Studies


Google+ All Figures and Tables are available here Figures & Tables A Cure for Type-1-Diabetes: Diabetes is “a silent epidemic which has immense human, social and economic costs” – said Ms Laoura Lazouras, on behalf of G77, United Nations Security Council Meeting 2006 in New York (Diabetes: the hidden pandemic, 2009, p3). According to the European Parliament, diabetes is “a major disease representing a significant burden across the EU” (Diabetes: the hidden pandemic, 2009, p3). Diabetes – also known as diabetes mellitus – is the most common metabolic disease in the world. Globally, 286 million people are suffering from diabetes (Diabetes Atlas, International Diabetes Federation (IDF), 3rd edition, 2006). 4.418 million deaths per year are caused by diabetes (Diabetes Atlas, International Diabetes Federation (IDF), 3rd edition, 2006). Diabetes worsens the life quality and expectations of the people suffering from it. People with diabetes need medical treatment a life long, and have a significantly increased risk of suffering serious complications, such as heart attack, stroke, kidney failure, blindness and ulcers leading to limb amputation. A diagnosis of diabetes means that managing the disease has to become a part of that person’s life. He/she has to manage the balance between diet, medication and exercise on a daily basis. This presents many problems and often leads to depression, so it is important that people with diabetes have access to full and accurate information, training in the practical skills they need, and psychosocial support to help them achieve control and confidence. The disorder of carbohydrate-metabolism is the source of the disease, but it also affects the fat- and protein- metabolism. The main cause of diabetes is the relative lack of insulin and its impact. In certain circumstances these divergences may occur together. Despite its rapidly growing prevalence, and the escalating costs of treating the disease, diabetes does not receive the urgent attention that it should. It remains seriously under-reported, partly because many people with type 2 diabetes do not realize they have it and do not seek help for what they see as minor symptoms until they have been established for years. Diabetes is also often not recorded as the cause of death, where the main cause of death may have been one of the typical diabetes complications mentioned above. (Diabetes: the hidden pandemic, 2009, p 1-40).


Medical treatment of diabetes and its complications put a severe burden both on patients and society. The International Diabetes Federation (IDF) estimates in 2007 the worldwide diabetes treatment and complication prevention strategies to cost around US-Dollar 232 billion, rising to US-Dollar 302,5 billion by 2025 (Diabetes: the hidden pandemic, 2009, p 1-40). Indirect costs by far exceed the costs of medical care. According to various studies (Diabetes: the hidden pandemic, 2009, p 1-40), 70% of the costs appear as direct healthcare costs, including hospital treatment, out-patient service and medicines. At least 30% of the expenses come from the indirect costs of lost productivity, like missed working days, limited-activity days, the loss of life, and permanent disability (Diabetes: the hidden pandemic, 2009, p 1-40). If we calculate the direct burden to the family and other social burdens of financial services through the social system, the amount will further increase. In case of a life long disease, another type of cost, the so called intangible charges appear (Diabetes: the hidden pandemic, 2009, p 1-40). 60-80% of the expenses calculated for the life-long treatment are not for the Medicare and the treatment of the basic disease, but for the treatment of its minor and major complications.

A new Treatment for Diabetes-Type-1: Reversing the islet cell damage
Type 1 diabetes is caused by the partial or total destruction of insulin producer beta-cells in the pancreas. This destruction of cells in the pancreas has the character of an auto-immune disease as the body’s-immune system, which is supposed to prevent attacks by infections, instead attacks the body’s own cells (Novel mechanisms of endothelial dysfunction in diabetes, JCDR 2010, 59-63). Type 1 diabetes can evolve at any age, even though it is more typical in childhood or in teenage years.

Various research groups on the immunology of type-l Diabetes (Novel mechanisms of endothelial dysfunction in diabetes, JCDR 2010, 59-63) have found that almost all diabetics carry either DR3 or DR4 or both. Moreover, most express hibernating viruses, bacteria or particles thereof in the surface membranes of their islet cells, which feed the autoaggressive T-cell response against islet cells and causes insulin dependent diabetes.

Up to now it is common ground to treat type 1 diabetes by insulin compensation. In double-blind-crossover studies spanning 18 years, involving 16,000 patients and including long-term follow-up (4 – 14 years), we were able to reverse the islet cell damage that causes insulin dependent diabetes. A combination of a multidose BCG vaccination scheme with low molecular weight Heparin, Interleukin-12, and Famiciclovir permanently abolished the need for insulin injections in 8 out of 10 insulin dependent diabetics within 9 – 12 months.


