Bacillus thuringiensis subsp. israelensis is the most important biological insecticide for the control of mosquito vectors of disease. This bacterium produces toxins during sporulation that cause larval death by lysis of cells in their midgut. The pBtoxis plasmid of B. thuringiensis subsp. israelensis encodes all the mosquitocidal toxins and a number of other coding sequences. The plasmid also carries potential germination genes organised in a single ger operon. Comparison of the germination responses of spores from strains with and without pBtoxis revealed that this plasmid could promote activation of the spores under alkaline conditions but not following heat treatment. Introduction of the ger operon on a recombinant plasmid to the plasmidless strain established this operon as the first with an identified role in alkaline activation.

Activation of innate immunity is regulated by a variety of signaling molecules within

the immune cells. The present thesis was aimed to improve our understanding

innate signaling mechanism and their possible use as bio-indicators of exposure and

disease. The first part of the thesis deals with the involvement of TOM1L1 (Target

of Myb1 like 1) in innate immune signaling and regulation of inflammatory cytokines

in immune cells (study I and II). The initial event of T-cells activation depend

on the recruitment of Src family kinases Fyn and Lck, leading to interleukine-2 (IL-

2) production in T cells. Understanding the regulatory aspects of IL-2 induction in

T-cells is of importance as IL-2 is a key regulator for T-cell proliferation and survival.

Interaction screening indicated the ability of TOM1L1 protein to interact with

Fyn, and Lck, that is important for IL-2 production in Jurkat T-cells. TOM1L1

silencing decreased the levels of CD3/CD28 dependent induction of IL-2 in Jurkat

T-cells, and LPS dependent induction of TNF-α in THP-1. Furthermore, overexpression

of TOM1L1 in Jurkat T-cells causes an increase of STAT3 expression. This

was accompanied by an increase in the levels of IL-1β dependent induction of IL-6

and TNF-α in THP-1 cells. These results indicate that TOM1L11 participate in

regulation of innate immune response. The second part of the thesis deals with

development of innate immune signaling responses used as a diagnosis tools for

disease and exposure (study III and IV). Inflammatory diseases are associated with

innate immune reactions. In response to inflammation, the immune cells release

inflammatory cytokines such as IL-1-β, IL-2, IL-6, IL-10, TNF-α and CXCL8. These

cytokines are regulated by stress related kinases include MAP kinase proteins such

as ERK1-2, JNK, and MAPK p38, through activation of transcription factors AP-1,

ATF-2, and NF-AT. In a clinical study, it was observed that activated MAPK p38

has a potential role in the regulation of IL-10 expression in intermittent claudication.

However, expression of IL-10 and MAPK p38 was opposed in stable angina

group. Therefore, targeting MAPK p38 in inflammatory disease such as cardiovascular

diseases, diabetes, and rheumatoid arthritis might be useful in development of

treatment strategies. Innate immune reactions can also be used to monitor stress

related inflammatory responses following environmental exposure of immune cells.

Inflammatory responses of exposure were studied by in vitro exposure to waters

from sewage treatment works and recipient waters. The analysis shows that exposure

to inland waters can result in activated immune responses and that these responses

are both site dependent and vary over time.

Keywords: Innate immunity, TOM1L1, inflammatory responses.

Differences in activation between spores from strains of Bacillus thuringiensis subsp. israelensis with and without the toxin-encoding plasmid pBtoxis are demonstrated. Following alkaline activation, the strain bearing pBtoxis shows a significantly greater germination rate. Expression of just three genes constituting a previously identified, putative ger operon from this plasmid is sufficient to produce the same phenotype and characterizes this operon as a genetic determinant of alkaline activation.

Diabetic retinopathy is one of the most common diabetic complications, and is a major cause of new blindness in the working-age population of developed countries. Progression of vascular abnormalities, including the selective loss of pericytes, formation of acellular capillaries, thickening of the basement membrane, and increased vascular permeability characterizes early nonproliferative diabetic retinopathy (NPDR). Capillary occlusion, as shown on fluorescein angiograms, is also one of the earliest clinically recognizable lesion of NPDR. In response to capillary non-perfusion, there is dilation of neighbouring capillaries, leading to early blood-retinal barrier breakdown, capillary non-perfusion, and endothelial cell injury and death. The resulting ischemia leads to increased production of growth factors, and the development of proliferative diabetic retinopathy (PDR), which is characterized by growth of new vessels and potential severe and irreversible visual loss. The exact pathogenic mechanism by which capillary non-perfusion occurs is still unclear but growing evidence now suggests that increased leukocyte-endothelial cell adhesion and entrapment (retinal leukostasis) in retinal capillaries is an early event associated with areas of vascular non-perfusion and the development of diabetic retinopathy. The leukocytes in diabetic patients are less deformable more activated, and demonstrate increased adhesion to the vascular endothelium. This review summarizes the current literature on the role of leukocytes in the pathogenesis of capillary occlusion, and discusses the potential of leukostasis as a new promising target in the treatment of diabetic retinopathy.

