Dermal bacterial LPS-stimulation reduces susceptibility to intradermal Trypanosoma brucei infection.

Infections with Trypanosoma brucei sp. are established after the injection of metacyclic trypomastigotes into the skin dermis by the tsetse fly vector. The parasites then gain access to the local lymphatic vessels to infect the local draining lymph nodes and disseminate systemically via the bloodstream. Macrophages are considered to play an important role in host protection during the early stage of systemic trypanosome infections. Macrophages are abundant in the skin dermis, but relatively little is known of their impact on susceptibility to intradermal (ID) trypanosome infections. We show that although dermal injection of colony stimulating factor 1 (CSF1) increased the local abundance of macrophages in the skin, this did not affect susceptibility to ID T. brucei infection. However, bacterial LPS-stimulation in the dermis prior to ID trypanosome infection significantly reduced disease susceptibility. In vitro assays showed that LPS-stimulated macrophage-like RAW264.7 cells had enhanced cytotoxicity towards T. brucei, implying that dermal LPS-treatment may similarly enhance the ability of dermal macrophages to eliminate ID injected T. brucei parasites in the skin. A thorough understanding of the factors that reduce susceptibility to ID injected T. brucei infections may lead to the development of novel strategies to help reduce the transmission of African trypanosomes.

Influence of the Draining Lymph Nodes and Organized Lymphoid Tissue Microarchitecture on Susceptibility to Intradermal Trypanosoma brucei Infection.

Infection of the mammalian host with African trypanosomes begins when the tsetse fly vector injects the parasites into the skin dermis during blood feeding. After injection into the skin, trypanosomes first accumulate in the draining lymph node before disseminating systemically. Whether this early accumulation within the draining lymph node is important for the trypanosomes to establish infection was not known. Lymphotoxin-ß-deficient mice (LTß-/- mice) lack most secondary lymphoid tissues, but retain the spleen and mesenteric lymph nodes. These mice were used to test the hypothesis that the establishment of infection after intradermal (ID) T. brucei infection would be impeded in the absence of the skin draining lymph nodes. However, LTß-/- mice revealed greater susceptibility to ID T. brucei infection than wild-type mice, indicating that the early accumulation of the trypanosomes in the draining lymph nodes was not essential to establish systemic infection. Although LTß-/- mice were able to control the first parasitemia wave as effectively as wild-type mice, they were unable to control subsequent parasitemia waves. LTß-/- mice also lack organized B cell follicles and germinal centers within their remaining secondary lymphoid tissues. As a consequence, LTß-/- mice have impaired immunoglobulin (Ig) isotype class-switching responses. When the disturbed microarchitecture of the B cell follicles in the spleens of LTß-/- mice was restored by reconstitution with wild-type bone marrow, their susceptibility to ID T. brucei infection was similar to that of wild-type control mice. This effect coincided with the ability to produce significant serum levels of Ig isotype class-switched parasite-specific antibodies. Thus, our data suggest that organized splenic microarchitecture and the production of parasite-specific Ig isotype class-switched antibodies are essential for the control of ID African trypanosome infections.

Development of in vitro enteroids derived from bovine small intestinal crypts.

Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host-pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or "mini gut", cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host-pathogen interactions in the bovine small intestine can be studied.

Runx1 Deficiency Protects Against Adverse Cardiac Remodeling After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown. METHODS: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1-deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels. RESULTS: Runx1-deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca2+-ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca2+-ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum-mediated calcium release, preserving cardiomyocyte contraction after MI. CONCLUSIONS: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI.

Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake.

It has long been established that the Trypanosoma brucei TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest Trypanosoma congolense paralogue, TcoAT1, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of TcoAT1 and show that in fact it is the syntenic homologue of another T. brucei gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The T. congolense genome does not seem to contain a syntenic equivalent to TbAT1. Two TcoAT1 alleles, differentiated by three independent SNPs, were expressed in the T. brucei clone B48, a TbAT1-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); TbAT1 was also expressed as a control. The TbAT1 and TcoAT1 transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only TbAT1 increased sensitivity to diamidines and to cymelarsan. Uptake of [(3)H]-diminazene was detectable only in the B48 cells expressing TbAT1 but not TcoAT1, whereas uptake of [(3)H]-inosine was increased by both TcoAT1 alleles but not by TbAT1. Uptake of [(3)H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in T. congolense.