The current status of research reveals that the endothelium, once considered a mere selectively permeable barrier between the bloodstream and the outer vascular wall, is now recognized to be a crucial homeostatic organ, fundamental for the regulation of the vascular tone and structure. [5] Therefore, endothelial dysfunction during diabetes has been associated with a number of pathophysiologic processes. [6] A considerable body of evidence in humans indicates that endothelial dysfunction is closely associated with the development of diabetic retinopathy, [7] nephropathy, and atherosclerosis in both type 1 and type 2 diabetes. [8] In this article, the recent findings on the mechanisms of endothelial dysfunction in diabetes, which could contribute to the development of new treatment options are discussed. The healthy endothelial monolayer is optimally positioned in order to respond to physical and chemical signals, by producing a wide range of factors that regulate vascular tone, cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation. The importance of the endothelium was first recognized by its effect in limiting the vascular tone. [9] The vascular endothelium also regulates blood flow and, limits leukocyte adhesion and platelet aggregation by producing nitric oxide (NO), prostacyclin, and ectonucleotidases. As such, inflammatory activity in the vessel wall is blunted. In addition, the endothelium regulates vascular permeability to nutrients, macromolecules, and leukocytes; limits activation of the coagulation cascade by the thrombomodulin/protein C, heparin sulfate/antithrombin, and tissue factor/tissue factor pathway inhibitor interactions; and regulates fibrinolysis by producing tissue activator of plasminogen (t-PA) and its inhibitor, PAI-1. [10] The term endothelial dysfunction refers to a condition in which the endothelium loses its physiologic properties and shifts toward a vasoconstrictor, prothrombotic, and proinflammatory state. [11] Endothelial dysfunctionhas been associated with a variety of processes, including hypertension, atherosclerosis, aging, heart and renal failure, coronary syndrome, obesity, vasculitis, infections, sepsis, rheumatoid arthritis, thrombosis, smoking as well as with type 1 and type 2 diabetes [6]. In diabetes, dysfunction of the vascular endothelium is regarded as an important factor in the pathogenesis of diabetic micro- and macroangiopathy. [8] There are three main sources contributing to endothelial dysfunction in diabetes: (1) hyperglycemia and its immediate biochemical sequelae directly alter endothelial function; (2) high glucose (HG), which influences endothelial cell functioning indirectly by the synthesis of growth factors and vasoactive agents in other cells and alters endothelial monolayer permeability; and (3) the components of the metabolic syndrome that can affect endothelial function. [8] There are many signaling molecules involved in the pathogenesis of endothelial dysfunction. In the following paragraphs, recent studies on this topic, mainly focusing on the roles of arginase and reactive oxygen species (ROS), protein kinase C (PKC), and tumor necrosis factor (TNF), are addressed. Conditions contributing to diabetic vascular remodeling and dysfunction include the effects of oxidative stress and decreased NO bioavailability. [16],[17],[18],[-19] NO production by endothelial NOS (eNOS) is critically involved in maintaining the integrity and stability of the vascular endothelium, preventing platelet aggregation and leukocyte adhesion, and maintaining blood flow. [20] Availability of the semi-essential amino acid l-arginine is required for eNOS activity and NO production and is therefore essential for vascular integrity and function. Arginase is a hydrolytic enzyme, which converts l-arginine into urea and ornithine and exists as 2 isoforms: arginase I and II. [21] Whereas arginase I is a cytosolic enzyme, expressed at high levels in the liver, arginase II is a mitochondrial enzyme expressed primarily in the extrahepatic tissues, especially in the kidney. Knockdown of arginase I has been shown to restore NO signaling in the vasculature of old rats. [22] Both arginase I and II have been found in endothelial cells, arginase I being the dominant isoform. [23]


Arginase and eNOS compete for their common substrate, L-arginine. As such, increased arginase activity can lead to eNOS dysfunction. [23],[24] We have shown that hepatic and vascular arginase activity is increased in diabetic rats and that arginase I expression and activity are increased in aortic endothelial cells exposed to HG. [24] TNF has also been shown to induce arginase activity. [25] Furthermore, arginase actions causing endothelial dysfunction, as indicated by decreased NO availability, are blocked by the Rho kinase inhibitor Y-27632. [26] Additionally, an inhibitor of arginase reversed diabetes-induced endothelial dysfunction in the coronary vessels of diabetic rats. [24] Also, arginase was found to mediate retinal inflammation in lipopolysaccharide (LPS)-induced uveitis. [21]

Taken together, these findings [21],[22],[23],[24],[25],[26] suggest that arginase and RhoA may be mediators of diabetes-induced inflammatory effects in vascular disease. Apart from arginase, ROS also play an important role in vascular dysfunction in diabetes, although the source of their generation remains elusive. Overproduction of superoxide can lead to scavenging of NO and to its reduced bioavailability. [27],[28] ROS have been implicated in increased arginase activity and expression. Indeed, arginase activation can cause uncoupling of eNOS by reducing the supply of l-arginine. The uncoupled eNOS uses molecular oxygen to produce superoxide, thereby further reducing NO and increasing ROS formation [Figure 1].

An important glucose-induced alteration in cellular metabolism that may account for endothelial dysfunction is activation of PKC. Hyperglycemia causes de novo synthesis of diacylglycerol, leading to the activation of PKC, a pathway now demonstrated in all vascular tissues involved in diabetic complications. [29] Of interest, the adverse effects of elevated glucose levels on acetylcholine-induced relaxation of rabbit aorta and rat pial arterioles were restored by the addition of PKC-inhibitors. [30],[31] Diabetes-induced translocation of PKC-alpha to renal membranes was associated with increased nicotinamide adenine dinucleotide phosphate oxidase-dependent superoxide generation. [32] It has been proposed that HG concentrations rather specifically activate the beta II isoform of PKC. [33] However, the PKC alpha isoform, which is activated by HG in bovine aortic endothelial cells, has also been suggested to play an important role in diabetes mellitus-associated endothelial dysfunction, since specific antisense or pharmacologic inhibition completely abolished the effects of HG on endothelial cell permeability. [34] The reported activity of PKC-alpha on endothelial permeability is at least partially mediated by inducing phosphorylation of p115RhoGEF, [35] a guanine nucleotide exchange factor (GEF) for Rho GTPase. [36] Because active RhoA is implicated in arginase induction, [24] it suggests that PKC-alpha might also be involved in regulation of arginase activity.

Human TNF is a 51-kDa homotrimeric protein. TNF is generated as a membrane-bound precursor that is cleaved by the metalloproteinase family member TNF-alpha converting enzyme, giving rise to the soluble protein. [37] The main sources of the cytokine are activated macrophages and T cells. TNF binds to 2 different TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), at least one of which is expressed in most somatic cells. [37] Soluble TNF has the highest affinity for TNF-R1, whereas membrane-bound TNF preferentially interacts with TNF-R2. [38] Apart from the ligand TNF, also the receptors exist as membrane-associated and soluble forms. [37] TNF-R1, but not TNF-R2, contains a death domain, which signals apoptosis upon the formation of the death-inducing signaling complex [37] Although not carrying a death domain, TNF-R2 has nevertheless been implicated in apoptosis regulation in microvascular endothelial cells. [39]

