Parasitologische und molekularbiologische Charakterisierung Isometamidium- empfindlicher und -resistenter Trypanosoma congolense und T. brucei brucei -Isolate aus Rindern Ost - und Westafrikas

Deckblatt-Impressum persönlicher Dank Contents Abbreviations Introduction Objectives Literature Review Material and Methods Results Discussion Conclusions Summary Zusammenfassung References Annexes Acknowledgements Curriculum Vitae SelbständigkeitserklärungResistance to isometamidium is a serious problem in many parts of sub-Saharan Africa. Several tests have been described for the detection of drug resistance in trypanosomes, but the techniques currently used suffer from a number of drawbacks. Therefore, faster, more sensitive and more reliable methods are required. The main objectives of the current studies were a) assessing the isometamidium sensitivities of Trypanosoma congolense clones derived from trypanosome stocks from naturally infected cattle in East and West Africa, b) inducing isometamidium resistance in a drug sensitive T. congolense clone, and c) characterising the putatively resistance-related nucleoside transporter gene (TbAT1) in isometamidium-sensitive and -resistant T. brucei brucei and T. congolense. Trypanosome clones were derived from T. congolense stocks collected from cattle in Ethiopia and Burkina Faso with different isometamidium sensitivity phenotypes. An overall cloning success rate of 20.30% was achieved, which ranged from 16.67 to 25.00% for the different stocks. The isometamidium sensitivities of the clones were assessed to multiple dosages of isometamidium in mice. The results demonstrated that all the T. congolense clones expressed high levels of resistance to isometamidium when compared to known isometamidium-sensitive T. congolense reference clones. The clones from Burkina Faso expressed significantly (p<0.05) higher levels of resistance in mice than the clones from Ethiopia. Analyses of variances of the mean relapse intervals showed that for most of the clones tested there were clear relationships between the time of relapses and the doses of isometamidium used; mice treated with lower doses relapsed after a shorter time than mice treated with higher doses (p=0.001). The CD50 values for the clones from Ethiopia ranged from 9.86 to 13.37 mg/kg bw, whereas, the clones from Burkina Faso had CD50 values ranging from 19.80 to > 20.0 mg/kg bw. There was no significant (p>0.05) variation in expression of resistance to isometamidium among three of the clones derived from a single stock (PA 77) from Ethiopia. Similarly, two of the clones derived from a stock of Burkina Faso (SA 95) expressed a similar (p>0.05) level of resistance to isometamidium. With the aim of understanding the developmental mechanism of resistance in trypanosomes, clones of T. congolense with different level of resistance to isometamidium were derived from a drug sensitive clone in mice. The levels of resistance of IL 2642 clone to isometamidium was increased at least 159-fold (from a CD50 of 0.0086 to 1.37 mg/kg bw) by repeated subcurative treatment of infected mice over a period of 16 months. This was associated with 2.2-fold increase (from a CD50 of 8.20 to 18.02 mg/kg bw) in resistance to diminazene aceturate, tested in mice. The results indicate that administering drugs below effective levels can produce drug resistance. The high level of resistance developed in immunosuppressed mice in the current study was stable and persisted when the clones were subsequently tested in immunocompetent mice. However, similar drug doses and similar protocols in normal immunocompetent mice failed to lead to development of isometamidium resistance in the IL 2642 clone. This shows that immunosuppression of the host may considerably reduce the efficacy of trypanocidal drugs and can lead to the rapid development of drug resistance. Analyses were made on the TbAT1 gene, which encodes for the P2 transporter, from T. b. brucei field stocks to investigate a possible link between the presence of mutations in this gene and isometamidium resistance. We have analysed the TbAT1 gene of T. b. brucei from 11 isometamidium-sensitive field stocks, two sensitive reference clones and two resistant reference clones. Sequence alignment showed that the