Y. Ha

High Dietary Inorganic Phosphate Increases Lung Tumorigenesis and Alters Akt Signaling

RATIONALE Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis. OBJECTIVES The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development. METHODS Experiments were performed on 5-week-old male K-ras(LA1) lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods. MEASUREMENT AND MAIN RESULTS A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-ras(LA1) mice. CONCLUSIONS Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.

Toxicity and clearance of intratracheally administered multiwalled carbon nanotubes from murine lung.

Carbon nanotubes (CNT) are known to have widespread industrial applications; however, several reports indicated that these compounds may be associated with adverse effects in humans. In this study, multiwalled carbon nanotubes were administered to murine lungs intratracheally to determine whether acute and chronic pulmonary toxicity occurred. In particular, pristine multiwalled carbon nanotubes (PMWCNT) and acid-treated multiwalled carbon nanotubes (TMWCNT) were used in this study. In broncheoalveolar lavage fluid (BALF) cell analysis, PMWCNT induced more severe acute inflammatory cell recruitment than TMWCNT. Histopathologically, both PMWCNT and TMWCNT induced multifocal inflammatory granulomas in a dose-dependent manner. The observed granulomas were reversible, with TMWCNT-induced granulomas diminishing faster than PMWCNT-induced granulomas. Although the area of granuloma reduced with time, hyperplasia and dysplastic characteristics such as mitotic figures, anisokaryosis, and anisocytosis were still observed. These findings demonstrate that MWCNT induces granulomatous inflammation, and the duration and pattern of inflammation seem to vary depending upon the types of MWCNT to which mice are exposed. Therefore, toxicity studies on various types of CNT are needed as the responsiveness to these compounds differs.

Outbreak of salmonellosis in pigs with postweaning multisystemic wasting syndrome

POSTWEANING multisystemic wasting syndrome (PMWS) is an infectious viral disease cause by porcine circovirus type 2 (PCV-2) (Allan and Ellis 2000). It is characterised by progressive weight loss, respiratory signs and jaundice (Clark 1997, Chae 2004). The disease occurs in herds that are usually otherwise in good health. PMWS is endemic in many pigproducing countries and continues to be a major cause of wasting disease in pigs. In the Republic of Korea, PMWS is commonly associated with respiratory diseases (Kim and others 2003). However, secondary infections with bacterial pathogens, particularly Salmonella Typhimurium and Salmonella Choleraesuis, typically occur concurrently with PMWS in many herds. Most herds undergo severe devastating diarrhoea. This short communication describes an outbreak of salmonellosis in pigs in herds with PMWS in the Republic of Korea. Between January 2003 and April 2004, there were 37 cases of severe diarrhoea and retardation of growth from 24 pig farms. Twenty-two cases were from 2003 and 15 cases occurred in 2004. All 37 cases were weaned pigs, aged from 28 to 63 days. Morbidity was 10 to 20 per cent but mortality was 70 to 80 per cent in all of the herds affected. The diarrhoea was initially yellowish in colour but progressed to black; antibiotic therapy was ineffective in treatment of the diarrhoea in all cases. Postmortem examinations were performed on the 37 pigs and samples of lung, liver, kidney, small and large intestines, spleen, tonsil, and inguinal lymph nodes were collected in neutral buffered formalin. Small and large intestines from all of the pigs were examined for viral pathogens such as PCV-2 and classical swine fever (CSF) virus, and for bacterial pathogens such as Salmonella species, Brachyspira pilosicoli and Brachyspira hyodysenteriae. Homogenates of the small and large intestines were inoculated into cultures of PCV-free porcine kidney 15 cells, MARC-145 cells, swine testicular cells, primary porcine fallopian tube cells and Vero cells. In situ hybridisation and immunohistochemistry were performed for PCV-2 and CSF virus, respectively, as described by Kim and Chae (2001) and Choi and Chae (2003). Positive and negative control tissues of each virus were included for each in situ hybridisation and immunohistochemistry procedure (Kim and Chae 2001, Choi and Chae 2003). PCR was carried out as previously described for Lawsonia intracellularis, B pilosicoli, B hyodysenteriae and salmonellae in porcine intestinal specimens (Elder and others 1997, Kim and others 1998, Choi and others 2002). The most consistent and predominant histopathological feature seen was lymphoid depletion in the lymphoid follicle and the paracortical zone of the lymph nodes, with the lymphoid follicles and the paracortical zone being replaced by fibrovascular stroma and occasionally macrophages. Multinucleated giant cells were scattered between stromal components, and grape-like clusters of intensely basophilic intracytoplasmic inclusion bodies appeared in these areas (Fig 1). Depletion of the medullary cords and empty medullary sinuses were also observed. A strong hybridisation signal for PCV-2 was detected in the cytoplasm of macrophages in lymph nodes, spleen and Peyer’s patches (Fig 2) in all 37 pigs tested. In the colon, necrosis frequently extended to involve the lamina propria (Fig 3), submucosa, muscularis mucosa and lymphoid follicles. Necrotic tissues were separated from the healthy mucosa by a marginal zone of leucocytes, and the deeper tissues were heavily infiltrated by neutrophils. The lamina propria and submucosa contained numerous neutrophils and macrophages. Salmonellae were detected in the small and large intestines from all 37 cases by PCR. Three pigs were also positive for L intracellularis and two pigs were also positive for B pilosicoli. B hyodysenteriae was not detected in the small and large intestines by PCR. S Typhimurium was isolated from 23 pigs and S Choleraesuis was isolated from two pigs. No other bacterium was isolated from the small and large intestines. Cytopathic viruses were not detected after five passages.

Expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 in lungs from pigs experimentally infected with Actinobacillus pleuropneumoniae.

The expression of mRNA encoding interleukin (IL)-10, IL-12p35 and IL-12p40 was studied, by reverse transcription–polymerase chain reaction and by in situ hybridization with a non-radioactive digoxigenin-labelled cDNA probe, in formalin-fixed, paraffin-wax-embedded lung tissue from pigs experimentally infected withActinobacillus pleuropneumoniae. Forty-eight 7-week-old colostrum-deprived pigs were randomly allocated to infected (n = 24) or control (n = 24) groups. Three pigs from each group were euthanized at 3, 6, 9, 12, 24, 36, 48 and 60 h post inoculation (hpi). IL-10 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-10 increasing at 36 hpi. IL-12p35 mRNA was detected in the lung at 3 hpi, numbers of cells positive for IL-12p35 increasing at 36 and 48 hpi and rapidly decreasing thereafter whereas IL-12p40 mRNA was constitutively expressed at low levels during the experiment. Hybridization signals for IL-10, IL-12p35 and IL-12p40 were always associated with inflammation, in particular with macrophages and neutrophils within alveolar spaces. Expression of these cytokines was minimal in non-lesional lung ofA. pleuropneumoniae-infected pigs and in normal lung from control pigs. In situ hybridization ofA. pleuropneumoniae and these cytokines in serial sections of lung tissues indicated close co-localization ofA. pleuropneumoniae and these cytokines in pleuropneumonia. The results suggest that the expression of IL-10 and IL-12 play a role in pathogenesis ofA. pleuropneumoniae infection.

Lack of evidence of porcine circovirus type 1 and type 2 infection in piglets with congenital tremors in Korea.

