The Polyamine Analogues PG11144 and PG11047 Inhibit Cell Proliferation and Decrease the Malignancy in LNCap-FGC Prostate Cancer Cells in Normoxia and Hypoxia

Abstract One of many new targets when looking for anti-cancer treatment is the polyamine pathway, which is essential in cell proliferation, gene regulation and cell death. A way to intervene with this pathway is to use compounds called polyamine analogues. Depletion in the intracellular polyamine pools results in decrease in cell proliferation, something that is desirable in cancer treatment. In this study the effect of two different polyamine analogues, PG11144 and PG11047 (10 µM concentration), on the prostate cancer cell line LNCap-FGC was investigated in both normoxia (21% O2) and hypoxia (1% O2) to better mimic the actual conditions in the tumor. Tumors often contain oxygen-deprived areas, which have been reported to be involved in the development of malignancy. When the cells were cultured in hypoxia, the colony forming efficiency in soft agar increased 100-fold compared to culturing in normoxia, indicating an increase in malignancy. PG11144 or PG11047 treatment decreased the colony formation efficiency of cells grown in hypoxia. The expression of β-catenin, a marker of malignancy, decreased in PG11144- or PG11047-treated cells grown in normoxia and hypoxia, with PG11144 being the most effective. Treatment with either compound reduced cell proliferation, especially in normoxia. Altogether, the data show promising anticancer activities of both compounds in LNCap-FGC cells grown in normoxia and hypoxia.Polyaminanaloger – nytt botemedel mot cancer? Prostatacancer är den vanligaste cancerformen hos män i Sverige. Idag behandlas sjukdomen med hjälp av kirurgi, strålning eller genom att sänka nivån av androgener, de manliga könshormonen, då prostatacancercellerna oftast är beroende av dessa för tillväxt. Så länge tumören endast är lokaliserad i prostatan är behandlingen oftast effektiv. Dock kan mer aggressiva cancerceller utvecklas efter hand. Dessa kan sprida sig till andra delar av kroppen, vanligtvis till benmärgen, och bilda nya tumörer, så kallade metastaser, vilket leder till betydligt allvarligare och mer svårbehandlad cancer. När tumören växer utvecklas blodkärl för att de snabbt växande cellerna ska kunna förses med näring och syre. Blodkärlssystemet är dock ofta oorganiserat i tumörer jämfört med normala vävnader och detta kan leda till dåligt blodflöde i vissa regioner, vilket kan göra att syrefattiga, hypoxiska, områden uppkommer. Celler som lever under dessa förhållanden växer dåligt och måste anpassa sig till sin stressfulla omgivning. Det har visat sig att dessa celler kan utveckla mer aggressiva egenskaper och att hypoxi även kan öka antalet så kallade cancerstamceller. En tumör innehåller celler med olika aggressiva egenskaper och cancerstamceller är de mest aggressiva. Cancerstamceller kan sprida sig ut i kroppen och bilda metastaser. De flesta behandlingar har visat sig ha dålig effekt på celler i hypoxi, och det är därför viktigt att utveckla nya läkemedel som även påverkar dessa celler och framför allt riktar sig mot cancerstamceller. Ett sätt att döda cancerceller är att sänka polyamin-nivåerna i cellen. Polyaminer är nödvändiga för att cancercellerna ska kunna dela sig. Genom att använda sig av konstgjorda polyamin-analoger som konkurrerar ut de naturligt förekommande polyaminerna kan man stoppa tillväxten av tumören. I den här studien har effekterna av de två polyaminanalogerna, PG11144 och PG11047, på prostatacancercellinjen LNCap-FGC, under både normal syretillgång och syrebrist, för första gången undersökts. Det visade sig att behandling med PG11144 eller PG11047 minskade antalet cancerstamceller när cellerna växte vid både normal syretillgång och vid syrebrist. PG11144 var den mest effektiva substansen, och den orsakade också kraftig celldöd och hämmade celldelningen. PG11047 minskade också antalet cancerstamceller och hämmade celldelningen, men orsakade inte celldöd. Att PG11144 och PG11047 minskade antalet cancerstamceller är mycket lovande för framtida cancerbehandlingar. Då dessa försök endast utfördes i cellkulturer vore det intressant att undersöka om analogerna har samma positiva effekt i djur och människor. PG11144 orsakade dock så kraftig celldöd att det finns risk att den även kan påverka normala celler i kroppen och ge svåra biverkningar om den används som läkemedel. Det är därför viktigt att även undersöka effekten på vanliga celler innan man övergår till djurförsök. PG11047 var inte alls lika giftig men minskade effektivt antalet cancerstamceller. Den skulle därför kunna vara mycket intressant för framtida behandlingar av cancer. Handledare: Stina Oredsson Examensarbete 30 hp i biologi 2013 Biologiska institutionen, Lunds universite

