During the last decades, development of specific agents affecting T helper cell subpopulations, i.e., Th1, Th2 and Th17, differentiation has drawn special attention. Many natural products were reported as a good agent for modulating the immune response by regulating of the differentiation of T helper cell subpopulations. These products are potential to be the immunostimulants for treatment of various diseases including infectious diseases, cancers, autoimmune diseases and also diabetes.

In this study, we aim to study the immunomodulatory effects of the Operation BIM product. We investigate the possible effect of the Operation BIM product on the controlling of T helper cell subpopulation differentiation.

Objectives

The objective of this study is to investigate the possible effects of the Operation BIM product on regulation of T helper cell subpopulations. The levels of various cytokines of blood collected from healthy donors before and after taking Operation BIM product for 15 days were compared.

Study approaches:

1. Study subjects

     • 12 healthy volunteers: 7 males and 5 females

     • Age: 20-50 years old

     • The recruited volunteers were separated into 2 groups:

          o Group 1: taking placebo; 6 subjects

          o Group 2: taking Operation BIM product; 6 subjects

2. Blood collections

Blood (5 ml. using heparin as anti-coagulant) were collected from each subjects at day 0. According to their group, subjects took Operation BIM product or placebo (4 capsule/day) everyday for 15 days. Afterwards, blood were collected (5 ml. using heparin as anti-coagulant) for the secon d time at day 16.

3. Study of the effect of the Operation BIM products on the regulation of T helper cell subpopulations

Peripheral blood mononuclear cells (PBMCs) were isolated from the collected blood by using Ficoll-Hypaque gradient centrifugation. PBMCs were in vitro stimulated with or without anti-CD3 monoclonal antibodies (clones OKT3) and cultured for 24 hours at 37oC in a CO2 incubator. The cell cultured supernatant were collected and centrifuged 20,000 rpm, 2 min. The cell free supernatants were separated and stored at -70°C for determination of cytokines.

4. Determination of T helper cell cytokines

Cytokines in the cell cultured supernatant were determined by Flow cytomixÒ (eBioscience, Inc. San Diego, CA, USA) followed the manufacturer’s protocol. Comparison of the cytokine levels from un-stimulated PBMC and stimulated PBMC in term of Stimulation Index on day 0 and d ay 15 were performed.

Results

The stimulation indexes of various cytokines in subjects taking the BIM product and placebo were calculated and compared. By normalization using subjects taking placebo, % enhancement and reduction of subjects taking BIM product were shown in Figure below. The results indicates that Operation BIM product reduced the production of TNF-a, IFN-g, IL-4, IL-5, IL-9, IL-10, IL-12 and IL-17.

In type 2 diabetes, which is usually associated with obesity or older age, is diabetes which mostly the result of insulin resistance. In these patients, the muscle or adipose cells do not respond adequately to normal levels of insulin produced by intact beta-cells. On other hand, Type 1 diabetes usually starts in people younger than 30 and is therefore also termed juvenile-onset diabetes, even though it can occur at any age. Type 1 diabetes is a chronic autoimmune disorder that precipitates in genetically susceptible individuals by environmental factors (1). The body's own immune system attacks the beta-cells in the islets of Langerhans of the pancreas, destroying or damaging them sufficiently to reduce and eventually eliminate insulin production. A combination of factors including a Th1-skewed CD4+ response as well as a deficiency of regulatory T cells are considered to be important hallmarks of disease progression (2).

There are several information suggesting that Th1 cells play a major role in diabetes, driving the development of disease via IFN-γ (3). This includes the observations that blockade of IFN-γ (4) or absence of STAT4 (5, 6) prevent disease, whereas IL-12 promotes accelerated diabetes (7). Tumor necrosis factor alpha (TNF-a) has well-described effects on lipid metabolism in the context of acute inflammation, as in sepsis. Recently, increased TNF- a production has been observed in adipose tissue derived from obese rodents or human subjects and TNF- a has been implicated as a causative factor in obesity-associated insulin resistance and the pathogenesis of type 2 diabetes. Thus, current evidence suggests that administration of exogenous TNF- a to animals can induce insulin resistance, whereas neutralization of TNF- a can improve insulin sensitivity (8). In addition, TNF-α shows a significant positive association with insulin secretory capacity when adjusting the effects of the confounding factors - age, sex and BMI in IGR subjects. Thus, there may be a causal relationship between TNF-α with insulin secretory defect in prediabetic and IGR subj ects (9).