Chae (2003). Positive and negative control tissues for each virus were included in each in situ hybridisation procedure (Kim and Chae 2001, Choi and Chae 2003). On microscopic examination of the tissues, mild to moderate cerebellar and spinal hypomyelination was detected in 33 of the 36 piglets. Neither PCV-1 nor PCV-2 nucleic acid was detected in any brain or spinal cord samples from the 36 piglets by in situ hybridisation; hybridisation signals of PCV-1 were seen occasionally in liver tissue from one piglet and lymph node from another piglet. PCV-2 DNA was also detected occasionally in the liver and spleen in one piglet, and in the spleen and lymph node in two other piglets with congenital tremor. No piglet had hybridisation signals of both PCV-1 and PCV-2. The granulomatous inflammatory reaction that is typical of lesions associated with PCV-2 infection in pigs with PMWS was not observed in the liver or lymph node from the PCV-1or PCV-2-positive piglets. CSF virus was not detected in any tissue of any of the piglets by in situ hybridisation. All the tests for PCV-1, PCV-2 and other cytopathic viruses were negative by the virus isolation procedure. In the present study of 36 cases of congenital tremor in piglets in Korea, no evidence of PCV-1 or PCV-2 nucleic acid was found in the central nervous system by in situ hybridisation. PCV-1 and PCV-2 were detected in non-neuronal tissues, such as the liver, spleen and lymph node, in two and three piglets, respectively. However, the identification of PCV-2 may not be significant, since PCV-2 can be detected in the lymph nodes of normal pigs by PCR and in situ hybridisation (Chae 2004). The detection of PCV-1 and PCV-2 in day-old piglets was interpreted as a consequence of infection in utero. However, it could not be ruled out that the piglets had been infected with PCV-1 or PCV-2 postnatally. The absence of CSF virus, the involvement of both male and female piglets, the absence of British Saddleback parentage and the lack of prenatal exposure to organophosphates excludes congenital tremor types A1, A3, A4 and A5, respectively. Therefore, the present cases with cerebellar and spinal hypomyelination may be classified as congenital tremor type A2; the two cases with no such histological lesions can be classified as congenital tremor type B. These results support those of researchers in Europe who failed to detect PCV infection in the central nervous system or non-neuronal tissues of piglets with congenital tremor (Kennedy and others 2003). These results are contradictory to those of a previous study, which found that PCV isolates from cases of congenital tremor type A2 can cause congenital tremor when inoculated into susceptible pregnant sows (Hines and Lukert 1994). Furthermore, PCV-2 was detected mainly in the brain and spinal cord from piglets with congenital tremor by Stevenson and others (2001). The apparent lack of consistency regarding the presence or absence of PCV infection in piglets with congenital tremor is difficult to explain. It is possible that a geographical difference exists in the involvement of PCV in congenital tremor; PCV may be associated with congenital tremor in pigs in the USA, but not in European and Asian countries. Further studies are therefore needed to determine the role of PCV in congenital tremor type A2.

Colostral transmission of porcine circovirus 2 (PCV-2): reproduction of post-weaning multisystemic wasting syndrome in pigs fed milk from PCV-2-infected sows with post-natal porcine parvovirus infection or immunostimulation.

Post-weaning multisystemic wasting syndrome (PMWS) was reproduced in pigs fed colostrum and milk from porcine circovirus 2 (PCV-2)-infected sows and infected post-natally with porcine parvovirus (PPV) or immunostimulated. Pregnant sows were inoculated intranasally with either PCV-2 (n=5) or PCV-2-free PK-15 cell lysates (control, n=10) 3 weeks before the expected farrowing date. Newborn piglets from five of the control sows were introduced to PCV-2-infected sows (n=6 for each sow) and allowed to feed on the colostrum for 12 h and then given 15 ml milk five times a day for 7 days. Newborn piglets from the other five control sows were fed colostrum and milk from their own sows. After 7 days, two piglets from each group were randomly selected to confirm PCV-2 infection. Twenty-one pigs fed by PCV-2-infected sows were randomly divided into three groups and subjected to post-natal PPV infection (group 1), immunostimulation (group 2) or no post-natal treatment (group 3). Twenty-one pigs fed by uninfected sows were also randomly divided and subjected to post-natal PCV-2 and PPV infection (group 4), post-natal PCV-2 infection (group 5) or no treatment (group 6, negative control). Body weight was significantly greater in group 6 than in groups 1, 2 and 4 at 49, 52, 56, 59 and 63 days of age. The typical granulomatous inflammatory reaction and lymphoid depletion of PMWS was observed in the lymph nodes of groups 1, 2 and 4 at 63 days of age. Group 3 had significantly fewer PCV-2-positive cells than groups 1, 2 and 4. In conclusion, PCV-2 shed from colostrum and milk is infectious and reproduces PMWS with post-natal PPV infection or immune stimulation.

Identification of porcine circovirus type 2 in retrospective cases of pigs naturally infected with porcine epidemic diarrhoea virus

Abstract The identification of porcine circovirus type 2 (PCV2) was studied in fresh intestinal tissues by polymerase chain reaction (PCR) and in formalin-fixed, paraffin-wax-embedded intestinal tissues by in situ hybridisation. The tissues came from pigs naturally infected with porcine epidemic diarrhoea virus (PEDV). A total of 35 (32.7%) of 107 small intestinal samples from pigs naturally infected with PEDV were found to be positive using PCR. Positive signals for PCV2 were detected in 32 (29.9%) of 107 small intestinal samples from pigs naturally infected with PEDV by in situ hybridisation. The distribution of positive cells in the jejunum and ileum was multifocal or patchy. Distinct positive labelling was found throughout the lamina propria in the small intestines. The results of this study indicate that PCV2 is highly prevalent in pigs naturally infected with PEDV.

