Disturbing the interaction between sympathetic nerves and macrophages in islets has been shown to protect from insulitis development. To study the intra-islet neuroimmune communications, we explored the interactions between macrophages and nerves in the islets by looking at their juxta-positioning in the islets of wild type and CX3CR1 knock out mice. We also cultured M1/M2 bone marrow-derived macrophages in order to understand basic expression profile changes during catecholaminergic stimulation. The results showed weaker colocalization between intra-islet macrophages and sympathetic nerves in CX3CR1-deficient mice as compared to the wild type showing that CX3CR1 played an important role in nerve-macrophage interactions. Also, low concentration of norepinephrine might induce pro-inflammatory effects in M1, as implied in how intra-islet macrophages responded to nerve signal excitation in type 1 diabetes development.

No description available.

AbstractSince the first discovery of COVID-19 in late 2019, several millions of people have beeninfected worldwide by the novel severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). Towards the end of the following year new mutant variants started emerging,namely 20I/501Y.V1, VOC 202012/01, or B.1.1.7, 20H/501Y.V2 or B.1.351 P.1(descendant of B.1.1.28), which were firstly discovered in United Kingdom, SouthAfrica, and Brazil, respectively. These variants contain several mutations located in thedifferent part of the virus genome. In this work, we focus mainly on commonly sharedmutations in the receptor binding domain (RBD) of the spike protein, E484K and N501Y.There is some evidence indicating that these mutations could increase diseasetransmissibility between humans. Here, we explore a concept of employing a diagnostictool to evaluate the importance of these mutations using short peptide epitopes containingthe mutations. A naturally occurring biologically stable cyclic peptide, sunflower trypsininhibitor 1 (SFTI-1), was used as a molecular scaffold to graft the epitopes. The epitopeswere tested against five serum samples and by using an indirect enzyme-linkedimmunosorbent assay (ELISA) the strength and/or response of the antibody reactivitywas determined. Linear versions of the cyclic peptides and RBD, as well as a longer nativelinear peptide containing both regions of mutation sites were used as controls. Initialresults suggest that short epitopes are insufficient to trigger antibody reactivity and thatepitope regions selection plays an important role. However, the insight presented in thiswork provides substantial information for future development of pharmaceuticalapproaches in COVID-19 therapy.

Inflammation is a defensive process that allows immune cells to be mobilized to help with infection removal and tissue regeneration. Inflammasomes are multiprotein oligomers in the cytoplasm and components of the innate immune system that have a role in inflammation. Glycogen synthase kinase 3 (GSK3) is a critical molecule involved in a wide range of inflammatory reactions. It has been reported to suppress the production of pro-inflammatory mediators in response to LPS when it is inhibited. The aim of this project was to study the effect of GSK3 inhibition in a concentration-dependent manner on the production of IL-6 as well as ASC-speck formation in THP1 ASC GFP cells stimulated with LPS and activated using nigericin. Using the cell culture supernatant ELISA was performed to quantify the IL-6 protein secreted by THP-1 macrophages. Using reverse-transcribed cDNA, qPCR was performed to measure the IL-6 gene expression. Finally, live-cell imaging was done to visualize the ASC-speck formation. It was found that upon stimulation of THP-1 cells a remarkable increase in the production of IL-6 was observed, however, the inhibitor did not suppress the production of IL-6 as hypothesized. This could be primarily due to the presence of another NF-κB pathway which is not mediated by GSK3 and therefore could not be inhibited using the GKS3 inhibitor. Future studies could decrease the LPS concentration to see if the uninhibited pathway can be observed at lower stimulation. Another probable solution could be lowering the FBS percentage to avoid potential inhibition.

