STUDY OF INTERFERONOGENOUS ACTIVITY OF THE NEW PROBIOTIC FORMULATION DEL-IMMUNE V ®

Context: Prior testing of Del-Immune V® has indicated effectiveness for immune system support; however, mechanisms of action and optimal doses have not yet been researched. Objective: To study the mechanisms of the immunomodulating effects of Del-Immune V and to investigate its dose-dependent effects on production of immunoregulatory cytokines in vivo and in vitro. Design, Setting, Participants, Interventions: One hundred forty nondescript laboratory mice with body mass ranging from 14 to 16 grams were divided into 7 test groups. Groups I, II, and III received 0.5 ml of aqueous solution of Del-Immune V by mouth in doses of 5, 50, and 500 μg per mouse respectively for 5 days at 24-hour intervals. Group IV mice received 0.5 ml Bifidim suspension by mouth in a dose of 50 μg/mouse on the same schedule. Group V mice (control group) received 0.15 M NaCl. Group VI and VII mice received a single dose of 50 μg/mouse Del-Immune V (Group VI) or Bifidim (Group VII) on day 1 of the test period. Eight hours after administration, and every 24 hours thereafter for 5 days, several mice from each group were killed by cervical dislocation; blood serum, peritoneal exudate macrophages, and splenocytes were obtained from each group of mice for testing. Main Outcome Measures: Interferonogenous activity of cultured splenocytes and serum levels of interferon. Results: Groups I-IV showed a marked increase of IFN levels in blood serum after administration of Del-Immune V or Bifidim; the optimal daily dose was found to be 50 Interferonogenous activity of Del-Immune V Page 3 μg/mouse. The highest serum IFN level was reported 24 hours after administration. The control group remained unchanged. Maintenance of elevated circulating IFN was possible only through repeated administration. STUDY OF INTERFERONOGENOUS ACTIVITY OF THE NEW PROBIOTIC FORMULATION DEL-IMMUNE V® Valentyn S. Pidgorskyy, Liubov N. Shynkarenko, Natalya A Timoshok, NykolayY. Spivak INTRODUCTION Among a large number of presently known therapeutic products that utilize lactobacilli, cell wall peptidoglycan is enjoying growing popularity as an immunomodulator that contains, among other things, fragments of DNA and cell peptidoglycan of the strain Lactobacillus rhamnosus V. Del-Immune V® (manufactured by Pure Research Products, LLC, Boulder, Colorado) was registered by the US Food and Drug Administration in 2002 as a food supplement for immediate immune system support. The biochemical structure of Del-Immune V and preliminary experimental and clinical data indicate that Del-Immune V may be highly effective in infectious diseases of viral (flu, hepatitis C), bacterial (bronchitis), and fungal etiology, allergies of all severity levels, asthma, chronic fatigue, and fibromyalgia. The mechanism of such a wide scope of biological activity of the formulation is still unclear. The goal of our research, therefore, was to study the mechanisms of the immunomodulating effects of Del-Immune V and to describe its dose-dependent effects on production of immunoregulatory cytokines in vivo and in vitro. The last 5 years have been marked by increasingly active study of the mechanisms of the immunobiological effects of probiotics and bacterial medications. As a result, bacterial medications such as liastenum (blasten), deodan, licopid, prodigiosanum, salmosanum, sodium nucleinate, MC (molecular composition – yeast DNA and Tilorone), biostim, BCG, rumurtide, ribomunyl, and lactolin are being used, in both trials and clinical practice, for different pathologies.(1-4) The adjuvant effect of BCG and the immunomodulating activity of formulations containing derivatives of lactobacilli, such as liastenum (blasten, Lactobacillus Interferonogenous activity of Del-Immune V Page 5 delbrueckii) and deodan (Lactobacillus bulgaricus), have been associated with peptidoglycans and their structural components, muramyl dipeptides (MDP). The most active analog of MDP, MurNac-L-Ala-D-Glu-NH2, has demonstrated adjuvant and pleiotropic effects and is capable of inducing a number of cytokines: IL-1, tumor necrosis factor (TNFα), IL-2, IL-6, IL-8, IL-12, and interferon gamma (IFN-γ). These cytokines in turn stimulate nonspecific cytotoxicity of normal and effector lymphocytes and natural killer cells (NK), and coordinate the body’s immune response, depending on the nature of the aggressive agent and the T-helper differentiation (Th1 or Th2). These properties of peptidoglycans indicate a basis for creating immunomodulating formulations for clinical use. Lactobacilli in the generally recognized as safe (GRAS) group are good sources of peptidoglycans. Toll-like receptors TLR4 and TLR2 for MDP and peptidoglycans have been identified on the surface of lymphocytes and macrophages. Fragments of probiotic bacterial DNA are interesting because of their capacity to stimulate production of cytotoxic lymphocytes and NKC, activate the complement system, heighten cytostatic and cytotoxic activity of macrophages, and regulate production of immunoregulatory cytokines. Owing to the ТТТСGТТТ DNA pattern of the strain, Lactobacillus rhamnosus GG was found to be a factor preconditioning immunobiological activity of the probiotic