isometamidium-sensitive T. b. brucei contained the wild-type sequence patterns. In contrast, the isometamidium-resistant T. b. brucei stocks showed the mutant-type sequence patterns that corresponded to the DNA sequence of the laboratory-derived melarsoprol-resistant STIB 777R stock. Six point mutations were detected in the isometamidium-resistant stocks, which were also described earlier in the laboratory-derived melarsoprol-resistant stock of T. brucei. Four of these nucleotide differences detected lead to amino acid substitutions: Ala178 -&gt; Thr (A178T), Gly181-&gt; Glu (Gl181E), Asp239-&gt; Gly (D239G) and Asn286-&gt; Ser (N286S). Furthermore, deletions of three nucleotides (TTC) that encode the amino acid phenylalanine were detected in the resistant stocks. The point mutation that led to the substitution of G by A at nucleotide position 532 (G532A) in the sensitive stock eliminated the Sfa NI restriction site. Whereas, the mutation at 857 bp that led to the substitution of A by G (A857G) generated a new Sfa NI restriction site. Consequently, in order to analyse the RFLP pattern of a fragment of TbAT1 (nucleotides 430-1108), the 677 bp PCR products from eight of the isometamidium-sensitive and two of the isometamidium-resistant T. b. brucei were subjected to digestion with Sfa NI. The results revealed two different banding patterns: the digest produced fragment sizes of 566 and 111 bp in the case of TbAT1 from isometamidium-sensitive stocks, whereas it produced fragment sizes of 435 and 242 bp in the case of TbAT1 from isometamidium- resistant stocks. Thus, the isometamidium-sensitive and resistant T. b. brucei could be successfully distinguished by digestion with the restriction endonuclease Sfa NI. It can therefore be concluded that there is a link between the presence of mutations in the nucleotide transporter gene (TbAT1) in T. b. brucei and isometamidium resistance. Furthermore, Sfa NI-RFLP, if validated with a large scale screening of field isolates, may serve as a convenient diagnostic tool for rapid identification of isometamidium-resistant T. b. brucei. The attempts made to amplify the TbAT1 homologous gene from the genomic DNA of T. congolense failed.Die Resistenz gegen Isometamidium ist in vielen südlich der Sahara gelegenen Regionen Afrikas ein ernstzunehmendes Problem. Zum Nachweis von Arzneimittel- Resistenz bei Trypanosomen sind mehrere Methoden beschrieben worden, allerdings haben die zurzeit angewendeten Techniken eine Reihe von Nachteilen. Daher ist der Bedarf an sensitiveren und verlässlicheren Methoden groß. Ziel der Studie war a) die Prüfung der Isometamidium Empfindlichkeit von Trypanosoma congolense Klonen, die aus Populationen von natürlich infizierten Rindern aus West- und Ostafrika stammten, b) die Resistenz gegen Isometamidium in einem sensiblen T. congolense Klon zu erzeugen und c) die Charakterisierung des vermutlich Resistenzabhängigen Nukleosid Transportgens (TbAT1) in Isometamidium-sensiblen und resistenten T. brucei brucei und T. congolense. Die Trypanosomen Klone stammen von T. congolense Populationen von Rindern aus Äthiopien und Burkina Faso, die unterschiedliche Phänotypen bezüglich Isometamidium-Sensibilität aufwiesen. Insgesamt lag der Klonierungserfolg bei 20.3%, der bei den einzelnen Populationen von 16,67% bis 25,0% schwankte. Die Sensibilität der Klone gegen Isometamidium wurde mittels Dosierungsversuchen an Mäusen erprobt. Es zeigte sich, dass alle T. congolense Klone im Vergleich mit den sicher Isometamidium-sensiblen Referenzstämmen einen hohen Grad an Resistenz gegen Isometamidium aufwiesen. Die Klone aus Burkina Faso zeigten in Mäusen signifikant (p<0,05) stärkere Resistenz als die Klone aus Äthiopien. Mittels einer Varianzanalyse der mittleren Intervallzeiten bis zum Rückfall der Mäuse konnte bei den meisten getesteten Klonen ein eindeutiger Zusammenhang zwischen dem Zeitpunkt des Rückfalls und der verwendeten Isometamidium Dosierung gezeigt werden; die Mäuse, die mit einer niedrigeren Dosierung behandelt worden waren, erlitten früher einen Rückfall, als die, die eine höhere Dosis erhielten (p=0.001). Die CD50-Werte der äthiopischen Klone lagen im Bereich von 9,86 bis 13,37 mg/Kg KGW während die Klone aus Burkina Faso CD50-Werte von 19,80 bis > 20,0 