In Vivo Activity of M3

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Introduction: M3 was evaluated for its effects upon murine leukocytes in vivo.
 

Methods: M3 precipitate was isolated by centrifugation and re-suspended, without drying, in phosphate buffered saline (PBS) at pH 8. This was administered daily per os (by mouth) to 5 mice for 4 weeks. A control group of 5 mice was given similar oral doses of PBS alone on the same schedule. Leukocyte counts and differential leukocyte counts were made on peripheral blood prior to administration of M3 and weekly thereafter for 4 weeks. Specific lymphocyte counts were measured by flow cytometry at 5 weeks and 8 weeks.

Background: Most CD4⁺ cells are helper T lymphocytes or are responsible for delayed-type hypersensitivity reactions. Most CD8⁺ cells are cytotoxic and/or suppressor cells.

Results: Administration of a single daily 0.2  mg oral dose of M3 per mouse did not cause a significant overall increase or decrease in circulating leukocytes compared to animals given PBS during the study period (Table 1). However, administration of M3 did result in an increase in lymphocytes in the circulating leukocyte population. Statistically significant increases in lymphocytes (compared to starting values) were noted at 2, 3, and 4 weeks of dosing (Table 2).

When lymphocytes were analyzed at 5 weeks with anti-T cell antibodies, mice receiving M3 had greater numbers of circulating CD4⁺ T-cells compared to animals receiving PBS (Table 3). Circulating CD8⁺ T-cells were also higher in the treatment group (Table 4). At 8 weeks (4 weeks after cessation of dosing), flow cytometry analysis indicated no differences between numbers of T-cells in M3-treated vs. control mice (Tables 3 and 4).

These results suggest that oral administration of M3 increases the relative numbers of lymphocytes, specifically increasing the relative numbers of CD4⁺ and CD8⁺ T-cells.

Discussion: Orally administered M3 clearly affects leukocyte levels by increasing CD4⁺ and CD8⁺ T-cell numbers. The effects on T-cells appear to be transitory as the relative numbers of CD4⁺ and CD8⁺ T-cells returned to control values after M3 dosing was discontinued.

The activation status of the lymphocytes (resting or activated) was not examined in this project but is worthy of study.

Conclusion: This study suggests that the immune system may be enhanced by the oral administration of M3.

   

TABLE 1 – Average Leukocyte Count

Time	PBS Group	M3 Group
Prebleed	4.66E6	5.22E6
Week 1 Bleed	15.6E6	10.3E6
Week 2 Bleed	9.15E6	8.29E6
Week 3 Bleed	11.5E6	9.84E6