Spatially distinct from its receptor binding sites, TNF carries a lectin-like domain, recognizing specific oligosaccharides, such as N, N′-diacetylchitobiose and branched trimannoses, [40] which can be mimicked by the 17-amino acid circular TIP peptide (amino acid sequence: CGQRETPEGAEAKPWYC). [41] Three residues, namely, T105, E107, and E110, appear to be crucial for this activity. The TIP peptide exerts a lytic activity toward bloodstream forms of African trypanosomes, [41] which occurs upon binding to the oligosaccharides expressed in the variant-specific glycoprotein of the parasites. More importantly, the TIP peptide also increases sodium transport in lung microvascular endothelial cells. [42] Interestingly, the activities of the lectin-like domain of TNF cannot be inhibited by the soluble TNF receptors. [41]

TNF is one of the key inflammatory mediators that is expressed during a variety of inflammatory conditions and initiates the expression of an entire spectrum of inflammatory cytokines ranging from many interleukins to interferons. [43] It is suggested that inflammation is an effector of not only endothelial dysfunction, but also insulin resistance and atherosclerosis. [44] Under inflammatory conditions, TNF can increase the expressions of adhesion molecules, such as vascular cell adhesion molecule (VCAM-1) and intercellular adhesion molecule (ICAM-1); and as such promote the adherence of monocytes. [45] Moreover, TNF can affect NO production by decreasing eNOS expression [46] and increase the production of ROS in neutrophils and endothelial cells through NAPH oxidase, [47] xanthine oxidase, [48] and uncoupled NOS. [49] The pivotal role of TNF in diabetes-induced endothelial dysfunction can also be manifested by the observation that endothelial function is close to normal in a TNF-knockout diabetic mouse model. [50]

The generation of TNF is increased during diabetes, and the cytokine has been shown to upregulate the expression of arginase in endothelial cells, which leads to endothelial dysfunction during ischemia reperfusion injury in mice. [25] Recent studies have indicated that TNF can affect endothelial barrier integrity. [51] by means of (1) inducing apoptosis of lung microvascular endothelial cells, [39] which can contribute to the disruption of the endothelial barrier during acute lung injury and acute respiratory distress syndrome; [52] (2) by inducing the production of ROS; [53] and (3) by directly increasing endothelial permeability in a RhoA/ROCK-dependent manner. [54] PKC-alpha activation was proposed to be involved in TNF-mediated increases in permeability of pulmonary microvessel endothelial monolayers. [55] On the other hand, the lectin-like domain of TNF, mimicked by the TIP peptide, can increase endothelial monolayer resistance in the presence of bacterial toxins, by means of inhibiting listeriolysin-induced PKC-alpha activation, which in turn inhibits RhoA activation and myosin light chain phosphorylation. [56] Moreover, the lectin-like domain of TNF can reduce ischemia-reperfusion-induced ROS generation in a lung transplantation model [57] As such, the lectin-like domain of TNF can potentially oppose the deleterious receptor-mediated activities of the cytokine on the endothelium. [58]

Faustman and others (Int J Biochem Cell Biol. 2010 Oct; 42(10):1576-9. Epub 2010 Jun 18.) have previously shown that immunotherapy with complete Freund’s adjuvant (CFA) or BCG is highly effective in the prevention and treatment of spontaneous insulin-dependent diabetes mellitus (IDDM) and in circumventing the rejection of syngeneic islet grafts in diabetic NOD mice. This protection is reversed by treatment with cyclophosphamide (Cy). BCG-induced protection against type 1 diabetes is attributed to the down-regulation of diabetogenic T cells both at the induction and effector phases of the disease. This treatment also induces regulatory cells that are sensitive to cyclophosphamide (8). CFA or BCG treatment in NOD mice has been shown to induce non-destructive insulitis (9).
Recent studies on NOD mice with cytokine gene deletions indicate that the immune response and cytokine switch after CFA or BCG therapy is probably an outcome rather than the cause of disease prevention (13). In mice, the patterns of cytokine production after infection or immunization with mycobacteria are dependent on many factors, such as the route, the nature of mycobacteria and the mouse strains used (14,15). Both TNF-{alpha} and IFN-{gamma} are the major Th1 cytokines produced early after mycobacteria infection, and this is followed by Th2-type cytokine production (16,77). The production of lL-4 after BCG immunization in syngeneic islet transplanted NOD mice occurs late and is maintained (12). IFN-{gamma} has been found to induce apoptosis of activated CD4 T cells in mice infected with mycobacteria (18). lt also inhibits the development of diabetes by down-regulating anti-islet effector cells (19). In addition, the exacerbation of autoimmune encephalomyelitis in IFN-{gamma} deficient mice is due to the failure of T cell apoptosis (20). Similarly, TNF-{alpha} has been shown to induce apoptosis in mature T cells (21) and in diabetogenic T cells of diabetic NOD mice (22). TNF-{alpha} suppresses spontaneous diabetes in NOD mice when given late but not early during the development of disease (23-25). Therefore, Th1-like cytokines might be a primary factor for CFA or BCG-induced down-regulation of destructive autoimmunity by activation-induced cell death (AICD) of diabetogenic T cells .

Fas-FasL pathway is well recognized as an efficient way to induce the apoptosis of activated Th1 and cytotoxic CD8 T cells (26-28). Moreover, TNF-{alpha} induces T cell apoptosis through TNFR, which plays a pivotal role in maintaining immune privilege of the eye through FasL-induced cell death promoted by TNF-{alpha} (29). In T cell receptor transgenic mice, it has been shown that both Fas and TNF are involved in AICD (30). Therefore, it was postulated that Th1 cytokines might contribute to the deletion of diabetogenic T cells in BCG therapy by AICD through both Fas and TNF pathways.

It is well recognized that standard Heparin has several antiinflammatory actions including the capacity to bind and neutralize products of the complement cascade, inhibition of chemoattractant-induced leukocyte adhesion invitro (26), and tissue accumulation of leukocytes in vivo (26), potentially through binding to CD11b/CD18 (26). Interestingly, an acculumating body of evidence indicated that also LMWH may exert anti-inflammatory effects in models of cutaneous anaphylaxis (26), endotoxemia (26), and inhibit TNF-α-induced leukocyte recruitment (26). It is noteworthy that potential mechanisms of LMWH in the Pancreas remain elusive and it is not yet known whether LMWH may reduce TNF-α production or interfer with leukocyte accumulation in the pancreas and thus attenuate the local inflammatory process leading to destruction of islet cells.