Aims/hypothesis: Increasing evidence suggests that chronic, subclinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We recently reported that a glycosylating enzyme, core 2 beta-1,6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T), is implicated in increased leucocyte-endothelial cell adhesion in diabetic retinopathy via an upregulation mechanism controlled by TNF-alpha.

Subjects, materials and methods: We examined the functional link between circulating TNF-alpha and the activity and phosphorylation of core 2 GlcNAc-T in polymorphonuclear leucocytes of patients with type 1 and type 2 diabetes.

Results: Plasma levels of TNF-alpha, although similar in patients with type 1 and type 2 diabetes, were significantly higher than in age-matched healthy controls, and correlated well with the severity of retinopathy. Core 2 GlcNAc-T activity followed the same trend and was associated with phosphorylation of the enzyme. Finally, the observation that TNF-alpha levels are also linked to glycaemic values suggests that in patients, as well as in vitro, the glycosylation-mediated cell adhesion process that plays a role in diabetic retinopathy may involve glucose- and TNF-alpha-induced protein kinase beta2 activation, and subsequently raise activity of core 2 GlcNAc-T through increased enzyme phosphorylation.

Conclusions/interpretation: Our results reveal a novel rationale towards a specific treatment of diabetic retinopathy, based on the inhibition of core 2 GlcNAc-T activity and/or the blockage of cognate glycans.

A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme core 2 beta 1,6-N-acetylglucosaminyltransferase (GlcNAc-T) through protein kinase C (PKC)beta2-dependent phosphorylation plays a fundamental role in increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. In the present study, we demonstrate that following exposure to plasma from diabetic patients, the human promonocytic cell line U937 exhibits a significant elevation in core 2 GlcNAc-T activity and increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by PKCbeta2-dependent phosphorylation of the core 2 GlcNAc-T protein, were found to be triggered by increased plasma levels of tumor necrosis factor (TNF)-alpha. Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of diabetic retinopathy, with the highest values observed upon treatment with plasma of patients affected by proliferative retinopathy. Furthermore, we noted much higher correlation, as compared with control subjects, between increased values of core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-alpha in the development of diabetic retinopathy, these observations further validate the significance of core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.

Increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy and atherogenesis in diabetic patients. We recently reported that raised activity of glycosylating enzyme [beta]1,6 acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. Here, we demonstrate that elevated glucose increases the activity of core 2 GlcNAc-T and adhesion of human leukocytes to retinal capillary endothelial cells, in a dose-dependent manner, through diabetes-activated serine/threonine protein kinase C beta2 (PKCbeta2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients. Inhibition of PKCbeta2 activation with the specific inhibitor, LY379196, attenuated serine phosphorylation of core 2 GlcNAc-T and prevented increased leukocyte-endothelial cell adhesion. Raised activity of core 2 GlcNAc-T was associated with a threefold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 on the surface of leukocytes of diabetic patients compared with age-matched control subjects. PKCbeta2-dependent phosphorylation of core 2 GlcNAc-T may thus represent a novel regulatory mechanism for activation of this key enzyme in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy.

The exact mechanism for capillary occlusion in diabetic retinopathy is still unclear, but increased leukocyte-endothelial cell adhesion has been implicated. We examined the possibility that posttranslational modification of surface O-glycans by increased activity of core 2 transferase (UDP-Glc:Galbeta1-3GalNAcalphaRbeta-N-acetylglucoaminyltr ansferase) is responsible for increased adhesion of leukocytes to vascular endothelium in diabetes. The mean activity of core 2 transferase in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients was higher compared with age-matched control subjects (1,638 +/- 91 [n = 42] vs. 249 +/- 35 pmol x h(-1) x mg(-1) protein [n = 24], P = 0.00013; 1,459 +/- 194 [n = 58] vs. 334 +/- 86 [n = 11], P = 0.01). As a group, diabetic patients with retinopathy had significantly higher mean activity of core 2 transferase compared with individuals with no retinopathy. There was a significant association between enzyme activity and severity of retinopathy in type 1 and type 2 diabetic patients. There was a strong correlation between activity of core 2 transferase and extent of leukocyte adhesion to cultured retinal capillary endothelial cells for diabetic patients but not for age-matched control subjects. Results from transfection experiments using human myelocytic cell line (U937) demonstrated a direct relationship between increased activity of core 2 transferase and increased binding to cultured endothelial cells. There was no relationship between activity of core 2 transferase and HbA(1c) (P = 0.8314), serum advanced glycation end product levels (P = 0.4159), age of the patient (P = 0.7896), and duration of diabetes (P = 0.3307). On the basis that branched O-glycans formed by the action of core 2 transferase participate in leukocyte adhesion, the present data suggest the involvement of this enzyme in increased leukocyte-endothelial cell adhesion and the pathogenesis of capillary occlusion in diabetic retinopathy.