Th17 immunity has been demonstrated in the development of autoimmune diabetes in animal models. In NOD mice, a model of spontaneous autoimmune diabetes, inhibition of Th17 cells has been shown to regulate autoimmune diabetes (10). IL-17 neutralization either by anti–IL-17 or by rIL-25 prevented development of autoimmune diabetes when given from 10 weeks of age to NOD mice (11). Earlier treatment from 5 weeks of age did not alter diabetes progression, which suggests that Th17 immunity contributes to the progression of autoimmune diabetes during the effector phase of the disease. Natalia et. al. differentiated islet-reactive BDC2.5 TcR transgenic CD4+ cells in vitro into Th17 cells and transferred them into NOD.scid and neonate NOD mice. NOD.scid recipient mice developed rapid onset of diabetes with extensive insulitic lesions, whereas in newborn NOD mice, despite extensive insulitis, most recipient mice did not develop diabetes. Surprisingly, BDC2.5+ cells recovered from diabetic NOD.scid mice, in comparison with those from neonate NOD mice, showed predominant IFN-γ over IL-17 expression, indicating conversion of donor cells into Th1 cells. Moreover, diabetes progression in NOD.scid recipients was dependent on IFN-γ while anti-IL-17 treatment reduced insulitic inflammation. These results indicate that islet-reactive Th17 cells promote pancreatic inflammation, but only induce IDDM upon conversion into IFN-γ producers.

In our study, we have demonstrated that the BIM product has immunomodulation effect. The product reduced the production of TNF-a, IFN-g, IL-12 and IL-17 which involved in the induction of diabetes and disease progression. Reduction of these cytokines may prevent the development of diabetes and bring the patients to have a better life.

References

1. Atkinson MA, Eisenbarth GS. Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 2001;358: 221–229.

2. Anderson MS, Bluestone JA. The NOD mouse: a model of immune dysregulation. Annu Rev Immunol 2005;23: 447–485.

3. Wang B, et al. Interferon-gamma impacts at multiple points durin g the progression of autoimmune diabetes. Proc. Natl. Acad. Sci. U.S.A. 1997;94:13844-13849.

4. Nicoletti F, et al. The effects of a nonimmunogenic form of murine soluble interferon-gamma receptor on the development of autoimmune diabetes in the NOD mouse. Endocrinology. 1996;137:5567-5575.

5. Boyton RJ, et al. Stat4-null non-obese diabetic mice: protectio n from diabetes and experimental allergic encephalomyelitis, but with concomitant epitope spread. Int. Im munol. 2005;17:1157-1165.

6. Yang Z, et al. Autoimmune diabetes is blocked in Stat4-deficient mice. J. Autoimmun. 2004;22:191-200.

7. Trembleau, S., et al. Interleukin 12 administration induces T helper type 1 cells and accelerates autoimmune diabetes in NOD mice. J. Exp. Med. 181:817-821, 1995 .

8. Moller DE. Potential role of TNF-alpha in the pathogenesis of insulin resistance and type 2 diabetes. Trends Endocrinol Metab. 11(6):212-7, 2000.

9. Mosaraf Hossain, M Omar Faruque, Golam Kabir , Naimul Hassan, Dwaipayan Sikdar, Quamrun Nahar, Liaquat Ali. Association of serum TNF-α and IL-6 with insulin secretion and insulin resistance in IFG and IGT subjects in a Bangladeshi population. International Journal of Diabetes Mellitus. Vol .2 issue 3 P.165- 168 December 2010

10. Jain R, et al. Innocuous IFN gamma induced by adjuvant-free ant igen restores normoglycemia in NOD mice through inhibition of IL-17 production. J. Exp. Med. 2008; 205: 207–218.

11. Emamaullee JA, et al. Inhibition of Th17 cells regulates autoimmune diabetes in NOD mice. Diabetes 2009;58:1302-1311.

12. Natalia MO, et al. Th17 cells promote pancreatic inflammation but only induce diabetes efficiently in lymphopenic hosts after conversion into Th1 cells. Eur J Immunol. 2009;39(1): 216–224.

Report by

Professor Dr. Watchara Kasinrerk, Ph.D.

Director; Biomedical Technology Research Center,

Faculty of Associated Medical Sciences,

Chiang Mai University,

Chiang Mai 50200, Thailand