Study on BCG vaccination in NOD mice
Between 1983 – 1985 we undertook a study at the University of Tübingen (The effects of BCG on murine endothelium, WO/1987/001385) to determine the effect of BCG vaccination on the progression of Cy-accelerated diabetes in NOD mice and to understand the mechanism of BCG immunotherapy. The time course of Cy and BCG administration showed that the progression of Cy-induced diabetes could only be blocked when BCG vaccination is given within 3 days of Cy administration. Mice given BCG 3 days before (-3 days) or 7 days after Cy treatment were not protected. BCG immunization 1 day after Cy treatment almost completely prevented insulitis in the islets of Cy-treated mice. Cy treatment reduced the endogenous production of anti¬GAD67 antibody, whereas BCG vaccination 1 day after Cy treatment restored the production of anti¬GAD67 antibody of lgG1 isotype. The comprehensive effect of BCG vaccination on cytokine production in Cy-treated mice was to increase lL-4 production and change the IL-4/tFN-gamma ratio in both serum and supernatant of spleen cell cultures. We found that BCG-induced protection was associated with increased splenic CD4 + CD45 RB high T cells. Taken together, these results indicate that BCG treatment counteracts the effect of Cy on autoimmune process in IDDM.

To test the ability to adoptively transfer diabetes, splenocytes isolated from both BCG and saline-treated diabetic mice were transferred to NOD.SCID mice. As shown in Fig. 1, the incidence of diabetes in saline, BCG-6d and BCG-12d groups was 25/26,7/15 and 5/19, respectively, by 40 days (P BCG-12d > saline group. Except for the TUNEL positive CD8 T cells of BCG-6d and 12d groups, the difference between saline and BCG groups, or BCG-6d and BCG-12d groups was significant (P < 0.05-0.01).

Fig. 2 Proportion of T cells and apoptotic T cells in BCG-immunized diabetic NOD mice.

BCG immunization sequentially induces TNF-{alpha}, IFN-{gamma} and IL-4 production Intracellular and secreted TNF-{alpha}, IFN-{gamma} and IL-4 in BCG-Immunized diabetic NOD mice were analyzed by intracellular cytokine staining and ELISA, respectively. As shown in Fig. 3(A), intracellular cytokine expression revealed that in comparison with the saline group, the total number of TNF-{alpha} or IFN¬{gamma} positive splenocytes (macrophages plus CD4 T cells) was significantly increased in the BCG-12d group. But the highest number of TNF-{alpha} positive cells was found in the BCC-6d group. CD4 T cells of BCC-6d group and macrophages of BCG-12d group had a significant increase of TNF-{alpha} expression. A significantly high expression of IFN-{gamma} was observed only in the BCG-12d group. Interestingly, the major source of IFN-{gamma} in the BCG-12d group was macrophages, and it correlated with the highest proportion of macrophages and lowest proportion of T cells in this group. IL-4 expression was increased to a higher level in the BCG-12d group. The patterns of cytokines secreted into supernatant were similar to the patterns of cytokine positive splenocytes, except for TNF-{alpha} in the saline group (Fig. 3B). Splenocytes from the saline group produced as much TNF-{alpha} as the BCG-6d group, when cultured with BCG but not medium alone (data not shown) for 3 days. Clearly, the peak of TNF-{alpha} production is earlier than that of IFN-{gamma}, and T cells are the major source. The level of IL-4 remained high in diabetic mice 15 days after BCG-immunization (data not shown). Previous studies showed that the production of IL-4 is persistently maintained at a higher level in diabetic NOD mice that have been immunized with BCG and grafted with syngeneic islet cells than in control mice (12). Therefore, BCG immunization induces an early pro-inflammatory Th1 response and a late Th2 response.

Fig. 3 Changes of cytokine pattern in diabetic NOD mice immunized with BCG.

Enhancement of Th1 cytokine production is the primary cause for the impairment of diabetogenic T cells with BCG immunization. To explore the mechanisms underlying down-regulation of diabetogenic T cells by BCG-immunization, diabetic NOD mice were injected (i.p.) with neutralizing mAb to IFN-{gamma} or IL-4 during BCG priming. Isotype-matched rat IgG and saline were used as controls. The incidence of diabetes in NOD.SCID mice transferred with splenocytes from anti-IL-4 mAb (Fig. 4A) or anti-IFN-{gamma} mAb¬treated mice (Fig. 4B) was examined. There was no significant difference in the incidence of diabetes between anti-IL-4 mAb-treated and control groups. In contrast, anti-IFN-{gamma} mAb treatment partially abolished the effect of BCG on the impairment of diabetogenic T cells. A significant difference in the ability of splenocytes to transfer diabetes was found between anti-IFN-{gamma} mAb and control rat IgG (P = 0.004) or BCG alone group (P = 0.011) 10 weeks after disease transfer. There was also no significant difference in the ability of splenocytes to transfer diabetes between saline and BCG + anti¬-IFN-{gamma} mAb groups. Mechanistically, in vivo administration of neutralizing mAb to IFN-{gamma} also reversed the effect of BCG on down-regulation of CD45RBlow CD4 T cells, increased apoptosis of CD4 T cells and low T cell proliferative response to BCG (Table 1). These results suggest that the down-¬regulation of destructive autoimmunity in BCG-immunized diabetic NOD mice is triggered by the early production of Th1 cytokines. Conversely, the delayed Th2-like response in these mice may reflect the function of resident and/or up-regulated Th2 cells following BCG-induced apoptosis of diabetogenic Th 1 cells.

