In experiment 2, mouse spleens and ears collected at the end of the experiment were cultured in MKP-F medium as described in detail previously [21]. In short: a 4 mm tissue biopsy was disinfected and placed in 7 ml Modified Kelly-Pettenkofer medium containing rifampicin, fosfomycin and amphotericin B as antibiotics. Cultures were incubated at 33 C and checked weekly for motile spirochaetes using a dark-field microscope at 250x magnification. After 3 weeks 500 μl of medium was inoculated into a new tube containing 7 ml of culturing medium. This was repeated three times. DNA from the cultures containing motile spirochaetes, tissue samples and moulted nymphs were extracted using the Qiagen DNeasy Blood & Tissue Kit [22]. DNA from questing larvae was extracted by alkaline lysis [23]. The presence of B. burgdorferi (s.l) DNA was detected with a duplex quantitative PCR using fragments of the outer membrane protein A gene and the flagellin B gene as targets [24]. In the same qPCR, B. miyamotoi could specifically be detected with primers and probe based on the flagellin gene for detection of the bacteria. The presence of Neoehrlichia mikurensis and Anaplasma phagocytophilum was detected as described [22]. Multi-Locus Sequence Typing on the Borrelia cultures was performed as described [25].
2011 17 M 32 Siberian Mouse
All mice were not infected with B. burgdorferi (s.l) and B. miyamotoi at the start of the experiments and all control mice were uninfected at the end of the experiments (Table 3). In experiment 1, 1 week after four challenges with field-collected larvae, 1 out of 10 mice was positive for B. afzelii and 2 out of 10 mice for B. miyamotoi. In experiment 2, 3 weeks after three challenges with field-collected larvae, 1 out of 10 mice was positive for both B. afzelii and B. miyamotoi. We were able to isolate and culture live spirochaetes for more than three passages from the ears of this infected mouse. The motile spirochaetes in this culture and the B. burgdorferi (s.l)-positive tissue samples and nymphs were all identified as B. afzelii by molecular typing. In addition, in experiment 2, 1 out of 10 mice was positive for B. miyamotoi. None of the rodent samples were infected with Anaplasma phagocytophilum or Neoehrlichia mikurensis (data not shown).
As far as we know, we also showed for the first time that larvae of I. ricinus can transmit B. miyamotoi. We found a B. miyamotoi infection rate of 2 % in larvae, which is comparable to the infection rate found in nymphs [32, 34]. This suggests a potentially higher contribution of larval tick bites to tick-borne relapsing fever in humans compared to larval contribution to Lyme borreliosis. Other studies have previously suggested an important role for larvae in the transmission of B. miyamotoi based on infections coinciding with larval activity and infestation [35, 36]. The contribution of larval transmission to rodents and from rodents to larvae to the enzootic life cycle of B. miyamotoi appears to be low. We did not find an increase in the infection rate of larvae with successive challenges, even in the two mice (2 and 3) that were B. miyamotoi positive after challenge 4. Infection rate of the emerged nymphs was not higher compared to the infection rate in questing larvae. In addition, mouse 9 was infectious to feeding larvae during challenge 1 and challenge 2, but was PCR negative after challenge 4, suggesting that, in contrast to B. afzelii, B. miyamotoi does not cause a persistent infection in rodents [16].
In breast cancer, glioblastoma and salivary gland cancer cells, a CBD-mediated downregulation of Id-1, an inhibitor of basic helix-loop-helix transcription factors, has been reported as the cause of the anti-invasive effect of this compound [98,99,100]. CBD also led to a downregulation of the sex-determining region Y (SRY)-Box 2 (Sox-2), a critical determinant of glioma tumour initiating cell growth and downstream target of Id-1, in glioblastoma cells [99]. Finally, the antimetastatic effect on breast cancer cells demonstrated for CBD in a mouse model [101] was directly linked to a downregulation of Id-1 in a later work [102]. In another investigation, siRNA, inhibitor and add-back experiments showed that the cannabinoid receptor- and TRPV1-dependent downregulation of plasminogen activator inhibitor (PAI)-1 in CBD-exposed lung cancer cells [103] is part of the anti-invasive effect of this cannabinoid, in addition to the upregulation of TIMP-1 mentioned above. Still other work revealed that the anti-invasive effect of THC on cholangiocarcinoma cells is associated with reduced activation of Akt and p42/44 MAPK [104]. Furthermore, a very recent work found that treatment with CBD in combination with THC or CBD alone inhibited bladder urothelial carcinoma cell migration independently of cannabinoid receptors [105].