Innate lymphocyte responses in inflammatory diseases

The immune system is a highly sophisticated system that has evolved to protect the host from external and internal threats. However, under some circumstances the immune response can get dysregulated and cause inflammatory diseases. In this thesis, I focus on three sets of disorders resulting from dysregulated immune responses: sepsis, toxic shock syndrome (TSS), and inflammatory bowel disease (IBD). They are highly heterogenous in their presentation and immune response profile and so far, there are no treatment options specifically targeting these conditions. The aim of this thesis is therefore to gain deeper insight into the immune mechanisms underlying these disorders. We have focused on two types of immune cells which are early responders in inflammation and share features of both the innate and adaptive immune system: group 3 innate lymphoid cells (ILC3) and mucosal-associated invariant T (MAIT) cells. The aim of paper I was to explore the role of the receptor GPR183 on ILC3s in the development of lymphoid structures in the colon at steady state and in promoting cell migration and tissue reorganization during colonic inflammation. Using mouse models and human IBD samples, we found that GPR183 on ILC3s senses oxysterols and is essential for the formation of cryptopatches and innate lymphoid follicles. In inflamed colon of both mice and humans, the oxysterol production in the surrounding tissue increased, resulting in migration of ILC3s out of the lymphoid tissues and towards the top of the colonic folds where they promoted recruitment of other immune cells. In paper II and III, we aimed to investigate the contribution of MAIT cells to the proinflammatory cytokine storm in TSS. In paper II, ex vivo stimulation experiments revealed that despite their low frequencies, MAIT cells were major producers of pro-inflammatory cytokines in streptococcal TSS (STSS). Superantigens produced by group A streptococci (GAS) bound to the b chain of the MAIT TCR, resulting in activation of a large fraction of the MAIT cell pool. MAIT cells were also found to be highly activated in patients with STSS. In paper III, we next explored the role of redox signaling in the strong MAIT cell response in TSS. We found that the MAIT cell cytokine response was inhibited by antioxidant treatment while other T cells were unaffected. Antioxidants reduced the total IL-18 production by PBMCs, and blocked IL-12 and IL-18-mediated MAIT cell activation. In paper IV, we investigated the MAIT cell phenotype in early samples from sepsis patients in the emergency room to evaluate the potential of MAIT cells as prognostic or diagnostic markers. MAIT cells were activated and reduced in frequency early during the sepsis response. Expression of the activation marker CD69 on MAIT cells was associated with lymphopenia, organ dysfunction and increased mortality. To summarize, this thesis reveals important novel insights on the roles of ILC3s in colonic inflammation and MAIT cells in sepsis and TSS

Robust T Cell Immunity in Convalescent Individuals with Asymptomatic or Mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.Fil: Sekine, Takuya. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Perez Potti, André. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Rivera Ballesteros, Olga. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Strålin, Kristoffer. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Gorin, Jean Baptiste. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Olsson, Annika. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Llewellyn Lacey, Sian. University Hospital of Wales; Reino UnidoFil: Kamal, Habiba. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Bogdanovic, Gordana. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Muschiol, Sandra. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Wullimann, David J.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Kammann, Tobias. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Emgård, Johanna. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Parrot, Tiphaine. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Folkesson, Elin. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Rooyackers, Olav. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Karolinska University Hospital; SueciaFil: Eriksson, Lars I.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Henter, Jan Inge. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Sönnerborg, Anders. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Allander, Tobias. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Albert, Jan. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Klingstrom, Jonas. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Gredmark Russ, Sara. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Björkström, Niklas K.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Sandberg, Johan K.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Price, David A.. Cardiff University School of Medicine; Reino UnidoFil: Ljunggren, Hans Gustaf. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Aleman, Soo. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Buggert, Marcus. Karolinska Huddinge Hospital. Karolinska Institutet; Sueci

Robust T cell immunity in convalescent individuals with asymptomatic or mild COVID-19

SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19

MAIT Cells Are Major Contributors to the Cytokine Response in Group A Streptococcal Toxic Shock Syndrome.

Streptococcal toxic shock syndrome (STSS) is a rapidly progressing, life-threatening, systemic reaction to invasive infection caused by group A streptococci (GAS). GAS superantigens are key mediators of STSS through their potent activation of T cells leading to a cytokine storm and consequently vascular leakage, shock, and multiorgan failure. Mucosal-associated invariant T (MAIT) cells recognize MR1-presented antigens derived from microbial riboflavin biosynthesis and mount protective innate-like immune responses against the microbes producing such metabolites. GAS lack de novo riboflavin synthesis, and the role of MAIT cells in STSS has therefore so far been overlooked. Here we have conducted a comprehensive analysis of human MAIT cell responses to GAS, aiming to understand the contribution of MAIT cells to the pathogenesis of STSS. We show that MAIT cells are strongly activated and represent the major T cell source of IFNγ and TNF in the early stages of response to GAS. MAIT cell activation is biphasic with a rapid TCR Vβ2-specific, TNF-dominated response to superantigens and a later IL-12- and IL-18-dependent, IFNγ-dominated response to both bacterial cells and secreted factors. Depletion of MAIT cells from PBMC resulted in decreased total production of IFNγ, IL-1β, IL-2, and TNFβ. Peripheral blood MAIT cells in patients with STSS expressed elevated levels of the activation markers CD69, CD25, CD38, and HLA-DR during the acute compared with the convalescent phase. Our data demonstrate that MAIT cells are major contributors to the early cytokine response to GAS, and are therefore likely to contribute to the pathological cytokine storm underlying STSS

The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

Objective. Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. Material and methods. Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). Results. GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. Conclusions. GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis