Diagnosis And Innovative Therapy Of Human Specific Enterocytozoon Bieneusi Genotype D Strain In Falcons (Falconiformes)
MÜLLER MG1
Abu Dhabi Falcon Hospital, Abu Dhabi, United Arab Emirates
Background: Out of 137 falcons of the same group, 24 falcons died from clinically unidentified abscesses in intestines, liver and kidneys within 6 weeks. 70 more falcons suffered from the same disease. Aims: 1) To identify the causative agent. 2) To establish an appropriate treatment regime and to reduce mortality rate of the sick falcons.
Methods: This study included 137 falcons of 5 breeds. Blood hematology and biochemistry, parasitology, radiography and endoscopy were performed in all birds as well as virological and serological tests. Necropsy, histopathology and immunohistochemistry of the dead falcons was performed. 6 liver and intestine samples were tested by universal diagnostic PCR for microsporidia followed by a second, species specific, PCR for confirmation of E. bieneusi. Gyr-Hybrid falcons revealed a higher morbidity rate of 59.2%. Intestinal abscesses were found in up to 50% of the sick birds. All falcons were treated with Dimetronidazole (Emtryl®) 50 mg/kg SID po for 10 days with 3-4 repetitions up to maximum 40 days. Individual treatment plans included the application of Nux vomica® 1ml/kg SID po and Mucosa compositum® 1ml/kg SID po in case of intestinal abscesses, Hepar compositum® 1ml/kg SID po and Legalon® 1 tab BID for 5 days for treatment of liver abscesses, and in case of kidney abscesses Berberis compositum® 1ml/kg SID po and Cantharis compositum® 1ml/kg SID po. Treatment progress was monitored through repeated endoscopies in 2-weeks intervals.
Results: After 5 months, the PCR tests revealed as causative agent the presence of Enterocytozoon bieneusi belonging to genotype D identical to the AF101200 strain (human). In total, 24 falcons died shortly after the initial examinations due to advanced disease stages. All other falcons responded well to the individual treatment plan. Repeated endoscopic examinations showed the continuous regression of the abscesses up to their full disappearance and complete recovery of the patients.
Conclusions: 1) Microsporidiosis caused by Enterocytozoon bieneusi as new parasitic disease in falcons. 2) Identification of Enterocytozoon bieneusi Genotype D Strain for the first time in raptors and falcons might indicate its possible zoonotic potential. 2) Individual treatment plans with Dimetronidazole supported by homeopathic medicines led to a full regression and survival of the diseased falcons.
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Alveolar Echinococcosis: The Impact Of Chemotherapy And Surgery On Survival Authors
MÜLLHAUPT B1,2, TORGERSON PR3, DEPLAZES P3, AMMANN RW4
1Gastroenterology and Hepatology and 2Swiss HBP Center University Hospital of Zurich, Zurich, Switzerland, 3Institute of Parasitology, University of Zurich, Zurich, Switzerland
Background/Aims: Alveolar echinococcosis (AE) is a serious liver disease. We have recently analyzed the long term prognosis of AE-patients, the burden of this disease in Switzerland and the cost effectiveness of treatment.
Methods: Relative survival analysis was undertaken using a national database with 329 patient records. 155 representative cases had sufficient details regarding treatment costs and patient outcome to estimate the financial implications and treatment costs of AE.
Results: For an average 54-year-old patients diagnosed with AE in 1970 the life expectancy was estimated to be reduced by 18.2 and 21.3 years for men and women, respectively. By 2005 this was reduced to approximately 3.5 and 2.6 years, respectively. Patients undergoing radical surgery had a better outcome, whereas older patients had a poorer prognosis than younger patients. Costs amount to approximately ?108,762 per patient. Assuming the improved life expectancy of AE patients is due to modern treatment the cost per DALY saved is approximately ? 6032.
Conclusions: Current treatments have substantially improved the prognosis of AE patients compared to the 1970s. The cost per DALY saved is low compared to the average national annual income. Hence, AE treatment is highly cost effective in Switzerland.
