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Psychostimulant abuse is a major public health concern yet no FDA-approved medications exist. Synthetic cathinones (“bath salts”) are a class of psychostimulants that have emerged relatively recently worldwide. One synthetic cathinone, MDPV (3,4-methylenedioxypyrovalerone) is mechanistically similar to cocaine but is over ten times more potent, possesses high abuse potential, and is relatively understudied. Recent studies have revealed involvement of inflammatory proteins called chemokines in the rewarding effects of MDPV and the mechanistically similar drug, cocaine. We and others have shown that the chemokine-receptor ligand pair CXCL12-CXCR4 is recruited in the rewarding effects of cocaine and MDPV. Humans and animal models of cocaine addiction have dysregulated CXCL12 and the commercially-available CXCR4 antagonist, AMD3100, can reverse cocaine use and relapse in preclinical models of addiction. Specifically, AMD3100 reduces self-administration and reinstatement to cocaine-seeking with concomitant alterations in CXCL12 gene expression in the midbrain. Here, I employ several complementary methods to demonstrate that AMD3100 also reverses MDPV-elicited behaviors. I demonstrate that (i) AMD3100 reverses MDPV-induced hyperlocomotion, conditioned place preference (preclinical model of drug reward), self-administration and reinstatement to MDPV-seeking behavior; (ii) AMD3100 can rescue MDPV-induced deficits in measures of anxiety and recognition memory shortly after a binge; and (iii) repeated MDPV exposure upregulates CXCL12 gene expression in the nucleus accumbens with concomitant downregulation of dendrite morphometrics and a related synapse scaffolding protein gene expression. These findings implicate CXCR4-CXCL12 signaling in the modulation of MDPV-elicited behaviors, suggesting that AMD3100 is a viable therapeutic option for the effects of this synthetic cathinone. / Psychology

Macrophage migration, an essential component of many biological processes and pathologic conditions, is mediated by integrated cellular signaling processes and cytoskeleton rearrangement. Recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. Furthermore, our lab has provided evidence demonstrating the involvement of a key innate immunity signaling kinase, IRAK-1, as a critical modulator of murine macrophage migration. Macrophage migration induced by a potent PKC activator, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) was significantly decreased in IRAK-1-/- murine macrophages compared with wild type cells. Mechanistically, we first demonstrated that IRAK-1 works downstream of PKCε and directly binds to VASP, a cytoskeleton regulatory protein, to regulate PMA-induced macrophage migration. Secondly, we proved that IRAK-1 is required for LPS-induced macrophage migration and expression of MCP-1, a chemotactic cytokine for macrophages, via transcription factor C/EBPδ instead of NFκB. IRAK-1 binds directly to IKKε and inhibition or knock-down of IKKε results in a significant decrease in C/EBPδ expression and MCP-1 mRNA expression. Lastly, we identified the direct association between IRAK-1 and Rac1, a member of the Rac subfamily in the Rho family of GTPases. These finding further confirmed the essential role of IRAK-1 during macrophage migration. Our research provides a novel facet regarding the molecular signaling processes regulating macrophage migration. / Ph. D.

The expression of CCR7 was evaluated in different cancer cell lines by using flow cytometry, western blot, Immunofluorescence and immunohistochemistry. We showed for the selected cell lines that the expression is maintained in cells grown as spheroids, and xenoplanted in mice. Furthermore, we showed the expression of CCR7 correlates with stage of the disease in patient derived head and neck cancer tissue. We also showed that expression of CCR7 in cancer cell lines correlates with migratory aptitude towards CCL21 in a scratch assay, Boyden chamber assay and spheroid invasion assay. We then showed that the expression of CCR7 is elevated under serum starvation and under hypoxia in cancer cell lines grown as monolayers and as spheroids; and that there is a correlation between hypoxia and CCR7 expression in spheroids, xenografted cells and clinical cancer tissue. However, we found that in cell line OSC-19, the increase in the expression of CCR7 did not correlate to increased migration. Our investigations following this observation showed that whilst hypoxia increases the expression of CCR7, it concurrently causes a decrease in reactive oxygen species (ROS) which strongly abrogates migratory aptitude in OSC-19, resulting in an overall loss of migration in OSC-19 cells. In addition, we characterised OSC-19 as a suitable model to evaluate small molecule CCR7 antagonists using a number of different assays. In particular, we showed that ICT13069 antagonised response of this cell line across a number of drivers of malignancy such as migration, invasion in 2D and 3D models. / Zarqa University / The full text was made available at the end of the embargo, 3rd December 2019

Yes / The CCR7 chemokine axis is comprised of chemokine ligand 21 (CCL21) and chemokine ligand 19 (CCL19) acting on chemokine receptor 7 (CCR7). This axis plays two important but apparently opposing roles in cancer. On the one hand, this axis is significantly engaged in the trafficking of a number of effecter cells involved in mounting an immune response to a growing tumour. This suggests therapeutic strategies which involve potentiation of this axis can be used to combat the spread of cancer. On the other hand, the CCR7 axis plays a significant role in controlling the migration of tumour cells towards the lymphatic system and metastasis and can thus contribute to the expansion of cancer. This implies that therapeutic strategies which involve decreasing signaling through the CCR7 axis would have a beneficial effect in preventing dissemination of cancer. This dichotomy has partly been the reason why this axis has not yet been exploited, as other chemokine axes have, as a therapeutic target in cancer. Recent report of a crystal structure for CCR7 provides opportunities to exploit this axis in developing new cancer therapies. However, it remains unclear which of these two strategies, potentiation or antagonism of the CCR7 axis, is more appropriate for cancer therapy. This review brings together the evidence supporting both roles of the CCR7 axis in cancer and examines the future potential of each of the two different therapeutic approaches involving the CCR7 axis in cancer.