mg/Kg KGW aufwiesen. Innerhalb von drei Klonen aus einer äthiopischen Population (PA 77) zeigten sich keine signifikanten Unterschiede im Resistenzgrad. Ebenso ähnelte sich die Ausprägung von Isometamidium Resistenz zweier Klone aus einer Population von Burkina Faso (p> 0,05). Um die Entwicklung des Resistenzmechanismus bei Trypanosomen besser zu verstehen, wurden mehrere T. congolense Klone mit unterschiedlichem Resistenzniveau aus einem Isometamidium-sensiblen Klon in der Maus gewonnen. Durch wiederholte Behandlung mit subtherapeutischer Dosierung über einen Zeitraum von 16 Monaten wurden der Resistenzgrad von Klon IL 2642 um mindestens das 159-fache gesteigert (von CD50= 0,0086 auf 1,37 mg/kg KGW). Dies war verbunden mit einem 2,2 fachen Anstieg einer an Mäusen getesteten Diminazen-Resistenz (von CD50= 8,20 auf 18,2 mg/Kg KGW). Die Ergebnisse deuten darauf hin, dass mit einer Verabreichung subtherapeutischer Dosierung Resistenzen gegen Medikamente hervorgerufen werden können. Der hier in immunsupprimierten Mäusen erzeugte hohe Resistenzgrad war stabil und erwies sich auch bei im Folgenden mit diesen Klonen infizierten immunkompetenten Mäusen als dauerhaft. Allerdings konnte mit ähnlichem Dosierungsschema und Studienprotokoll bei normalen immunkompetenten Mäusen keine Isometamidium- Resistenz bei dem Klon IL 2642 hervorgerufen werden. Dies zeigt, dass eine Immunsuppression des Wirts die Wirksamkeit von trypanoziden Wirkstoffen beträchtlich herabsetzen und so zu einer schnellen Entwicklung von Resistenz beitragen kann. Um einen möglichen Zusammenhang zwischen dem Auftreten von Genmutationen und Isometamidium-Resistenz zu finden, wurde das TbAT1 Gen des T. b. brucei Feldstammes analysiert, welches für den P2 Transporter kodiert. Insgesamt wurde das TbAT1 Gen von T. b. brucei von 11 Isometamidium-sensiblen Feldstämmen sowie von zwei sensiblen und zwei resistenten Referenzklonen analysiert. Im Sequenzvergleich zeigte der Isometamidium-sensible Stamm von T. b. brucei das Sequenzmuster des Wildtyps. Im Gegensatz dazu wies der Isometamidium-resistente Stamm von T. b .brucei ein mutiertes Sequenzmuster auf, welches mit der DNA Sequenz des experimentell gewonnenen Melarsoprol- resistenten Stammes STIB 777R übereinstimmte. In diesen Isometamidium- resistenten Stämmen konnten sechs Punktmutationen nachgewiesen werden, die auch schon bei dem experimentell gewonnenen Melarsoprol-resistenten Stamm STIB 777R gefunden worden waren. Vier dieser Nukleotid-Unterschiede führten zum Austausch einer Aminosäure: Ala178 -&gt; Thr (A178T), Gly181 -&gt; Glu (Gl181E), Asp239 -&gt; Gly (D239G) und Asn286 -&gt; Ser (N286S). Des Weiteren wurden bei den resistenten Stämmen drei Nukleotid-Deletionen (TTC) entdeckt, die für die Aminosäure Phenylalanin kodieren. Die Punktmutation, die im sensiblen Stamm an der Position 532 (G532A) zu einem Austausch von G durch A führte, zerstörte so die Schnittstelle für das Enzym Sfa NI. Im Gegensatz dazu generierte die Punktmutation an Position 857 bp, welche den Austausch von A durch G (A857G) verursachte, eine neue Schnittstelle für das Enzym Sfa NI. Im Folgenden wurden die 677 bp-großen PCR-Produkte von acht der Isometamidium- sensiblen und von zwei der Isometamidium-resistenten T. b. brucei mit Sfa NI verdaut, um das RFLP Muster des Fragments von TbAT1 (Nukleotid 430-1108) zu analysieren. Das Ergebnis enthüllte zwei verschiedene Bandmuster: Bei TbAT1 von Isometamidium-sensiblen Stämen ergab der Verdau Fragmente von 566 bp und 111 bp, während die Fragmente bei TbAT1 von Isometamidium-resistenten Stämmen bei 435 und 242 lagen. Demzufolge konnten Isometamidium-sensible und Isometamidium-resistente Stämme von T. b. brucei erfolgreich durch eine Restriktionsverdau mit Sfa Ni unterschieden werden. Daraus kann geschlossen werden, dass es einen Zusammenhang zwischen der Präsenz von Punktmutationen im Nukleotid Transportergen (TbAT1) von T. b. brucei und dem Auftreten von Isometamidium-Resistenz gibt. Des Weiteren ist der RFLP von Sfa NI nach einer Validierung mittels umfangreicher Untersuchungen von Feldisolaten möglicherweise eine geeignete Methode für eine schnelle Diagnose von Isometamidium-resistenten T. b. brucei. Der Versuch, das homologe TbAT1-Gen von T. congolense zu amplifizieren, blieb erfolglos