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Calcium Signaling And Angiogenesis
MUNARON L, FIORIO PLA A
Department of Animal and Human Biology, Nanostructured Interfaces and Surfaces Centre of Excellence (NIS), and Center for Complex Systems in Molecular Biology and Medicine – SysBioM, University of Torino, Italy
Background: The proliferation and motility of vascular endothelial cells (EC) are critical steps in angiogenesis and are strictly controlled by different extracellular signals. Proangiogenic factors (VEGF, bFGF) generate cytosolic calcium rises through calcium entry from extracellular medium: this event is due to the opening of calcium-permable channels in the plasmamembrane, mainly activated by arachidonic acid (AA) and nitric oxide (NO). Even if some calcium entry blockers are under clinical trial at present with encouraging results, a better knowledge about the molecular nature of calcium channels and their intracellular regulation could lead to new and more specific strategies in therapeutical approach to cancer progression and angiogenesis.
Methods: Single cell calcium measurements by fluorescent probes, patch clamp, tubulogenesis assay, wound healing, time lapse, PCR, siRNA, proteomics.
Results: Here we describe proangiogenic intracellular calcium signals in endothelial cells derived from human breast carcinoma (B-TEC). AA, released upon EC stimulation with VEGF or FGF, promotes B-TEC proliferation, migration and organization of vessel-like structures in vitro. AA induces Ca2+ entry in the entire capillary–like structure during the early phases of tubulogenesis in vitro: no such responses are detectable in B-TECs organized in more structured tubules. An inhibitor of Ca2+ entry and angiogenesis, Carboxyamidotriazole (CAI), significantly and specifically decreases AA-induced B-TECs tubulogenesis, as well as AA-induced Ca2+ signals in B-TECs. Finally, preliminary results suggest that at least part of AA-dependent calcium entry could be due to the opening of TRPV4, a well known calcium-permeable channel.
Conclusions: 1) AA-activated Ca2+ entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation, motility and tubulogenesis, and it is downregulated during the reorganization of tumor-derived endothelial cells in capillary-like structures, 2) inhibition of AA-induced Ca2+ entry may contribute to the antiangiogenic action of CAI.
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The Antitumor Action Of Neurokinin-1 Receptor Antagonists As New “Magic Bullets” Called “Intelligent Bullets”
MUÑOZ M, ROSSO M
“Virgen del Rocío“University Children’s Hospital, Seville, Spain
Background: In the 21st century, the era of “molecularly targeted” anticancer therapy, the Paul Ehrlich’s concept of the “Magic Bullets” for cancer cells, can be useful for neurokinin-1(NK-1) receptor antagonists. Substance P (SP) is a neuropeptide belong to tachykinin family, SP after binding to the NK-1 receptor regulates many biological functions and it is implicated in behaviour emotional, inflammation, pain, mitogenesis, angiogenesis and migration of tumour cells. NK-1 receptor antagonists (L-733,060/L-732,138) were used in the treatment of inflammatory diseases; produce analgesia, antidepressive, anxiolytic and antiemetic effects in vivo studies. We have carried out in vitro studies of the growth inhibition capacity of these NK-1 receptor antagonists against glioma, neuroblastoma, melanoma, retinoblastoma, pancreas, larynx and gastrointestinal carcinoma cell lines.
Methods: Coulter counter was used to determine viable cell numbers followed by application of the tetrazolium compound MTS. An immunoblot analysis was used to determine the NK-1 receptors, and the DAPI staining method was applied to demonstrate apoptosis.
Results: We have demonstrated the presence of several NK-1 receptor isoforms in all human tumor cells studies. Nanomolar concentrations of SP increased the growth of all cell lines and micromolar concentrations of L-733,060/L-732,138 inhibited the growth of such cell lines in a dose-dependent manner, with and without previous administration of SP. After administration of L-733,060/L-732,138 apoptosis was observed in all tumor cell lines studies.