Low dietary inorganic phosphate stimulates lung tumorigenesis through altering protein translation and cell cycle in K-ras(LA1) mice.

Recent surveys indicate that Pi intake has increased steadily as Pi-containing foods have increased. Our previous study demonstrated that high dietary Pi strongly stimulated lung tumorigeneis. In order to answer the issue whether low Pi may be chemopreventive, we examined the effects of low Pi on lung cancer. Eighteen 5-wk-old male K-ras LA1 lung cancer model mice were randomly allocated to 2 groups. One group was fed a normal diet (0.5% Pi) and other group was fed low Pi (0.1% Pi) diet for 4 wk. Lung cancer development was evaluated by histopathological examination, Western blot, kinase assay, and immunohistochemistry. Low Pi increased the expression of sodium-dependent phosphate co-transporter 2b, and activated Akt signal with decreased PTEN expression in the lungs of K-ras LA1 mice. Low Pi increased the Akt/mTOR-mediated protein translation through upregulating the phosphorylation of p70S6K and 4E-BP1. In addition, low Pi stimulated cell cycling as evidenced by altered cell cycle regulators such as cyclin D1 and D3. Finally, low Pi increased lung tumorigenesis in K-ras LA1 mice compared to the normal diet group. Our results clearly demonstrated that low Pi also promoted lung tumorigenesis, thus suggesting that an appropriate intake of dietary Pi may be critical for lung cancer prevention as well as treatment.

Tellurium recovery from cemented tellurium with minimum waste disposal

Abstract A waste minimization hydrometallurgical process with near zero-discharge has been developed for the treatment of cemented tellurium from copper refining. Tellurium is recovered by a series of leaching, precipitation and electrowinning steps, and the barren electrolyte is recycled to the leaching step of fresh cemented tellurium feed with a replenished NaOH solution. In the leaching step, more than 95% of tellurium is selectively extracted in the form of TeO32− within 10 min at 80°C and 200 g/l pulp density. The leach residue is leached with H2SO4 solution and can be sent to the existing Cu electrowinning circuit. In the next step, most of the remaining Cu and Pb impurities are precipitated with Na2S. Then, tellurium of 99.9% purity is electrowon from the purified pregnant solution, leaving most of Se and As behind in the solution. Impurity levels of Se and As can be lowered by the addition of hydrazine hydrate at every fifth cycle, and the purified NaOH solution is recirculated to the leaching step.

Pathogenesis and Chronologic Localization of the Human Influenza A (H1N1) Virus in Cotton Rats.

Objectives We aimed to evaluate the pathogenesis and chronologic localization of human influenza A (H1N1) virus in experimentally infected cotton rats. Methods The animals were intranasally inoculated with 107 plaque-forming units of A/Solomon Islands/3/2006 (H1N1) influenza virus and evaluated for pathogenicity for a period of 28 days. Virus replication kinetics and pathological properties were assessed chronologically. Acute antiviral responses were evaluated by mean of real-time polymerase chain reaction. Results Cotton rats infected with A/Solomon Islands/3/2006 virus lost weight until 6 days post-inoculation (DPI) and showed decreased activity until 3 DPI. At necropsy, focal areas of redness and consolidation of lungs were evident at 1, 2, and 3 DPI. Lung histopathology showed moderate to severe interstitial pneumonia, alveolitis and bronchiolitis. Influenza A specific viral protein was detected in bronchiolar epithelial cells, alveolar septa and pneumocytes. Influenza viruses were recovered from the lungs during the early period of infection and the titer peaked at 1 DPI. Viral proteins were detected from 4 hours to 6 hours DPI. These trends correlate with the up-regulation of mRNA expression of the IFN-α, Mx1, and Mx2 genes that play critical roles in the anti-influenza response at the early stage of infection. Conclusion Our results provide evidence that supports the use of cotton rats for the study of influenza virus pathogenesis and the immune response.