Chemokines are a family of chemotactic cytokines that play a multifaceted role in human biology. Chemokine receptor CCR7, activated by its ligands CCL19 & CCL21, is known to play a significant role in cancer metastasis. In view of the wider roles that chemokines play in the biology of the cell, we hypothesised that CCR7 could influence cancer progression through other mechanisms in particular increased proliferation and/or increased chemo- and radioresistance, and whether these effects may have a physiological/clinical relevance. Interestingly, CXCR4 involvement in cancer recurrence, metastasis and proliferation is already well established. Thus, it was used as a point of reference to compare whether CCR7 axis effect on cancer proliferation and chemoresistance is similar to that observed in CXCR4 axis. It is proven that hypoxia is a driving factor in increased CCR7 expression. This study shows that CCR7 expression is upregulated as a response to a number of other stress factors, in particular that caused by different chemotherapeutic treatments. In addition, we showed that CCL21, one of the two endogenous ligands for CCR7 is similarly produced under these conditions We used several techniques to establish the expression and functionality of CCR7 and CXCR4 in different cancer cell lines. We then showed that activation of the CCR7 axis by physiologically relevant concentrations of CCL21 induces cancer cell proliferation and chemo- and radio-resistance. Furthermore, these effects are abrogated by small molecule antagonists (ICT13069), neutralizing monoclonal antibody, or CCR7 knockdown. Our findings support the hypothesis that antagonising CCR7 receptor will not only inhibit cancer metastasis, as it is well-illustrated in the literature, but it would also lead to alternative therapeutic approaches as well as potential clinical endpoints. / University of Tobruk, and the Ministry of Higher Education of Libya

Leukocyte extravasation is mediated in part by tissue specific chemotactic cytokines (chemokines) and specific chemokine receptors expressed on the surface of circulating cells. C-C chemokine ligand CCL25 is expressed exclusively in the intestine and thymus and mediates chemotaxis by cells expressing receptor CCR9. This chemokine and receptor pair may be relevant in the pathogenesis of intestinal inflammation, in diseases such as Crohn’s disease (CD) and coeliac disease. In this thesis I investigated CCR9 expression in situ, in tissues affected by intestinal inflammation, and also examined the effects of CCR9 antagonist treatment in patients. In vitro I investigated CCR9 function using human peripheral blood T cells enriched for CCR9 by cell sorting or all-trans retinoic acid treatment. Using tissues collected as part of a clinical trial in CD testing CCR9 antagonist, CCX282-B, I investigated ways of measuring if treatment reduced the number of CCR9 expressing cells in the intestinal mucosa. However, in situ staining for CCR9 by immunohistochemistry was unsuccessful, and in this thesis, I explored reasons why this might be the case. Treatment with CCX282-B did however, show a tendency to reduce T cell density in the intestinal mucosa, although results were highly variable between individuals. In an examination of human CCR9 function in vitro I demonstrate for the first time that CCL25 stimulates CCR9 surface internalization. These data clarify the observation that CCR9 staining by IHC produces poor results in tissues where ligand is abundant, such as the intestine and thymus. I describe a novel technique for measuring calcium flux in two populations simultaneously by flow cytometry, which confirmed that in a heterogeneous population of cells, only CCR9 expressing cells respond to CCL25 by calcium flux. Variability in clinical trials is partly created by the use of concomitant medications, and in CD, corticosteroids are widely used. For the first time I show that glucocorticoids (GC) impair CCR9 mediated chemotaxis, calcium flux and intracellular signalling without changes to CCR9 mRNA and surface protein expression. Reduced CCR9 mediated signalling was accompanied by an enhanced expression and function of co-expressed CXCR4, demonstrating that the effects of GC were receptor-specific and not mediated by non-specific toxicity or inhibition of cell signalling. In a second study CCX282-B was tested in patients with coeliac disease, and in this trial, there was no reported concomitant use of GCs. It was confirmed that dietary gluten stimulates significant T cell recruitment to the intestinal mucosa with a pronounced accumulation of intraepithelial lymphocytes (IEL) and a rise in the frequency of FoxP3 expressing cells. Patients on CCX282-B had lower IEL counts, and an equivalent proportion of FoxP3 expressing T cells, suggesting that CCR9 blockade restricted the recruitment of effector T cell subsets. This thesis confirms that the accumulation of T cells is central to inflammation in the intestine and that modulating chemokine receptor function may affect this. Furthermore, this thesis demonstrates that the function of CCR9 is suppressed by GCs, which are widely used therapeutically and therefore could identify a novel mechanistic basis for their activity in CD.