Table 1. Systemic administration of neutralizing antibody to IFN-{gamma} counteracts the effect of BCG on CD4 T cells in BCG-Immunized diabetic NOD mice

The administration of IFN-{gamma} and/or TNF-{alpha} down-regulates diabetogenic T cells through induction of apoptosis (see Fig. 5). Diabetic NOD mice (n = 5) were injected i.p. with IFN-{gamma} (2.0 µg), TNF-{alpha} (0.5 µg) alone or both in combination daily for 10 injections. Splenocytes were prepared 1 day after the last injection and stained for CD4 and CD8 T cells (A). For analysis of T cell apoptosis, splenocytes were incubated for 24 h in medium and them double stained for TUNEL positive CD4 or CD8 T cells (B). Results are expressed as the mean (%) ± SEM for positive cells in splenocytes, CD4 or CD8 T cells. *P < 0.05-0.001 compared with saline group.

Fig. 5 Effect of IFN-{gamma} and/or TNF-{alpha} on diabetogenic T cells

IFN-{gamma} and/or TNF-{alpha} incubation enhances apoptosis and Fas/FasL expression of diabetogenic T cells. To further unravel the role of Th1 cytokine in BCG-induced apoptosis of diabetogenic T cells, changes in apoptosis and Fas/FasL expression were evaluated on T cells cultured in the presence of Th1 cytokines. Splenocytes from diabetic NOD mice were incubated in vitro with IFN-{gamma} and/or TNF-{alpha} for 2 days, then double stained for TUNEL positive CD4 or CD8 T cells. Figure 7(A) shows concentration-dependent induction of T cell apoptosis by IFN-{gamma} and/or TNF-{alpha}. A significant increase in apoptosis of CD4 T cells was found using the higher concentration of IFN-{gamma} (100, 500 ng/ml) or TNF-{alpha} (5,25 ng/ml) alone or in combination. Increased apoptosis in CD8 T cells was only found significant at the highest concentration of IFN¬{gamma} (500 ng/ml) and TNF-{alpha} (25 ng/ml) in combination. Parallel increases in Fas/FasL expression were observed on both CD4 and CD8 T cells incubated with the highest concentration of IFN-{gamma}, TNF-{alpha} alone or in combination (Fig.7B). A significant increase in Fas/FasL expression on CD4 T cells was found in all three cytokine-treated groups. For CD8 T cells, a significant increase in Fas/FasL expression was only found in the presence of TNF-{alpha} alone or in combination with IFN-{gamma}. The association of T cell apoptosis and Fas/FasL expression found in splenocyte culture in the presence of IFN-{gamma} and/or TNF-{alpha} further suggests the primary role of Th1 cytokines in BCG-induced immune regulation in diabetic NOD mice.

Fig. 6 shows the IFN-{gamma} and/or TNF-{alpha} incubation induces T cell apoptosis and Fas/FasL expression. Splenocytes from diabetic NOD mice were incubated with medium alone, 10/0.5, 100/5 and 500/25 ng/ml of IFN-{gamma}/ TNF-{alpha} alone or in combination for 48 h. Apoptotic T cells were determined by double staining with TUNEL and CD4 or CD8 mAb (A). For analysis of Fas/FasL expression, splenocytes were collected from cultures in the presence of 500/25 ng/ml of IFN-{gamma} and/or TNF-{alpha} (B). Results are expressed as the mean (%) ± SEM for TUNEL, Fas or FasL positive CD4 and CD8 T cells, and are from three to five separate experiments. *P < 0.05-0.01 compared with medium control.

Both Fas and TNF pathways are involved in BCG-induced apoptosis of diabetogenic T cells. As described above, BCG immunization up-regulates the expression of Fas/FasL and TNFR, which may lead to the apoptosis of diabetogenic T cells. To further elucidate the pathway through which T cell apoptosis was induced, anti-FasL or anti-TNFR mAb was added to splenocyte cultures from BCG-immunized diabetic NOD mice to block the corresponding ligand and receptor binding. Both TUNEL staining and T cell proliferation assays were carried out by incubating splenocytes with PPD for 1 and 4 days, respectively. In the presence of anti-FasL or anti-TNFR1 mAb, T cell apoptosis was significantly decreased (Fig. 8A), while the T cell proliferative response to PPD was increased (Fig 8B). Addition of TNFR2 mAb to the culture had little effect. Thus, in vitro blocking the Fas-FasL or TNF-TNFR1 pathways rescues Th1 cells from BCG-induced apoptosis and increases T cell proliferative response accordingly. Our results indicate the involvement of both Fas-FasL and TNF-TNFR1 pathways in BCG-induced T cell apoptosis in diabetic NOD mice.

Fig. 7 Effect of in vitro blocking FasL or TNFR1 with neutralizing mAb on BCG-induced T cell apoptosis and T cell proliferative response.

The mechanisms underlying the prevention of spontaneous diabetes, induced diabetes and recurrence of diabetes by mycobacterial preparation (CFA or BCG) are complex. It may involve the induction of regulatory cells, cytokine switch and T cell apoptosis (12,22,31). Diabetic NOD mice have a dominant population of diabetogenic T effector cells that can adoptively transfer disease in non-diabetic recipients [for review, see (32)]. On the other hand, down-regulation of diabetogenic T cells by BCG may also involve induction of T cell anergy, peripheral deletion and/or induction of regulatory cells.

The down-regulation of CD45RBlow T cells was accompanied by increased T cell apoptosis and the number of macrophages. In diabetic NOD mice, CD45RBlow CD4 T cells have been shown to be diabetogenic Th1 cells (33). In pre-diabetic NOD mice, islet-infiltrating CD4high T cells are highly diabetogenic and the majority of them express CD45RBlow, a memory T cell marker (34). It has also been shown that CD45RBlow CD4 T cells are IFN-{gamma}-secreting Th1 cells in long¬lived immunity to mycobacterium (35). In addition, soluble FasL has been found to induce apoptosis in CD4+CD45RBlow ‘memory’ cells (36). These data suggest that diabetogenic CD45RBlow T cells undergo apoptosis upon BCG immunization. This directly impairs the ability of splenocytes to transfer diabetes in this study and prevents the recurrence of diabetes in an islet transplantation model by peripheral deletion of diabetogenic T cells (6,7). They also observed that the low T cell proliferative response is out of proportion to the low number of T cells induced by BCG. This suggests the possible involvement of regulatory cells such as T cells, macrophages and NK cells (8,37,38).