The epithelial cells lining the intestinal mucosa separate the underlying tissue from components of the intestinal lumen. Innate immunity mediated by intestinal epithelial cells (IECs) provides rapid protective functions against microorganisms. Innate immunity also participates in orchestrating adaptive immunity. Key components in innate defence are defensins. To study the production of defensins and how it is affected by intestinal inflammation IECs were isolated from the small and large intestines of patients suffering from ulcerative colitis (UC), Crohn´s disease (MbC), celiac disease (CD), and from controls, and analyzed by quantitative RT-PCR (qRT-PCR) and immunoflow cytometry. Defensin expressing cells were also studied by in situ hybridization and immunohistochemistry. Normally, only small intestinal Paneth cells express human α-defensin 5 (HD-5) and HD-6. In UC colon IECs, HD-5, HD-6, and lysozyme mRNAs were expressed at high levels. In Crohn´s colitis colon the levels of HD-5 and lysozyme mRNAs were also increased although not to the same extent as in UC. No increase was detected in MbC with ileal localization. Metaplastic Paneth cell differentiation in UC colon was primarily responsible for the expression of the antimicrobial components. Human β-defensin 1 (hBD-1) mRNA was more abundant in large than in small intestine of controls, and remained unchanged in UC and MbC. hBD-2 mRNA was barely detectable in normal intestine and was induced in UC IECs but not in MbC IECs. mRNAs for the recently discovered hBD-3 and hBD-4, were detected in IECs from both small and large intestine. Both hBD-3 and hBD-4 mRNA were significantly increased in IECs of UC patients but not of MbC patients. Bacteria and IL-1β induced hBD-2 but not hBD-1 mRNA in colon carcinoma cell lines. IFN-γ, but not TNF-α or IL-1β, augmented hBD-3 expression in these cells, while none of the agents induced hBD-4. High antimicrobial activity of IECs in UC may be a consequence of changes in the epithelial lining, which permit the adherence of microorganisms. Unexpectedly, in situ hybridization revealed expression of hBD-3 and hBD-4 mRNAs by numerous lamina propria cells in colonic tissue from UC patients. These cells were identified as plasma cells (CD138+). hBD-3 and hBD-4 mRNAs were also demonstrated in the plasmacytoma cell line U266. This is the first demonstration of defensins in plasma cells. The four prominent constituents of the intestinal glycocalyx, carcinoembryonic antigen (CEA), CEA cell adhesion molecule 1 (CEACAM1), CEACAM6 and CEACAM7 all seem to play a critical role in innate defence of the intestinal mucosa by trapping and expelling microorganisms at the epithelial surface. The inducibility of these molecules in colonic epithelial cell lines was analyzed by qRT-PCR, immunoflow cytometry, and immunoelectron microscopy. IFN-g but not bacteria, LPS, TNF-α, or IL-1β modified the expression of CEA, CEACAM1 and CEACAM6. None of these agents modified CEACAM7 expression. IFN-γ was shown to have two effects: a direct effect on CEACAM1 transcription, and promotion of cell differentiation resulting in increased CEA and CEACAM6 and decreased CEACAM7 expression. Scanning electron microscopy of jejunal biopsies from children with CD revealed the presence of rod shaped bacteria in ~40% of patients with active CD, but only in 2% of controls. 19% of treated CD patients still had adhering bacteria. Presence of bacteria is not due to lack of antimicrobial factors. In fact, HD-5, HD-6, and lysozyme mRNA levels were significantly increased in IECs of patients with active CD. hBD-1 and hBD-2 were unchanged. Lack of induction of hBD-2 may reflect disturbed signalling in IECs of CD patients. Analysis of CEA and CEACAM1 mRNA/protein expression showed no differences between CD patients and controls. Analysis of the mucins MUC2 and MUC3 revealed significantly increased MUC2 levels in active disease and unchanged MUC3. Immunohistochemistry demonstrated goblet cell metaplasia as well as staining of the apical portion of absorptive cells. Glycosylation status of proteins was studied by lectin histochemistry. Goblet cells in the mucosa of CD patients were stained by the lectin UEAI. This was not seen in controls. The lectin PNA stained the glycocalyx of controls but not that of CD patients. Thus, unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients and may allow bacterial binding. We conclude that the intestinal epithelium is heavily involved in the innate defence of the mucosa and that its reactive pattern is affected by intestinal inflammation. Keywords: human intestinal mucosa; epithelial cells; innate immunity; defensin; ulcerative colitis; Crohn´s disease; celiac disease; glycoαcalyx; mucin