O líquen plano oral (LPO) é uma doença inflamatória crônica que afeta em torno de 1 a 2% da população mundial adulta, entre 30 e 60 anos, principalmente mulheres. As lesões podem estar presentes em diversos sítios, porém tem predileção pela mucosa jugal, gengiva e bordas laterais da língua, todas bilateralmente. As manifestações de LPO são frequentes, e podem se caracterizar clinicamente em seis tipos: reticular, em placas, papular, atrófico, erosivo-ulcerativo e bolhoso, porém o mais comum é o reticular. O presente estudo teve como objetivo avaliar a expressão de genes relacionados à resposta inflamatória e autoimunidade no líquen plano oral (LPO) em pacientes com a variante exclusivamente reticular comparado a controle saudável. Foram incluídos consecutivamente 10 pacientes com LPO que preencheram os critérios de inclusão adotados, sendo 9 mulheres e um homem, e 6 indivíduos controle, sendo 4 mulheres e 2 homens pareados por sexo, idade e uso de medicações sistêmicas. Amostras de mucosa bucal foram coletadas por meio de biópsia incisional em ambos os grupos de pacientes e submetidas à extração de RNA para posterior análise da expressão gênica por PCR-array selecionada para este estudo. Os resultados obtidos foram o aumento da expressão de 8 genes, CXCL 9 e CXCL 10 vão de acordo com a literatura, corroborando com nosso estudo e de certa forma reafirmar mais necessidade de estudos bem desenhados . / The oral lichen planus (OLP) is a chronic inflammatory disease that affects around 1-2% of the adult population between 30 and 60 years, mainly women. The lesions may be present in several sites, but has a predilection for the buccal mucosa, gums and lateral borders of the tongue, all bilaterally. The manifestations of OLP are frequent, and may be characterized clinically in six types: reticular, plaque-like, papular, atrophic, erosive-ulcerative and bullous, but the most common is the reticular . The present study aimed to evaluate the expression of genes related to inflammatory response and autoimmunity in oral lichen planus (OLP) in patients with exclusively reticular variant compared to healthy control. 10 consecutive patients with OLP who met the inclusion criteria, 9 women and one man, and six control subjects were included were 4 women and 2 being proportionally matched by sex, age and use of systemic medications men. Samples of oral mucosa were collected by incisional biopsy in both groups of patients and subjected to RNA extraction for analysis of gene expression by selected for this study-PCR array. The results were the overexpression of genes 8, where only two of them go according to the literature, corroborating our study and somehow reaffirm need for more well-designed studies.

O líquen plano oral (LPO) é uma doença inflamatória crônica que afeta em torno de 1 a 2% da população mundial adulta, entre 30 e 60 anos, principalmente mulheres. As lesões podem estar presentes em diversos sítios, porém tem predileção pela mucosa jugal, gengiva e bordas laterais da língua, todas bilateralmente. As manifestações de LPO são frequentes, e podem se caracterizar clinicamente em seis tipos: reticular, em placas, papular, atrófico, erosivo-ulcerativo e bolhoso, porém o mais comum é o reticular. O presente estudo teve como objetivo avaliar a expressão de genes relacionados à resposta inflamatória e autoimunidade no líquen plano oral (LPO) em pacientes com a variante exclusivamente reticular comparado a controle saudável. Foram incluídos consecutivamente 10 pacientes com LPO que preencheram os critérios de inclusão adotados, sendo 9 mulheres e um homem, e 6 indivíduos controle, sendo 4 mulheres e 2 homens pareados por sexo, idade e uso de medicações sistêmicas. Amostras de mucosa bucal foram coletadas por meio de biópsia incisional em ambos os grupos de pacientes e submetidas à extração de RNA para posterior análise da expressão gênica por PCR-array selecionada para este estudo. Os resultados obtidos foram o aumento da expressão de 8 genes, CXCL 9 e CXCL 10 vão de acordo com a literatura, corroborando com nosso estudo e de certa forma reafirmar mais necessidade de estudos bem desenhados . / The oral lichen planus (OLP) is a chronic inflammatory disease that affects around 1-2% of the adult population between 30 and 60 years, mainly women. The lesions may be present in several sites, but has a predilection for the buccal mucosa, gums and lateral borders of the tongue, all bilaterally. The manifestations of OLP are frequent, and may be characterized clinically in six types: reticular, plaque-like, papular, atrophic, erosive-ulcerative and bullous, but the most common is the reticular . The present study aimed to evaluate the expression of genes related to inflammatory response and autoimmunity in oral lichen planus (OLP) in patients with exclusively reticular variant compared to healthy control. 10 consecutive patients with OLP who met the inclusion criteria, 9 women and one man, and six control subjects were included were 4 women and 2 being proportionally matched by sex, age and use of systemic medications men. Samples of oral mucosa were collected by incisional biopsy in both groups of patients and subjected to RNA extraction for analysis of gene expression by selected for this study-PCR array. The results were the overexpression of genes 8, where only two of them go according to the literature, corroborating our study and somehow reaffirm need for more well-designed studies.