Antigen-Specific Immunomodulatory Effects of Transfer Factor

Research Article

Austin J Clin Pathol. 2020; 7(1): 1062.

Antigen-Specific Immunomodulatory Effects of Transfer Factor

Vetvicka V* and Vetvickova J

Department of Pathology, University of Louisville, USA

*Corresponding author: Vaclav Vetvicka, Department of Pathology, University of Louisville, 511 S. Floyd, Louisville, KY 40202, USA

Received: May 20, 2020; Accepted: June 13, 2020; Published: June 20, 2020

Abstract

The aim of this study was to evaluate the antigen-specific immunomodulatory effects of colostrum and egg yolk-derived transfer factors. All mice were immunized with ovalbumin during the administration period. To evaluate the immunological effects, proliferation, surface markers, antibody formation, serum immunoglobulins and cytokine secretion were measured. The immunomodulatory effects of the Transfer Factor Plus Tri-Factor Formula were shown by evaluating ovalbumin-specific immune cell proliferation, cytokine release, and formation of specific antibodies. Our data showed potentiation of IL-2 and IFN-γ secretion, higher proliferation of spleen cells and increased production of ovalbuminspecific IgG2a antibodies. This study confirmed that these transfer factors have significant potential to modulate specific immune responses.

Keywords: Transfer factors; Cytokines; Antibodies; Immunoglobulins; Proliferation

Introduction

Transfer Factors (TF), sometimes known as Lawrence TF, was originally described and developed by Sherwood Lawrence, who observed that antigen-specific cell-mediated immunity can be transferred from sensitive individuals to naïve individuals via a dialyzable leukocyte extract [1]. At that time, TF represented a revolutionary hypothesis, as the prevailing dogma favored antibodies.

Originally, TF was thought to be present in colostrum material that was smaller than 20 kDa. Later it was better characterized to be around 2.5 kDa. Originally applied by subcutaneous injection, further studies found that long term oral administration yielded positive effects too [2]. Fear of HIV infection via blood-borne products and a scandal involving two Harvard researchers working with TF [3] had a negative impact on further TF research during that period. Despite this setback, new data showing antigen-specific extracts from CD8-positive lymphocytes renewed interest in TF research [4]. More detailed studies followed using different preparations of bovine and murine TF and found the same conserved amino acid sequence in all of these isolates [5]. Current knowledge of TF preparation and characterization allows not only the use of TF prepared from cow colostrum or chicken egg yolks, but also specifically specific TF, such as Staphylococcus aureus-specific TF [6].

The current study is a continuation of the evaluation of the biological effects of Transfer Factor Plus Tri-Factor Formula combining TF isolated from chicken egg yolk (Ovo-Factor) and bovine colostrum (Ultra-Factor XF and Nano-Factor, proprietary filtration method), as well as other ingredients. As the original study found significant effects on phagocytosis, proliferation of splenocytes, cytotoxicity of NK cells and production of some cytokines [7], we expanded our study to further test the potential stimulation of the immune system.

Material and Methods

Animals

Female, 6 week old BALB/c mice were purchased from the BioLASCO (Taiwan). Ten mice/group were used in this study. Each animal was weighed once a week during the study period.

Transfer factor

Transfer Factor Plus Tri-Factor sample was kindly donated by 4Life Research, Sandy, UT, USA. Three different doses, 273 mg/kg/ day (low), 546 mg/kg/day (medium) and 1,365 mg/kg/day (high) were used. Individual samples and a negative control (sterile water) were administered daily by oral gavage for 8 weeks.

Sample collection

Blood samples were collected at the end of experiments by heart puncture. Serum was obtained after centrifugation at 2,200 x g for 15 minutes and stored at -30o C. Upon use of isoflurane as anesthesia, spleens were removed and gently ground with fine steel mesh.

Material

Ovalbumin (OVA) and complete and incomplete Freund’s adjuvant were purchased from Sigma (St. Louis, MO, USA).

Cytokine analysis

Splenocytes at 1-2x106 cells/well concentration were treated with OVA for 72 hrs at 37o C. After centrifugation, supernatants were collected and levels of IL-2, IL-4, IL-5, IL-10, TNF-α, and IFN-γ were evaluated by ELISA using appropriate ELISA kits (Biolegend, San Diego, CA, USA).

Immunization

Mice were immunized with 25μg Ovalbumin (OVA) in complete Freund’s adjuvant ip. Four weeks after the supplementation with tested samples. Two weeks later, mice were challenged with a second ip. Injection of the same dose of OVA in incomplete Freund’s adjuvant. Anti-OVA antibodies of the IgG1, IgG2a, and IgE class were determined by an indirect ELISA.

Proliferation

Splenocytes were cultivated in a 95-well plate at a concentration of 1x105 cells/well. After 72 hrs of cultivation with OVA, the proliferation was measured using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) at 490 nm using the following formula:

Stimulation index SI = OD490 nm of OVA-stimulated cells/OD490 nm of unstimulated cells

Serum immunoglobulins

Whole blood was centrifuged at 2,200 x g for 15 minutes and serum samples were analyzed for levels of OVA-specific IgG1, IgG2a, and IgE by indirect ELISA using OVA-coated plates and HRP-conjugated anti-mouse IgG1, IgG2a, and IgE. The amounts of antibodies were calculated after detection of optical density at 450 nm with an ELISA reader and expressed as ELISA Units (EU).

Surface markers

Splenocytes were labeled with fluorescence-conjugated monoclonal antibodies at the concentration of 2x105 cells/well. Antibodies recognizing T lymphocytes (CD4+/CD3+), cytotoxic T lymphocytes (CD8+/CD3+), B lymphocytes (CD19+/CD45+) and NK cells (PanNK+/CD45+) were used and the individual subpopulations were evaluated by flow cytometry.

Statistics

One way ANOVA followed by Duncan’s test were used to statistically analyze the data.

Results

No clinical signs of illness were observed during the study. Mean body weight at the beginning of the study was 16.1-18.9 g, and mean weight at the end of the study was 18.7–23.5 g (Table 1). Shows that the body weight or spleen-to-body weight ratio did not statistically differ among tested groups (Table 1). The growth ratio in each group was about the same. The mean spleen-to-body weight ratio was not statistically different among all studied groups (data not shown). Our data showed no differences in IL-2 and IFN-γ secretion among negative control and treated groups under unstimulated basal level (data not shown).But in the case of OVA-sensitized animals, we found a significant dose-dependent stimulation of IL-2 (Figure 1) and IFN-γ secretion (Figure 2). No significant TF effects were found for IL-4, IL-10 and TNF-α secretion (Table 2). None of the tested conditions changed the production of IL-5 (Table 2). With respect to antibody formation, the production of IgG1 and IgE was not changed (Table 3). However, the production of IgG2a was significantly increased by low, medium and high doses of tested material (Figure 3). Lastly, proliferative response to OVA stimulation was significantly increased when compared to the blank control. Among the OVAsensitized groups, the proliferation induced by OVA was significantly enhanced in low, medium, and high dose groups in comparison with the negative control (Figure 4).