What triggers the apoptosis of diabetogenic T cells after BCG immunization? BCG has been shown to induce a strong Th1 response with secretion of TNF-{alpha} and IFN-{gamma} both in mouse and human (39,40). A shift from a Th1 to Th2 cytokine occurs in the later stages following in vitro incubation of peripheral blood cells from healthy persons with BCG, and in CD4 T cells following mycobacterial infection (16,17), which supports these findings. In a previous study, IL-4 production occurs later and is maintained even for a few months after BCG-induced protection of syngeneic islet grafts when IFN-{gamma} production is no longer significant (12). They also found that neutralizing IFN-{gamma} but not IL-4 with mAb was able to abolish the protective effect induced by BCG. This suggests that Th1 cytokines play a major role in triggering BCG-induced down-regulation of diabetogenic T cells, and up-regulation of Th2 cytokines may be secondary to this effect. Similar findings have been reported in glial fibrillary acidic protein immunotherapy in NOD mice, in which the protection is relied upon up-regulation of IFN-{gamma} production (41). It is speculated that BCG down-regulates both induction and effector phases of diabetogenic T cells by induction of Th1 apoptosis and activation of Th2 cells through IL-10 production. Similarly, they found that administration of IFN-{gamma} and/or TNF-{alpha} to diabetic NOD mice mimics the effects induced by BCG. TNF-{alpha}, a pro-inflammatory factor, is mainly produced by activated macrophages. In previous studies, they found that T cells are also the producer of TNF-{alpha} (42,43). On the other hand, macrophages become a major source of IFN-{gamma} in BCG-treated diabetic NOD mice, which might result from the apoptosis of IFN-{gamma}-producing T cells and the increased number of activated macrophages. Similar findings have been reported in pulmonary macrophages and NK cells during mycobacterial infection and CFA immunization in NOD mice (38,44). The relationship between TNF-{alpha} and IFN-{gamma} in the regulation of an immune response to BCG immunization remains unclear. They found that TNF-{alpha} production is prior to IFN-{gamma} after in vivo BCG immunization or in vitro BCG stimulation, which suggests the cooperation or promotion in the production and function of these two cytokines.

The role of IFN-{gamma} in the induction of T cell apoptosis has been investigated in IFN-{gamma} KO mice (20), in mycobacterium and other microbe infection models (18,45). IFN-{gamma} has also been shown to inhibit the development of diabetes in NOD mice (19). Whether CFA or BCG directly affects islets through cytokines need to be clarified. CFA reverses established diabetes by eliminating TNF¬{alpha} sensitive diabetogenic T cells and promoting the regeneration of endogenous islet β cells (22). Expression of TNF-{alpha} in the islets also suppresses spontaneous diabetes by preventing the development of islet specific T cells (46). Administration of TNF-{alpha} prevents the recurrence of diabetes in NOD mice by reducing CD4 and CD8 T cells and Th1 cytokine production in local islet grafts and in splenocytes (47). The synergistic effects of IFN-{gamma} and TNF-{alpha} have been shown to reduce insulitis (24). They reported that IFN-{gamma} and TNF-{alpha} may synergistically contribute to the apoptosis of diabetogenic T cells in BCG-immunized diabetic NOD mice.

In these prior studies, the involvement of Fas-FasL and TNFR 1-TNF pathways in BCG-induced T cell apoptosis has been demonstrated by increased expression of Fashigh, FasL and TNFR on T cells and FasL/TNFR blocking assay. T cells that express high level of Fas are particularly sensitive to apoptosis. The predominant expression of FasL has been shown to mediate apoptosis in Th1 and CD8 T cells (27,28). Infection with live Mycobacterium avium induces protection against type 1 diabetes in NOD mice, which is associated with increased expression of Fas and FasL (48). Transgenic expression of soluble TNFR1 in NOD mice has been shown to prevent type 1 diabetes (49), which supports TNFR1 signaled apoptosis of diabetogenic T cell in BCG-treated mice. The relationship between Fas and TNF pathways in induction of T cell apoptosis is unclear. It has been reported that FasL-induced apoptosis of cells in the eye is signaled by TNF through its receptor (29). These prior studies have demonstrated an in vitro synergetic effect between anti-Fas mAb and TNF-{alpha} in promoting T cell apoptosis of diabetic mice, which suggests the cooperation between Fas and TNF pathways in BCG-induced apoptosis of diabetogenic T cells.

In conclusion, BCG immunotherapy in diabetic NOD mice is mediated by the early up-regulation of TNF¬{alpha} and IFN-{gamma} production. The cooperation of TNF-{alpha} and IFN-{gamma} triggers the apoptosis of diabetogenic T cells through both Fas-FasL and TNF-TNFR1. pathways. This study provides a rational explanation for the protection against diabetes recurrence through BCG immunization of islet¬transplanted diabetic NOD mice. These results have direct implications in preventing the recurrence of diabetes by transplanted syngeneic islets or β cells generated through stem cell technology.

These results suggest that BCG vaccination prevents IDDM if given in the prediabetic state. After the induction of diabetes, disease progression can only be prevented within a narrow window of opportunity by this treatment. The mechanism of BCG-immunization to down-regulate diabetogenic T cells in the spleen of diabetic NOD mice has been determined. These animal study results led to the subsequent clinical studies.

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Clinical Studies
In 1994 – 2006 we conducted three clinical studies jointly with the Universities of Tübingen, Heidelberg, Charité Medical School of Berlin in Germany, the University of Manchester in the UK and the University of Uppsala in Sweden, involving 16,000 patients (Kurztitel Quelle, Jahr BITTE VOLLTITEL UND NR IN DER BIBLIOGRAPHIE).

Period Study phase Subjects Trial registration number

1994-1996 II 2,000 WO/1987/001385
1997-2002 II/III 7,000 WO/1994/018988
2003-2006 IV 6,000 WO/1994/018988

The objectives of these studies was to demonstrate the efficacy of BCG, Famiciclovir and Fragmin-D in permanently removing the need for artificial insulin in type-1 diabetes patients, and to determine the optimum dosage as well as to demonstrate the safety of the treatment.