producer. Thus, CpG DNA are identified with the help of TLR9 and TLR10 expressed in the intercellular (endosomes) cell compartments. CpG DNA identification with TLR9 and TLR10 results in activation of neutrophils and cytokine production. MATERIALS AND METHODS The study examined the dose-dependent effect of Del-Immune V on production of immunoregulatory cytokines in nondescript mice with body mass of 14-16g. One hundred forty animals were selected on the basis of the analogue principle, and were divided into 7 Interferonogenous activity of Del-Immune V Page 6 groups of 20. The animals were fed balanced rodent food and water ad libitum. Group I, II, and III mice received 0.5 ml of aqueous solution of Del-Immune V orally in doses of 5, 50, and 500 μg/mouse respectively for 5 days at 24-hour intervals. Group IV mice were administered 0.5 ml Bifidim suspension (control probiotic medication) orally in a dose of 50 μg/mouse on the same schedule. The Bifidim was a dry mass of antagonistic bifidus bacteria immobilized on enterosorbent in combination with ascorbic acid (Intervetmed Ltd., Kiev, Ukraine). Group V mice were administered 0.15 М NaCl. Group VI and VII mice were used to study the interferonogenous activity of a single administration of 50 μg/mouse of DelImmune V (Group VI) or Bifidim (Group VII). Cytokine production by IFN and TNF was examined in intact and treated mice 8 hours after initial administration and then every 24 hours for the next 5 days. For this purpose, several mice from each group were killed by cervical dislocation ; blood serum, peritoneal exudate macrophages (PEM), and spleen, from which splenocytes were harvested from each group of mice for testing. The optimal dose of Del-Immune V was also tested via in vitro induction of immunoregulatory cytokines in splenocytes and PEM (1х10 cell/ml) of treated and intact mice by culturing cells with the formulation in final concentrations of 5, 50, and 500 μg/ml. Interferonogenous activity of the tested formulations was assessed in comparison with Bifidim 50 μg/ml and standard inductors (IFN-α; Newcastle Disease Virus, NDV–10 TCD50/cell; IFN-γ; phytohemagglutinin, PHA–20 μg/ml; Difco; TNF, LPS E. Coli 0111–4 μg/ml–Sigma USA). Levels of cytokine production (IFN and TNF) were determined 6, 24, and 48 hours after incubation of the cell with the formulations. Biological activity of TNF was assessed by cytotoxicity in the passaged culture of murine fibroblasts L-929. The result was recorded on a multiscanner (Dynatech, Switzerland) with a wavelength of 540 nm. The cytotoxicity index was calculated using the formula CI = К-О/Кх100%, where К and О represent optical density values for the cell in the Interferonogenous activity of Del-Immune V Page 7 culture medium (RPMI 1640 with 10% FСS). The calibration curve based on standard recombinant TNF formulation Sіgma was used for standardization of the cytotoxicity index. IFN levels in cell cultures and serum were measured using standard microtitration in the passaged cell culture L-929 against 100 TCD 50 indicator virus (vesicular stomatitis virus, Indiana VSV) with constant СО2 level(15). The significance of the results was analyzed by Student-Fisher t-test. Differences of P < .05 were considered to be significant. RESULTS Daily oral administration of Del-Immune V or Bifidim to Groups I-III in the course of 5 days in doses of 5, 50, or 500 μg/mouse resulted in a marked increase in IFN levels in blood serum (see Figure 1). The optimal interferonogenous dose was found to be 50 μg/mouse (Group II). After 24 hours of observation, circulating IFN levels in Group II reached 4.5 log2 U/ml (P > .05). After repeated administrations, levels reached 5.5±0.7 log2 U/ml (P > .05), in comparison with 2.0±0.7 log2 U/ml in the control group (Group V). Further administration of Del-Immune V in a dose of 50 μg/mouse on day 3 allowed for maintenance of the 5.5±0.5 log2 U/ml level. Administration of the formulation on days 4 and 5 resulted in nonsignificant decreases in circulating IFN levels. When Del-Immune V was administered in doses of 5 and 500 μg/mouse (Groups II and III), findings were similar, although maximum interferon levels were not as high. Insert Figure 1 about here. One-time oral administration of Del-Immune V or Bifidim to mice in a dose of 50 μg/ml resulted in increased circulating IFN level 8 hours after administration. The highest serum IFN level was reported 24 hours after administration, while levels in control group animals remained unchanged (see Table 1). Insert Table 1 about here. Interferonogenous activity of Del-Immune V Page 8 Forty-eight hours after administration of Del-Immune V, serum IFN levels in all active groups remained reliably enhanced in comparison with the control group, but IFN was later eliminate


INTRODUCTION
Among a large number of presently known therapeutic products that utilize lactobacilli, cell wall peptidoglycan is enjoying growing popularity as an immunomodulator that contains, among other things, fragments of DNA and cell peptidoglycan of the strain Lactobacillus rhamnosus V. Del-Immune V® (manufactured by Pure Research Products, LLC, Boulder, Colorado) was registered by the US Food and Drug Administration in 2002 as a food supplement for immediate immune system support.