Conclusions: We demonstrated that: 1) The NK-1receptors were expressed in all these tumor cell lines, 2) SP is a potent mitogen in these tumor cell line, 3) the antitumor action of the NK-1 receptor antagonists L-733,060/ L-732,138 against these human tumor cell lines occurs through the NK-1 receptor and 4) the cell death is apoptosis pathway. This new findings suggests that the NK-1 receptor antagonists are a new and promising antineoplastic agents, called “Intelligent Bullets” concept goes beyond that “Magic Bullets” are to attack the tumour cells invaders antitumor action, but in addition, in the host they present/display beneficial effects such as: anti-inflammatory, analgesic, anxyolitic, antidepressant, antiemetic, hepatoprotector and neuroprotector.
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Antibiotic Resistance Of Psychrotrophs Spoiling Raw Milk
MUNSCH-ALATOSSAVA P, ALATOSSAVA T
University of Helsinki, Dept. of Food Technology, Helsinki, Finland
Background: Prolonged cold storage of raw milk favors the growth of psychrotrophic bacteria, which produce heat-resistant exoenzymes of considerable spoilage potential. We evaluated the distribution of antibiotic resistant traits among isolates originating from conventional dairy farming sytems, and compared then isolates from organic and conventionally managed farms.
Methods: Two studies were performed: 1) 60 isolates originating from farms, lorries, and silos (conventional farming) were analysed with ATB®PSE strips (BioMérieux, France); 2) The susceptibility tests were performed for 36 isolates from 6 samples (conventional farming), and for 43 isolates from 9 samples (organic farming) with 6 antibiotics in discs (Ceftazidim, Ciprofloxacin, Gentamycin, Imipenem, Minocycline, Trimethoprim+ Sulfamethoxazole).
Results: 1) Study 1: The ATB®PSE strips (designed to investigate the pseudomonal susceptibility/resistance), revealed that many psychrotrophs (ascribed to Pseudomonas, Stenotrophomonas, Acinetobacter, Burkholderia genera), besides exhibiting spoilage features were also multiresistant. Respectively, 42.3 %, 52.9 % and 94.1 % of the isolates, retrieved from farm, lorry and silo tanks, presented resistance to at least three classes of antibiotics (Microbiol. Res. 162, (2007), 115-123).
2) Study 2: The isolates retrieved from milk, from conventional dairy farms, expressed higher prevalence of resistance to 4 of the 6 considered antibiotics; contrarily to gentamycin and trimethoprim-sulfamethoxazole, for which both categories of isolates presented similar resistance frequencies.
Conclusions: The observations from Study 1 suggest an accumulation of antibiotic multiresistant traits among psychrotrophs along the cold chain of raw milk storage and transportation. In Study 2, dairy management practices seem also to affect the level of susceptibility of psychrotrophs to antimicrobial agents. However, more milk samples have to be analysed to confirm this preliminary data. The question of whether psychrotrophs spoiling raw milk may play a role as reservoir for antibiotic resistance genes needs to be considered. In our recent studies, we showed at laboratory scale, that flushing raw milk with pure N2 gas constitutes an interesting perspective for limiting the spoilage and pathogenic potential of psychrotrophs; we believe the treatment may be of interest to control the antibiotic resistance potential of raw milk psychrotrophs as well.
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Optimized Productions Of Recombinant Human Proteins, An Enzyme And Two Cytokines, In Fermentor Cultures Of The Yeast, Pichia Pastoris
MURASUGI A
Product Development Department, Meiji Dairies Corporation, Tokyo, Japan
Background: The expression system of methylotrophic yeast, Pichia pastoris, is now widely used in order to express various proteins in host cells or to secrete them into the culture medium. By using this system, we tried to express three human proteins, bile-salt stimulated lipase (BSSL), and two cytokines, midkine (MK) and pleiotrophin (PTN), in high cell density fermentor cultures of Pichia pastoris to obtain the active or intact proteins for the investigations of these proteins.