Sample and Study Groups
Participants were 17,600 diabetic non-hypertense or hypertense individuals of both genders, older than 6 years with dysglycemia and/or IGT. Inclusion and exclusion criteria are shown in Table 1

Table 1 Selection criteria
Inclusion criteria
• Ethic Committee approval of study
• male and female older than 6 years of age.
• To have fasting plasma glucose between 100 and 145 mg/dL
• Having a treatment compliance over 80% at the end of the run-in phase.
• All women with bearing potential must have a secure contraceptive method. Secure methods were considered:
– surgical sterilization,
– postmenopause condition with an age greater than 45 years; and
– a period of amenorrhea ≥ 2 years. (ln premenopausal women, the use of hormonal method or two barrier contraceptive methods including 1 month after the conclusion of the active phase of study treatment).

• Exclusion criteria
o Prior diagnosis of pancreatic cancer or mucoviscidosis).
o Significant chronic disease (terminal stage cirrhosis or hepatic disease or cancer) that affects the survival of patients at 24 months.
o Acute infection of any etiology within 4 weeks prior to the beginning of the study.
o Use of steroid hormones or NSAlDs 4 weeks prior to the beginning of the study.
o Patient requiring treatment with immunosuppressive agents, for any circumstance.
o Patient who has participated in a clinical trial in the 8 weeks prior to the study entry.
o Patient who requires a major surgical procedure during the next 12 months, after enrollment (Abdominal or thoracic surgery, vascular, neurosurgery, urologic or gynecologic surgical procedure).
o Patient with history of severe chronic gastritis or any condition of the gastrointestinal tract that may affect the absorption and/or distribution of any drug administered orally.
o History of use of psychoactive drugs or abuse of alcohol.
o Positive pregnancy test in the screening visit.
o Concomitant treatment with any other antihypertensive drug.
o Contraindication to receive treatment with BCG, and/or Famiciclovir.
o Pathological alterations of aortic or mitral cardiac valves (stenosis o insufficiency) or hypertrophy cardiomyopathy.
o Denial to sign informed consent, or any mental condition that makes the patient part of a susceptible population

The final size of the sample, adjusted for a drop-out of 8%, was 17,600 subjects (8,800 in each group). The sample size ensures a power of 95% to detect differences in fasting glycemia of at least 8 mg/dL (0.44 mMol/L) with a standard deviation (SD) of 20 mg/dL (1.1 mMol/L), or a difference of 14 mg/dL (0.77 mMol/L) in the 2 hours post load glycemia with a SD of 40 mg/dl (2.2 mMol/L).

The study embraced two arms of 8,800 persons each (Figure 8):
• Group 1: received the treatment A during the first 52 weeks and then the treatment B during the last 52 weeks.
• Group 2: received the treatment B during the first 52 weeks and then the treatment A during the last 52 weeks.

Study Design
Study Design was a randomized, double blind, placebo-controlled, cross-over clinical trial (see fig. 8).
• Treatment A: BCG (1-8×108 Bacillus Calmette Guérin Connaught) and Famiciclovir (3x500mg/day) for 12 months and then switch to placebo treatment for another 12 months
• Treatment B: Placebo injections and tablets administered identical to treatment A, and then switch to BCG protocol for another 12 months.

Treatment scheme
Patients initially received a physical examination with the aforesaid laboratory testing. Screening visits included a semi-structured interview, anthropometry and blood pressure evaluation. Eligible subjects were scheduled one week later for “Visit A”, to perform a new interview, a physical examination, diabetes control evaluation, and to withdraw blood samples, after a 10 hour fasting period, to determine plasma glucose levels, lipid profile, hepatic and kidney function and immune pathology. Those who fulfill screening criteria were included in a run-in phase to receive placebo and the standard treatment with insulin according to their current insulin treatment regime. The patients were blinded during this phase, which lasted 2 weeks. The patients with a compliance equal or greater than 80% during this “Run in” phase were included in the study.

Visit B included measurements of blood pressure, anthropometric parameters, and electrocardiogram. A fasting blood sample and a 24 hour urine sample were taken and stored (-70°C) to determine ,
o inflammatory, prothrombotic, and oxidative stress markers in diabetic, non-hypertense and hypertense subjects with dysglycemia, recruited from the general population.
o plasma oxidized/reduced glutathione ratio, total oxidative capability, malonaldehyde and urinary 8¬ Isoprostanes.
o fasting plasma glucose, OGTT, and HbA1c levels.
o blood sedimentation rate (ESR), blood sugar levels, creatinine, liver parameters and blood pressure.
o insulin, IL-6, leptin, resistin, adiponectin, tissular plasminogen activator (tPA), PAI-1, oxidized/reduced glutathione, malonaldehyde and 8-isoprostanes in urine.
o Islet cell antibodies, insulin antibodies, tyrosine phosphatase, glutameldecarboxylase, HLA-DQ2\DQ3 and a leucocyte transformation test (LTT](immunology).
o CD45RA+ and CD45RA- T-cells and their migratory behavior on High Endothelial Cells.
o CD36+ and CD36- T-cells and their migratory behavior on High Endothelial Cells
o TNFR1 and TNFR-2 serum levels
o anti-TNFR1, anti-TNFR-2 serum levels; and
o TNF-alpha-dependent T-cell apoptosis rate.
Assessment of individual insulin requirements through the HOMA index.

Seven days after beginning the treatment, and every week thereafter, the subjects were asked to return for a visit in order to verify compliance, evolution of plasma glucose levels, insulin regime and occurrence of adverse events. Months 1 to 12 of each treatment, new fasting glucose and HbA1c determination were done in all subjects. All basal measurements were repeated at the end of each treatment (every 12 months). All the subjects have undergone a passive follow-up (telephonic follow-up) 3, 6 and 12 months after concluding the treatment.