<p>Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.</p>

<p>Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. </p><p>An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. </p><p>In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified.</p><p>The thrombin inhibitor melagatran completely blocked the IBMIR in an <i>in vitro</i> tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. </p><p>These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.</p>

<p>Antibodies have the ability to influence the antibody response against the very antigen they are specific for, in a process called antibody feedback regulation. Depending on the nature of the antigen, the antibody response can be either enhanced or almost completely inhibited. This thesis focuses on the underlying mechanisms of antibody feedback regulation <i>in vivo</i>. </p><p>Antigen-specific IgG can inhibit the antibody response to a particulate antigen. Based on its ability to inhibit B cell activation, the inhibitory FcγRIIB (low affinity receptor for IgG) has been suggested to be involved. Here we show that although FcγRIIB is required for efficient suppression<i> in vitro, </i>it is not required <i>in vivo</i>. Therefore, even though FcγRIIB can inhibit antibody responses, other mechanisms (such as epitope masking and enhanced antigen clearance) play a more dominant role<i> in vivo</i>.</p><p>The antibody response to soluble antigen is greatly enhanced when it is introduced to the immune system in complex with antigen-specific IgG or IgE. We found that FcγRIIB attenuates the magnitude of IgG-mediated enhancement. In mice lacking FcγRIIB, IgG enhanced the antibody response much more efficiently than in normal mice.</p><p>Since B cells require CD4⁺ T cell help in order to become antibody-producing cells, we examined the CD4⁺ T cell response to immune complexes <i>in vivo</i>. Using an adoptive transfer strategy with transgenic ovalbumin (OVA)-specific CD4⁺T cells, we could show that the enhanced OVA-specific IgG response to IgG2a/OVA and IgE/OVA complexes was preceded by a potent OVA-specific CD4⁺ T cell response. IgG2a-mediated enhancement was dependent on activating Fcγ receptors, whereas IgE-mediated enhancement was dependent on CD23, the low affinity receptor for IgE. We identified CD23⁺ B cells as the responsible effector cells for IgE-mediated enhancement<i> in vivo</i>. Taken together, these results show that Fc receptor-mediated antigen presentation is a major mechanism underlying antibody feedback enhancement. </p>

<p>Prostasomes are extracellularly occurring submicron, membrane-surrounded organelles produced by the epithelial cells of the prostate and present in semen. Their precise physiological role is not known, although some of their properties assign them to important physiological and patho-physiological functions. In this thesis, some new properties of seminal and malignant cell line (DU145, PC-3 and LNCaP) prostasomes have been identified. </p><p>Differences in the expressions and activities of prostasomal CD59, ATPase, protein kinases and tissue factor (TF) have been characterized. The transfer of prostasomal CD59 to CD59-deficient erythrocytes (rabbit and human PNH erythrocytes) has been established. CD59, protein kinases and TF were overexpressed by malignant cell prostasomes. ATPase activity was highest on seminal prostasomes with minimal expression by malignant cell prostasomes resulting in more residual ATP available for phosphorylation reactions. Several proteins were phosphorylated by prostasomal protein kinases, <i>viz.</i> complement component C3, fibrinogen, vitronectin and E-cadherin. Furthermore, TF was identified as the main endogenous phosphorylation substrate on prostasomes. In addition, prothrombotic effects of prostasomes were established. DU145 and PC-3-derived prostasomes exerted a higher clotting effect on whole blood and plasma compared to LNCaP and seminal prostasomes.</p><p>In conclusion, malignant cell prostasomes showed higher ability to interact with the biological system in favor of prostate cancer cell promotion and survival. The roles played by prostasomes in this context may improve the understanding of the mechanisms that help the prostate cancer cells to avoid the complement attack (CD59 transfer and phosphorylation of C3), to promote angiogenesis (TF) and to metastasize. It may also provide a better understanding of some of the complications usually seen in some terminal prostate cancer patients like thrombotic events and tendency to develop disseminated intravascular coagulation.</p>

Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.