Methods: Fermentation was started with 3.5 L medium in a 10 L vessel, and it was performed in various conditions. The amount of BSSL was determined by its lipase activity. The amounts of MK and PTN were determined by liquid chromatography analyses. The cell proliferation activity of MK was assayed using NIH3T3 cells.
Results: In the expression of BSSL, yeast invertase secretion signal was used. When the BSSL expression was induced by methanol at low cell density in the fermentor culture, almost no enzyme was accumulated in the medium. Therefore, the time of induction, and then the cell growth condition were studied. Finally, approximately 1 g/L BSSL was accumulated in the medium. In the expression of MK, when its own secretion signal was used, a half of secreted MK received yeast specific mannosylations. Thus, yeast α-mating factor (α-MF) secretion signal was used. MK secreted in the medium received no mannosylation, and the amount was 640 mg/L. However, about 70% of the product was truncated. Therefore, pep4 Pichia pastoris host was used, and MK was expressed at 20°C and at pH 3. At the end of one week induction, 360 mg/L of authentic MK was obtained. For the expression of PTN, α-MF secretion signal was also used, and the PTN was expressed in a fermentor at 20°C and at pH 5. At forth day after induction, 260 mg/L of intact PTN was obtained.
Conclusion: The expression system of Pichia pastoris is powerful one for the production of therapeutic proteins. As described above, three human proteins were successfully expressed in their suitable conditions, respectively. For the industrial production of recombinant therapeutic proteins, optimization of the fermentor production would sometimes be an indispensable and laborious work. Optimization processes described in detail at the meeting will be helpful for the fermentor expressions of other proteins.
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Citrate Transport Mechanism In Prostate And Its Changes In Malignant Transformation. Implications For Fatty Acid Synthesis In Cancer
MYCIELSKA ME, DJAMGOZ MBA
Division of Cell and Molecular Biology, Neuroscience Solutions to Cancer Research Group, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London, UK
Background: Prostate is a unique organ that produces, stores and releases large amounts of citrate. Excess citrate accumulation is possible because of a unique regulation of mitochondrial aconitase (m-ACNT). The level of citrate in prostatic lumen can reach 180 mM. Importantly, citrate level drops significantly when prostate becomes metastatic.
Methods: The present study was conducted on two human prostate cell lines: normal epithelial PNT2-C2 and strongly metastatic PC-3M. Citrate transport mechanisms were characterised using whole-cell patch clamp. Citrate metabolism in cancer cells was studied using spectrophotometric methods, real-time PCR and adhesion, motility and membrane secretory assays.
Results: Normal human prostate PNT2-C2 cells expressed a K+-dependent citrate transport mechanism designed primarily to release citrate into the lumen. It was electrogenic with the estimated stoichiometry of 1 cit3- : 4 K+. Strongly metastatic prostate PC-3M cells were shown to express not only the same K+-dependent citrate release mechanism but also electrogenic, Na+-dependent citrate uptake mechanism. Citrate preincubation of prostate cells resulted in the increased metastatic cell behaviour in cancer cells but had no effect on normal PNT2-C2. Extracellular citrate in concentrations as low as 50 M was sufficient to increase free fatty acids (FFA) synthesis in cancer cells. FA synthesis could be also decreased by (1) inhibiting fatty acid synthase (FAS) with cerulenin or (2) NADPH production through c-aconitase (c-ACNT)/c-isocitrate dehydrogenase (c-ICD) with oxalomalate. Intracellular Fe chelator (inhibiting c-ACNT activity) also reduced FA synthesis.
Conclusions: 1. Citrate release mechanism in prostate is electrogenic and K+-dependent. 2. Prostate cancer cells express additionally Na+-dependent uptake mechanism. 3. Extracellular citrate in low concentrations can be taken-up by cancer cells and increase their FA synthesis. 4. In cancer, NADPH for FA synthesis is supplied mainly by c-ACNT/cICD. 5. c-ACNT is overexpressed and upregulated in cancer through intracellular high levels of Fe.
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