K+ channel opener; nilvadipine, a Ca2+ antagonist; and propranolol, a beta-adrenoceptor antagonist. KRN4884 (50 microg/kg, i.v.) and levcromakalim (300 microg/kg, i.v.) inhibited the ST segment elevation and the development of arrhythmias induced by ET-1 (5 microg, i.c.) and decreased the incidence of death. Nilvadipine (300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.) each prevented the ST segment elevation, but the suppressions of the occurrence of arrhythmias produced by nilvadipine and propranolol were less than that shown by KRN4884. KRN4884 (30 and 50 microg/kg, i.v.), levcromakalim (100 and 300 microg/kg, i.v.) and nilvadipine (100 and 300 microg/kg, i.v.) significantly decreased the mean blood pressure in a dose-dependent manner, but propranolol did not. The heart rate was decreased by nilvadipine (100 and 300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.), but was not affected by KRN4884 (30 and 50 microg/kg, i.v.) or levcromakalim (100 and 300 microg/kg, i.v.). These results suggest that pretreatments with KRN4884 and levcromakalim are more effective on ET-1-induced electrocardiogram changes than those with nilvadipine and propranolol.

Colonial growth and differentiation of epithelial cells derived from abattoir adult porcine livers.

The parenchymal cell fraction was isolated from abattoir adult porcine livers and cultured in Dulbecco's Modified Eagles' medium/Ham's F12 medium (DMEM/F12; 1:1) medium supplemented with 5% foetal calf serum, 10 ng/mL glucagon, 10 microg/mL insulin, 60 ng/mL hydrocortisone and eight other factors (NAIR-1 medium). The fraction contained a number of epithelial cells other than hepatocytes, some of which attached to the culture plates as cell clusters and began to grow after 3 days in culture. These epithelial cells growing as colonies were found to express cytokeratin 18 by immunocytochemistry. After 7-8 days, duct-like structures emerged in the central parts of the colonies. The cells constituting the duct-like structures and some cells located outside the structures were positive for cytokeratin 19 and gamma-glutamyltransferase (GGT). The albumin-positive cells were located in the outer parts of the colonies rather than their central parts. Albumin was also detectable in the cells surrounded by the duct-like structures. Moreover, cytochrome P450 IA1 was induced by 3-methylcholanthrene (3-MC) on day 16. These results suggest that porcine liver epithelial cell clusters may contain stem-like cells which can differentiate into mature hepatocytes or bile duct epithelial cells.

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface.

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03-3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver.

The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.

Interferon-gamma promotes the survival and Fc epsilon RI-mediated histamine release in cultured human mast cells.

We examined the effects of interferon-gamma (IFN-gamma) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-gamma in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-gamma was blocked by neutralizing antibodies to IFN-gamma or to IFN-gamma receptor (IFN-gamma R). When mast cells were incubated with IFN-gamma in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the alpha-chain of IFN-gamma R (IFN-gamma R alpha) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gamma R monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gamma R on their surface. These findings suggested that IFN-gamma activates human mast cells via specific receptors in certain aspects of inflammatory reactions.

Comparative pharmacokinetics of KRN8602, a new morpholino anthracycline, and adriamycin in tumor-bearing mice.

It has been reported that KRN8602 shows antitumor effects similar or superior to those of Adriamycin (ADM) against several murine and human cell lines and has been found to be effective against multidrug resistant tumor cells. We investigated the pharmacokinetics of KRN8602, a new morpholino anthracycline, in comparison with ADM in mice bearing colon26 adenocarcinoma. After intravenous administration, both drugs disappeared triexponentially from the plasma and KRN8602 was eliminated faster than ADM. The rate of elimination of KRN8602 from tissues was also faster than than of ADM. The relative order of the area under the curve (AUC) of KRN8602 was spleen > tumor > small intestine > lung > kidney > heart > liver > brain > plasma, while that of ADM was spleen > kidney > lung > liver > heart > small intestine > tumor > plasma. ADM was not detectable in the brain. The AUC of KRN8602 was higher than that of ADM in the tumor and brain, but it was lower in other tissues. The tissue-to-plasma concentration ratio (KPapp) of KRN8602 was higher than that of ADM in the tumor, spleen, small intestine and brain. KRN8602 was metabolized to several metabolites. The concentrations of M1 and M2 (glycoside-type metabolites) was relatively high in the spleen. M3 (aglycone-type metabolite) showed a very high AUC ratio in the liver (34%). In tumor, M1 and M2 concentrations were low and M3 was not detected. KRN8602 had a greater activity than ADM and M2 had a cytotoxic activity similar to KRN8602 against colon26 cells in an MTT assay. These results suggest that the strong antitumor effect of KRN8602 against colon26 is due not only to its strong cytotoxic activity but also to its marked transferability into tumors. KRN8602 shows better selective toxicity than ADM, because KRN8602 is more selective for tumors than ADM and less is transferred to normal tissues.

Effects of T-helper 2-type cytokines, interleukin-3 (IL-3), IL-4, IL-5, and IL-6 on the survival of cultured human mast cells.

Although stem cell factor (SCF) has been identified as a critical cytokine for the development of human mast cells from their progenitors, the effects of other cytokines on human mast cells are less well understood. We examined the effects of several cytokines on the survival of human mast cells of 100% purity generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of 100 ng/mL recombinant human (rh) SCF and interleukin-6 (IL-6). Mast cells suspended in conventional serum-containing medium died over a period of 2 to 6 days after the withdrawal of SCF and IL-6. The cells became pyknotic and underwent DNA fragmentation characteristic of apoptosis. The addition of SCF, IL-3, IL-4, IL-5, or IL-6 to the cultures in both serum-containing and serum-free medium prolonged their survival in a dose-dependent manner. Some other cytokines, such as IL-2, IL-9, IL-10, IL-11, tumor necrosis factor-alpha, transforming growth factor-beta 1, and nerve growth factor, had no survival-promoting effect at 100 ng/mL. Preincubation of mast cells with SCF, IL-4, IL-5, or IL-6 for 24 hours during sensitization with IgE enhanced IgE/anti-IgE antibody-induced histamine release from mast cells, whereas IL-3 showed a negligible effect. Polymerase chain reaction amplification of alpha-chains of IL-3 receptor (R), IL-4 R, IL-5 R, and IL-6 R yielded products of the correct size predicted from the sequence of each receptor. The binding assay using 125I-labeled IL-3 indicated that these mast cells bear receptors for IL-3. These findings suggest that IL-3, Il-4, IL-5, and IL-6, which are mainly produced by T-helper 2 lymphocytes, might regulate the functions of human mast cells in vivo via specific receptors in allergic reactions.

p53 gene mutation and integrated hepatitis B viral DNA sequences in human liver cancer cell lines.

A G:C-->T:A mutational hotspot at codon 249 of the p53 tumor suppressor gene has previously been identified in hepatocellular carcinoma (HCC) of patients from Qidong, China and southern Africa in which aflatoxin B1 (AFB1) and hepatitis B virus (HBV) are known synergistic risk factors. We have examined p53 mutation patterns of HCC from geographic areas in which the risk factors vary. Nine HCC lines and four hepatoblastoma lines (HB) were examined for p53 gene mutations and the relationship with HBV infection. Five of the nine HCC lines had homozygous mutation or deletion randomly distributed in exons 6-8, whereas none of the four HB cell lines had p53 mutations. One of the four HB lines (HepG2) had an N-ras mutation at codon 61 position 2. The p53 point mutations in the three HCC cell lines from Japan resulted in the amino acid changes of cysteine for tyrosine in cell line HuH 7 at codon 220 (A:T-->G:C), alanine for glycine in cell line HLF at codon 244 (G:C-->C:G), and serine for arginine in cell line HLE at codon 249 (G:C-->C:G). In addition, the deletion of 18 base pairs from codon 264 position 3 to codon 270 position 1 has resulted in the deletion of Leu-Gly-Arg-Asn-Ser-Phe from the amino acids sequences 256-270 in the Japanese cell line HuH 4. The cell line PLC/PRF/5 that showed p53 mutation at codon 249 (G:C-->T:A) with substitution of serine for arginine was derived from a South African patient. Our results indicate that whereas the p53 gene is not mutated in the HB cell lines, the HCC cell lines frequently contain an abnormal p53 gene. In addition, p53 point mutations were not detected in the four Japanese HCC cell lines that were positive for genomic integration of HBV X-gene and surface antigen gene. The three Japanese HCC cell lines with p53 mutations did not contain HBV sequences, indicating that hepatocarcinogenesis associated with p53 mutation does not require the genomic integration of HBV sequences.

Primary culture of liver cancer tissues with or without transcatheter arterial embolization and establishment of a cell strain.

More epithelial-like cells are found in primary cultures of transcathetel arterial embolization (TAE)-untreated human liver cancer tissues than in those of TAE-treated tissues. A new cell strain, HUH-33, established from the former, grows slowly and is untransplantable into nude mice and secretes alpha-fetoprotein, albumin and some other tumor markers. Chromosome numbers are widely distributed. HUH-33 expresses hepatocyte type keratin and contains desmosome- or intermediate filament bundle-like structures.