The biochemical structure of Del-Immune V and preliminary experimental and clinical data indicate that Del-Immune V may be highly effective in infectious diseases of viral (flu, hepatitis C), bacterial (bronchitis), and fungal etiology, allergies of all severity levels, asthma, chronic fatigue, and fibromyalgia. [1][2][3][4][5][6] The mechanism of such a wide scope of biological activity of the formulation is still unclear. The goal of our research, therefore, was to study the mechanisms of the immunomodulating effects of Del-Immune V and to describe its dose-dependent effects on production of immunoregulatory cytokines in vivo and in vitro.

MATERIALS AND METHODS
The study examined the dose-dependent effect of Del-Immune V on production of immunoregulatory cytokines in nondescript mice with body mass of 14-16g. One hundred forty animals were selected on the basis of the analogue principle, and were divided into 7 groups of 20. The animals were fed balanced rodent food and water ad libitum. Group I, II, and III mice received 0.5 ml of aqueous solution of Del-Immune V orally in doses of 5, 50, and 500 µg/mouse respectively for 5 days at 24-hour intervals. Group IV mice were administered 0.5 ml Bifidim suspension (control probiotic medication) orally in a dose of 50 µg/mouse on the same schedule. The Bifidim was a dry mass of antagonistic bifidus bacteria immobilized on enterosorbent in combination with ascorbic acid (Intervetmed Ltd., Kiev, Ukraine). Group V mice were administered 0.15 М NaCl. Group VI and VII mice were used to study the interferonogenous activity of a single administration of 50 µg/mouse of Del-Immune V (Group VI) or Bifidim (Group VII). Cytokine production by IFN and TNF was examined in intact and treated mice 8 hours after initial administration and then every 24 hours for the next 5 days. For this purpose, several mice from each group were killed by cervical dislocation ; blood serum, 30 peritoneal exudate macrophages (PEM), 31 and spleen, 32 from which splenocytes were harvested 33 from each group of mice for testing.
The optimal dose of Del-Immune V was also tested via in vitro induction of Biological activity of TNF was assessed by cytotoxicity in the passaged culture of murine fibroblasts L-929. 30 The result was recorded on a multiscanner (Dynatech, Switzerland) with a wavelength of 540 nm. The cytotoxicity index was calculated using the formula CI = К-О/Кх100%, where К and О represent optical density values for the cell in the culture medium (RPMI 1640 with 10% FСS). The calibration curve based on standard recombinant TNF formulation Sіgma was used for standardization of the cytotoxicity index. 34 IFN levels in cell cultures and serum were measured using standard microtitration in the passaged cell culture L-929 against 100 TCD 50 indicator virus (vesicular stomatitis virus, Indiana VSV) with constant СО 2 level (15). The significance of the results was analyzed by Student-Fisher t-test. Differences of P < .05 were considered to be significant. 35

RESULTS
Daily oral administration of Del-Immune V or Bifidim to Groups I-III in the course of 5 days in doses of 5, 50, or 500 µg/mouse resulted in a marked increase in IFN levels in blood serum (see Figure 1). The optimal interferonogenous dose was found to be 50 µg/mouse (Group II). After 24 hours of observation, circulating IFN levels in Group II One-time oral administration of Del-Immune V or Bifidim to mice in a dose of 50 µg/ml resulted in increased circulating IFN level 8 hours after administration. The highest serum IFN level was reported 24 hours after administration, while levels in control group animals remained unchanged (see Table 1).
After administration of Del-Immune V or Bifidim in a dose of 50 µg/ml, serum TNF was 0.6 ng/ml (P < .05) and 0.8 ng/ml (P < .05), respectively, while in the control group it did not In vitro trials showed that adding Del-Immune V or Bifidim in concentrations of 5, 50, or 100 µg/ml to macrophages of experimental and intact mice resulted in TNF production peaking 8 hours after adding these formulations ( Figure 5). TNF production potential of PEM was dose-dependent. The optimal in vitro concentration of Del-Immune V and Bifidim was 50 µg/ml.