Several studies have shown that cannabinoid compounds inhibit tumour neovascularisation in mouse models with xenografts (reviewed in ref. [117]). However, data on the exact mechanisms underlying these effects, especially with regard to tumour-stroma interactions, are still scarce. Early reports on the effect of cannabinoid compounds on tumour formation found a regressive effect based on anti-angiogenic effects through the downregulation of a number of proangiogenic parameters such as vascular endothelial growth factor (VEGF), placental growth factor (PlGF), angiopoietin-2 (Ang-2) [118, 119] and MMP-2 [119]. The same group later revealed that the CB2 receptor agonist JWH-133 modulates several hypoxia-related angiogenesis markers, most of which are associated with the VEGF pathway [120]. Thus, downregulation of VEGF-A and -B, hypoxia-inducible factor-1α (HIF-1α), connective tissue growth factor (CTGF), midkine, Id-3, Ang-2 and its receptor tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (Tie-1), and HO-1 was detected, while upregulation was found for the type I procollagen α1 chain (COL1A1) [120]. Remarkably, the authors of the latter study found VEGF receptor (VEGFR)-2 downregulation in response to JWH-133 in experimental glioma xenografts in mice. In another paper, inhibition of MMP-2 expression in human umbilical vein endothelial cells (HUVEC) was confirmed as part of the anti-angiogenic effects of CBD [121]. Consistent with the aforementioned direct anti-angiogenic effects, a further investigation identified the hexahydrocannabinol analogues LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] as inhibitors of VEGF-induced proliferation, migration and capillary-like tube formation of human endothelial cells as well as VEGF-induced blood vessel formation in the chorioallantoic membrane assay and VEGF release from cancer cells [122]. Thereby, the effects of the test substances on cell proliferation and tube formation were not abolished by cannabinoid receptor antagonists, suggesting a cannabinoid receptor-independent mechanism.
Regarding the effect of ECs, one study investigated the influence of 2-AG on different subpopulations of immune cells involved in the progression of pancreatic ductal adenocarcinoma using an orthotopic mouse model. Here, 2-AG increased the proportion of CD83+, CD86+ and MHCII+ cells in CD11C+ cell populations in the spleen of mice [134]. Dendritic cells promoted to maturation by 2-AG exhibited higher expression of proinflammatory cytokines (IL-6, IL-12, interferon-α) mediated by activation of the CB1 receptor and subsequent upregulation of the phosphorylated form of signal transducer and activator of transcription 6 (STAT6). A concomitant activation of T cells in the spleen was not observed. On the other hand, in spleen and tumour tissue of mice, 2-AG also induced the proliferation of myeloid-derived suppressor cells, which are known to suppress the T cell response thereby promoting an immunosuppressive microenvironment. Nevertheless, the antiproliferative effect of 2-AG on cancer cells ultimately led to an overall tumour-regressive effect in vivo [134].
Although some newly approved anticancer drugs are also used as monotherapy for certain indications, it seems more likely that cannabinoid compounds will be used as a combination and add-on option with currently employed cytostatics, assuming successful clinical trials. Against this background, THC and CBD, which are currently being tested in some studies as combination, have been preclinically shown to enhance the effect of various cytostatics, such as for vinca alkaloids, cytarabine, doxorubicin, mitoxantrone, carmustine, temozolomide, bortezomib, carfilzomib and cisplatin (reviewed in ref. [111, 141]). Thereby, combined administration of CBD and temozolomide in patient-derived neurosphere cultures and orthotopic mouse models was demonstrated to exert a significant synergistic effect in both reducing tumour size and prolonging survival [85]. Of particular importance for the use of cannabinoids in the treatment of glioblastoma is that the aforementioned booster effect on temozolomide action was previously confirmed in elaborate in vivo mouse models [59]. In glioblastoma cells, CBD has also been shown to enhance the effect of cisplatin [142]. Recently, a synergistic effect was also confirmed for the tumour regressive effect of CBD and cisplatin in a murine model of squamous cell carcinoma of the head and neck as well as for the in vitro cytotoxicity of CBD in combination with cisplatin, 5-fluorouracil or paclitaxel on human squamous cell carcinoma cells of the head and neck [52]. However, the mechanisms of these synergies are not yet fully understood. In this context, one study has shown that cannabinoid-mediated enhancement of the effect of vinblastine in resistant leukaemia cells was accompanied by THC- and CBD-induced downregulation of P-glycoprotein [143], while the synergistic cannabinoid effect over mitoxantrone in embryonic fibroblasts occurred via inhibition of ATP-binding cassette transporters (ABC)G2 [144]. Another study focusing on the effect of THC on the sensitisation of leukaemia cells to treatment with cytarabine, doxorubicin and vincristine showed reduced p42/44 MAPK activity as the underlying mechanism of THC-induced enhancement of the respective cytostatic effect [145]. In addition, a number of mechanistic studies have found a CBD-mediated increase in tumour cell susceptibility to the proteasome inhibitor bortezomib [146, 147], doxorubicin [148, 149] as well as temozolomide and carmustine [148]. There has also been a report that CBD increases the uptake into and toxicity on glioma cells of doxorubicin, temozolomide and carmustine via an increase in TRPV2 activity and associated increased calcium influx [148], with these results also confirmed for doxorubicin in triple negative breast cancer cells [149]. With regard to the synergistic effect with bortezomib, it was also shown that the combination of CBD and THC inhibits the expression of the immunoproteasome subunit β5i in multiple myeloma cells [147]. In addition, the synergistic effect of the combination of CBD and THC should also be mentioned here, which for example induces autophagy-dependent necrosis in multiple myeloma cells and inhibits cellular migration by downregulating the expression of the chemokine receptor CXCR4 and the plasma membrane glycoprotein CD147 [147]. However, in contrast to the results of these studies, which showed an enhancement of cytostatic effects when combined with cannabinoid compounds, a recently published paper did not find a survival benefit in the cannabinoid treatment group in combination with cyclophosphamide in an in vivo medulloblastoma model [84]. 2ff7e9595c
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