The BH3-only protein Bik/Blk/Nbk inhibits nuclear translocation of activated ERK1/2 to mediate IFNγ-induced cell death

IFNγ induces cell death in epithelial cells, but the mediator for this death pathway has not been identified. In this study, we find that expression of Bik/Blk/Nbk is increased in human airway epithelial cells (AECs [HAECs]) in response to IFNγ. Expression of Bik but not mutant BikL61G induces and loss of Bik suppresses IFNγ-induced cell death in HAECs. IFNγ treatment and Bik expression increase cathepsin B and D messenger RNA levels and reduce levels of phospho–extracellular regulated kinase 1/2 (ERK1/2) in the nuclei of bik+/+ compared with bik−/− murine AECs. Bik but not BikL61G interacts with and suppresses nuclear translocation of phospho-ERK1/2, and suppression of ERK1/2 activation inhibits IFNγ- and Bik-induced cell death. Furthermore, after prolonged exposure to allergen, hyperplastic epithelial cells persist longer, and nuclear phospho-ERK is more prevalent in airways of IFNγ−/− or bik−/− compared with wild-type mice. These results demonstrate that IFNγ requires Bik to suppress nuclear localization of phospho-ERK1/2 to channel cell death in AECs

Economic Contribution of Gum and Resin Resources to Household Livelihoods in Selected Regions and the National Economy of Ethiopia