Insert Figure 5 about here.
TNF production by the macrophages of the experimental mice after administration of a specific LPS inductor, Del-Immune V, or Bifidim was more intensive than by PEM of the intact mice. Both Del-Immune V and Bifidim induced a higher immune response in macrophage cells of experimental mice, resulting in enhanced production of IFN and TNF.
Cell-mediated immune regulation and stimulation of effector function by macrophages are indicators of the immunomodulating activity of the above formulations. The dose-dependent responses of mice to these immunomodulators should be tested in human subjects to determine whether similar effects will be found. induce monocytic production of IL-10, inhibiting cytotoxicity activation of IFN-γ and secretion by Т-and NK-cells. 56 Since clinical applications of LPS and gram-negative bacteria are limited because of high toxicity, finding selective immunomodulators is one of the main conditions for improving the efficacy of immunostimulating therapy.

DISCUSSION
In this study, Del-Immune V stimulated the functional activity of monocytemacrophagal murine cells. However, higher dosages did not always result in higher efficacy.
The success of immune active therapy can be enhanced not only by new medications but also by their rational use.
The living cells of Bifidim stimulated in vitro TNF production more intensively than Del-Immune V. Cytokine production in vitro induced by Del-Immune V and Bifidim was compared with cytokine production in vivo. 57 Induction of pro-inflammatory cytokines IFN and TNF by Del-Immune V and Bifidim in vitro suggests that these formulations stimulated a nonspecific immune response in vivo. On the basis of these results documenting the potential of oral Del-Immune V and Bifidim to stimulate synthesis of IFN-α/β and -γ as well as TNF, it should also be noted that IFN-γ can induce expression of TNF-α receptors on macrophages. 58 These cytokines synergistically stimulate macrophage cells that, in turn, intensify killing activity. Intercellular cooperation of epithelial and immunocompetent cells in response to cytokine and chemokine molecules is effected via МНС (major histocompatibility complex) class І and ІІ molecules, 45 which are receptors for IFN-γ, IL-1, TNF-α, TGF-β, IL-2, IL-4, IL-7 and IL-10. 55,59 Interferonogenous activity of Del-Immune V ® Page 13 The synergistic activity of cytokine (IFN and TNF) production induced by Del-Immune V and Bifidim helps to demonstrate some therapeutic effects of these formulations.
The comparative study of Del-Immune V and Bifidim demonstrated that both formulations had a stimulating effect on cytokine secretion activity of the splenocytes and macrophages necessary for production of IFN and TNF. Bifidim contains living cells of bifidus bacteria, while active substances of Del-Immune V are MP (muramyl peptides) and nucleoproteids of the probiotic strain Lactobacillus rhamnosus V. Del-Immune V demonstrated higher interferonogenous activity in vivo and in vitro than Bifidim (Figures 1 and 3). However, in vitro, Bifidim stimulated higher levels of TNF in comparison with Del-Immune V ( Figure 5).
The choice of probiotic formulation (live probiotics cells or structural derivatives of probiotic cells) depends on a large number of factors, including potential, mechanism, mode of administration, and desired immune response. The mechanisms of action of this group of formulations are most likely multifactorial and include a number of signals, cell types, and receptors. One characteristic of probiotic activity is selective effects on the immune system of the macro-organism, whereby only those parts of the natural immune response that require correction are altered. [60][61][62][63][64] Probiotics demonstrate a variety of influences on immunological processes, depending on the type and strain of the bacteria. For example, bacteria L. fermentum and L.
plantarum stimulate B-cell proliferation, while L. acidophilus mainly causes induction of Тcell immune response. 65 Incubation of different strains of lactobacilli with human peripheral blood mononuclears showed that L. brevis, L. reuteri, L. lactis, L. casei and L. plantarum stimulate, to varying degrees, production of IL-1, IL-12, TNF-α, and IFN-γ. 66 Similar findings show that L. plantarum, L. rhamnosus and L. paracasei ssp. paracasei, when cultured with peripheral blood mononuclears, intensify secretion of IL-12. 56 Interferonogenous activity of Del-Immune V ® Page 14 Certain structural components of lactobacilli, including peptidoglycans and DNA fragments, can also influence the secretion activity of human monocytes in vitro through intensified production of IL-1, IL-6 and TNF-α; in vivo they can activate synthesis of Е2 prostaglandin and activate the system of complement and maturation of T-cell precursors. 67 In this study, the new probiotic formulation Del-Immune V, in a dose of 50 µg/mouse, was shown to actively induce IFN and moderately stimulate the production of tumor necrosis factor, showing significant promise as an immunomodulating preparation. Its natural origin, interferonogenous activity, safety, usability, and the possibility of oral administration secure Del-Immune V a worthy place among modern immunomodulating medications.