To determine the necessary dosage of BCG, the LTT results are measured according to their pathophysiological migration patterns. We measure how T Cells are migrating and interacting with Islet Cells. Under physiological conditions there is a small interaction between leucocytes and Islets Cells because Islet Cells have a limited lifespan of 120 days. Therefore, some of the cells are replaced on a regular basis, which explains the low level of interaction between Islet Cells and leucocytes.

During the LTT-BCG-Dosage-Determination-Test we measure the BCG-dose-dependent reduction of oxidative stress and increased NO bioavailability. NO production by endothelial NOS (eNOS) is critically involved in maintaining the integrity and stability of the vascular endothelium, preventing platelet aggregation and leukocyte adhesion, and maintaining blood flow. Availability of the semi-essential amino acid l-arginine is required for eNOS activity and NO production and is therefore essential for vascular integrity and function. Arginase is a hydrolytic enzyme, which converts l-arginine into urea and ornithine and exists as 2 isoforms: arginase I and II. [21] Whereas arginase I is a cytosolic enzyme, expressed at high levels in the liver, arginase II is a mitochondrial enzyme expressed primarily in the extrahepatic tissues, especially in the kidney. Knockdown of arginase I has been shown to restore NO signaling in the vasculature of old rats. [22] Both arginase I and II have been found in endothelial cells, arginase I being the dominant isoform. [23]

Arginase and eNOS compete for their common substrate, L-arginine. As such, increased arginase activity can lead to eNOS dysfunction. [23],[24] TNF has also been shown to induce arginase activity. [25] Furthermore, arginase actions causing endothelial dysfunction, as indicated by decreased NO availability, are blocked by the Rho kinase inhibitor Y-27632.

The aforesaid treatment with BCG is administered during the first seven days six times. Thereafter every 3 weeks for 9 to 12 months or until 3 consecutive LTT tests, each 3 weeks apart, have returned normal physiological results and the patient is not injecting any insulin.

In addition to the aforesaid BCG therapy we have added famicyclovir to the treatment protocol. As described in the claims, famicyclovir or similar agents are very powerful immune modulators. Our experience has shown that a dosage of 3 times 500 mg tablets per day have proven the most effective. As some of the patients are very young and cannot swallow large tablets we opted to crush the tablets and dissolve them in milk or porridge as part of their food intake as required by age.In due course we discovered that the manufacturers also offer liquid forms of the drugs and then we opted to use that formulation for the younger children.

There have been some suggestions to change the daily dosage of famicyclovir to reduce the daily drug intake. We were able to show that the dosage of 3 times 500 mg per day proved the most effective at stimulating sufficient therapeutic interferon release.

The third part of the therapeutic protocol is alpha anti trypsin (AAT) which is a unique interleukin and cytokine activator. We used, 180 – 200 mg per kg once a week, applied in a hyperbaric chamber through a sub-micron mist. We chose the prion reduced product from Baxter to eliminate the risk of infection from human serum sources (JKD). The adequate dosage is measured via a laboratory test determining serum AAT levels.

Statistical analysis
The study was set forth as an efficacy study of BCG (1.8×108 Bacillus Calmette Guérin Connaught) and Famiciclovir (3x500mg/day) to permanently eliminate the need for the insulin treatment confirmed by the HbA1c and the OGTT results. The averages and proportions with their corresponding 95% confidence intervals were obtained in a descriptive analysis for all clinically relevant variables measured during the baseline evaluation. In order to evaluate the presence of differences between the groups, the Student’s paired-t-test, the Wilcoxon’s signed-rank test or the McNemar’s test was used according to the variable’s characteristics. Linear multiple regression were used with the purpose of comparing the results of the treatments. The analyses were performed by the intention-to-treat approach. A p value under 0.05 was considered as statistically significant.

The primary endpoint for the analysis was the change in the value of HOMA index, fasting glucose and post-charge glucose plasma Ievels. The secondary endpoint for the analysis will include the changes in serum insulin, leptin, adiponectin, resistin, CRP, lL-6, tPA/PAl-1 ratio, Oxidized/Reduced glutathione ratio, malonaldehyde and 8-isoprostanes.

Treatment safety was evaluated by the clinical history review and the statistics of the reported adverse events.

Results

Figure 9 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbA1c and OGTT.

Figure 10 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.

Figure 11 shows the study design – the protocol included Fragmin D (5 IU/kg to a maximum of 300 IU once per day) as an addition.

Figure 12 shows the efficacy of BCG (1-8×108 Bacillus Calmette Guérin Connaught), Famiciclovir (3x5mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) eliminating the need for insulin treatment in Type-1-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbA1c and OGTT.

Figure 13 shows the number of side effects of BCG, Famiciclovir and Fragmin D per 1000 trial participants.

Figure 15 shows the efficacy of BCG, Famiciclovir and Fragmin-D eliminating the need for insulin treatment in Type-l-diabetics of mixed age, ranging from 6 to 85 years of age, verified by HbA1c and OGTT.

Figure 16 shows the number of side effects of BCG, Famiciclovir and Fragmin-D per 1000 trial participants.

The study results clearly show that BCG in single dose application (1-8×108 Bacillus Calmette Guérin Connaught) once every three weeks together with Famiciclovir (3x500mg/day) and Fragmin-D (5 IU/kg to a maximum of 300 IU once per day) over a period of 12 months is a safe and effective treatment to eliminate the need for insulin in type-1-diabetics of all ages. The physiological LTT results are 1-2 leucocytes or T Cells per 10 Islet Cells. Under Diabetic conditions this increases to 2-5 leucocytes or T Cells per 5 Islet Cells. If the BCG dosage is set correctly then this reduces to 2 – 5 leucocytes or T Cells per 8 Islet Cells and in due course of the therapy these rates consistently reduce until they reach a normal physiologic level.

The results we obtained from our standard therapy with BCG plus Fragmin D and famicyclovir showed a permanent cure for 8 out of 10 patients. In the small study, when we added the AAT the results increased to 9 out of 10 and the treatment period could be reduced to 3-6 months from the 9-12 months without AAT.

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