Ethiopia has one of the largest dry forest and woodland resource bases in the Horn of Africa, predominated by diverse Acacia, Boswellia, Commiphora, and Sterculia species, with an estimated annual production potential of over 300,000 tonnes of commercial gums and resins. However, until recently, less than 1% of this potential has been tapped and traded while the resource bases are degrading fast. Shortage of locality-specific case studies typifying the state of gum and resin production and marketing systems and nationwide socio-economic significance of the resources has delayed development of value-added commercialization of the commodities and integrated management of the resource bases. A study aimed at exploring the value chain of traded gums and resins and their contribution to rural livelihood and national economy was conducted in 11 purposively selected localities in five National Regional States within the major gum-belts in Ethiopia. Two major cities, central for product processing and marketing, were also assessed. A questionnaire survey was administered to 135 randomly selected households, and key stakeholder interviews, group discussions, and field observations were carried out following the value chain (from producers to exporters). Results showed that one or more of the seven gums and resins (frankincense, myrrh, opopanax, hagar, gum arabic, gum talha, and gum gumero) were produced and traded at the studied districts. While frankincense marketing dominated the northern part, gum arabic, myrrh, and opopanax are most popular in the south and southeastern part of the country. About 93% of the interviewed households engaged in collecting, marketing, or both activities. Gums and resins contributed up to 14% of the average annual cash income of the households. However, a significant difference (P < 0.001) was found in the amount collected and income generated per household and locality. Strong correlation was observed between cash income from gums and resins and off-farm activities (R = 0.74) and other types of non-timber forest products like honey (R = 0.72, α = 0.01). However, weak correlation was observed between incomes from gums and resins and crop and livestock production. Despite the observed inefficient value chain, the gum and resin resources have considerable contributions to the national economy. For instance, the annual average revenue from three districts in Tigray National Regional State was USD 882,000 in 2010. Between 2002 and 2010, about 2,306 tonnes of different gums and resins were traded and average revenue of USD 3,220,542 was obtained in one district in the same region. At the national level, between 1997 and 2010 about 6,174 tonnes of gum arabic and about 33,865 tonnes of other gums and resins were exported, and more than USD 72 million were generated. Responding to what sort of institutional arrangement governs the value chain and use of gums and resins resources at the present situations, about 41% of the respondents asserted customary and national legal arrangements, while 56% mentioned alternative systems as means of conflict resolution. Key policy and development interventions that could enhance the socio-economic importance of the gum and resin value chain at the local and national levels, while also increasing responsibility and commitment towards long-term management of the resource bases, have been recommended

A genetic variant of p53 restricts the mucous secretory phenotype by regulating SPDEF and Bcl-2 expression

Despite implications for carcinogenesis and other chronic diseases, basic mechanisms of p53 and its variants in suppressing Bcl-2 levels, are poorly understood. Bcl-2 sustains mucous cell metaplasia, whereas p53−/− mice display chronically increased mucous cells. Here we show that p53 decreases bcl-2 mRNA half-life by interacting with the 5’ untranslated region (UTR). The p53-bcl-2 mRNA interaction is modified by the substitution of proline by arginine within the p53 proline-rich domain (PRD). Accordingly, more mucous cells are present in primary human airway cultures with p53Arg compared with p53Pro. Also, the p53Arg compared with p53Pro displays higher affinity to and activates the promoter region of SAM-pointed domain-containing Ets-like factor (SPDEF), a driver of mucous differentiation. On two genetic backgrounds, mice with targeted replacement of prolines in p53 PRD show enhanced expression of SPDEF and Bcl-2 and mucous cell metaplasia. Together, these studies define the PRD of p53 as a determinant for chronic mucus hypersecretion

Deacetylation of p53 induces autophagy by suppressing Bmf expression

Interferon γ (IFN-γ)–induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-γ on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-γ down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death–stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-γ did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-γ–induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53’s proline-rich domain. Suppression of Bmf facilitated IFN-γ–induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf−/− but not in bmf+/+ cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy

Intracellular Insulin-like Growth Factor-1 Induces Bcl-2 Expression in Airway Epithelial Cells

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and IGF-1 coincided with induced Bcl-2 expression compared to controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using shRNA showed that intracellular (IC)-IGF-1 was increasing Bcl-2 expression. Blocking EGFR or IGF-1R activation also suppressed IC-IGF-1, and abolished the Bcl-2 induction. Induced expression and co-localization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and EGFR pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases

Does the BCL-2 family member BIK control lung carcinogenesis?

Hyperplastic airway epithelial cells may be the cause for increased risk for lung cancer in patients with chronic lung diseases. The B-cell lymphoma 2 (Bcl-2) family member, Bcl-2-interacting killer (BIK), triggers cell death specifically in these hyperplastic cells because of adequate presence of Death-associated Protein Kinase 1 (DAPk1), BCL-2 Antagonist Killer (BAK), and Extracellular Signal-regulated Kinase 1/2 (ERK1/2). Therefore, BIK may be a useful tool to control the development of lung cancer in patients with chronic diseases

T cells suppress memory-dependent rapid mucous cell metaplasia in mouse airways

Abstract Background Airway epithelial cells (AECs) are crucial for mucosal and adaptive immunity but whether these cells respond in a memory-dependent manner is poorly studied. Previously, we have reported that LPS intratracheal instillation in rodents causes extensive neutrophilic inflammation and airway epithelial cell hyperplasia accompanied by mucous cell metaplasia (MCM). And the resolution process required a period of 40 d for the inflammation to subside and the lung epithelia to resemble the non-exposed condition. Therefore, the present study investigated the memory-dependent response of airway epithelial cells to a secondary LPS challenge after the initial inflammation was resolved. Methods Airway epithelial and mucous cells were assessed in response to a secondary LPS challenge in F344/N rats, and in C57BL/6 wild-type (Foxn1WT) and T cell-deficient athymic (Foxn1nu) mice that were instilled with LPS or saline 40 d earlier. Epithelial expression of TLR4, EGFR, and phosphorylated-ERK1/2 (pERK) were also analyzed. Results LPS-pretreated F344/N rats responded with elevated numbers of AECs after saline challenge and with 3-4-fold increased MCM following the LPS challenge in LPS- compared with saline-pretreated rats. LPS-pretreated rats showed 5-fold higher number of AECs expressing TLR4 apically than saline-pretreated rats. Also, the expression of EGFR was increased in LPS-pretreated rats along with the number of AECs with active or nuclear pERK, and the levels were further increased upon LPS challenge. LPS-pretreated Foxn1nu compared with Foxn1WT mice showed increased MCM and elevated levels of TLR4, EGFR, and nuclear pERK at 40 d after LPS instillation. LPS challenge further augmented MCM rapidly in Foxn1nu compared with Foxn1WT mice. Conclusion Together, these data suggest that AECs preserve an ‘innate memory’ that drives a rapid mucous phenotype via spatiotemporal regulation of TLR4 and EGFR. Further, T cells may suppress the sustained elevated expression of TLR4 and EGFR and thereby the hyperactive epithelial response

Exploring the functional and metabolic effects of adding garra fish meal to a plant-based broiler chicken diet

The present study evaluated the metabolic and functional effects of adding garra meal to a broiler chicken diet. Three hundred twenty Sasso-breed day-old chicks were randomly assigned to four dietary treatments with either 0, 10, 20 or 30% garra meal added on top of formulated starter and grower basal diets. The experiment lasted for 42 days. Feed intake and body weight gain increased at the starter and grower phases of broilers with garra meal addition (P < 0.05). Broiler chickens fed 30% garra meal were more efficient in converting feed to body weight and yielded the highest carcass weight (P < 0.05). Crude protein ileal digestibility coefficient was higher with 20% (76.2%), and crude fat with 20 (92.1) and 30% (92.6%) garra meal receiving groups (P < 0.05). The increase in individual and total esterified carnitine concentrations in dried blood spots demonstrated the elevated metabolic rate with garra meal addition (P < 0.05). A better supply of glucogenic substrate to the citric acid cycle was seen with garra meal addition due to the increase of propionylcarnitine to acetylcarnitine ratio (P < 0.05) without any apparent effect on ketogenesis in terms of serum 3-hydroxybutyrylcarnitine to acetylcarnitine ratio. Yet, it likely showed that part of the amino acids from garra meal were used as glucogenic substrate (P < 0.05). Histomorphometry data showed 20% garra meal addition elevated villus height, crypt depth and their ratio in the proximal parts of the small intestine (duodenum and jejunum) with the opposite results observed in the more distal part (ileum) with the highest for the control group (P < 0.05). It can be concluded that garra meal improved broiler performance when added to a plant-based diet and only few parameters warranted for caution when using more up to 30% garra meal addition. Beyond growth performance, garra meal generated a shift to a more efficient digestion, absorption and nutrient metabolism