Patient Selection

Twenty sequential patients with previously known stage IV malignancies were selected to participate in this study at the Immune Institute. After informed consent was obtained. 20 patients with stage IV, end-stage cancer were enrolled (one bladder, five breast cancer, two prostate cancer, one neuroblastoma, two non-small cell lung, three colon cancer, 1 mesothelioma, two lymphoma, one ovarian cancer, one gastric, one osteosarcoma). Therapy and surgery within 1 month of the procedure were exclusion criteria. The protocol was explained to them and consent forms were signed as required by the local Institutional Review Board. Inclusion criteria included age 18 to 75; 1 month to 6 month survival diagnosed by a Board Certified Oncologist; palliative therapy only planned in the future and no evidence of acute organ failure. Exclusion criteria included patients with severe leukopenia; poor respiratory or renal function; heart failure above New York heart association grade II; ventricular arrhythmias; autoimmune disorders; and coagulation abnormalities (Table 3).

Table III. Patient Demographics

• Average age; 49.3+/- 15.6

• Gender; 12 males, 8 females^[20]

• Previous surgery; 13

• Previous chemotherapy; 15

• Previous radiation; 14

• Stage; All stage IV

• Liver involvement; 9

• Avg. length of diagnosis; 6.9 +/- 6.1 months

• Oncologist prognosis median survival; 3.7 +/- 3.0 months

• Further standard therapy planned; 3/20 (all palliative)

Peripheral blood monocytic cells (PBMC) were isolated from the 400 ml of heparinized whole blood by density gradient centrifugation using Lymphocyte Separation Media (LSM), Cellgro^Ⓡ (Mediatech, Herndon, VA). The PBMC were washed three times with phosphate-buffered saline (PBS; Mediatech, Herndon, VA) to remove platelets and any traces of LSM. The washed cells were then used fresh for laboratory studies.

Laboratory Studies

NK Cell Activity

MOLT-4 cells (5x10⁶, ATCC, Manassas VA) were incubated with 30 μCi of 51-Chromium (⁵¹Cr) for 1 h at 37℃ with 5% CO₂. The cells were washed once with PBS to remove excess chromium and transferred to a 96 well microplate. Each assay was done in triplicate and the results of the three wells averaged. Wells to assess total release contained 50 μL of the labeled cells, 50 μL of 3% triton-X and 100 μL of RPMI. Wells to determine spontaneous release consisted of 50 μL of labeled cells and 150 μL of RPMI. Test wells contained 100 μL of the PBMC (5x10⁵/plate), 50 μL (5000 cells) of the labeled MOLT-4 cells and 50 μL of RPMI. The micro plate was then incubated for 4 h at 37℃ with 5% CO₂. Following the incubation, the plate was centrifuged at 4000 rpm for 15 min. A 100 μL sample of the supernatant from each well was transferred into a test tube and radioactivity was counted for one minute with a gamma counter. A standard formula was used to calculate the Natural Killer activity.

Tumor Necrosis Factor-a

A dilution series for TNF-a standard was prepared as specified by the manufacturer's package insert (Sigma, St. Louis MO). 200 μL of each standard and samples were pipetted into the designated wells of a microplate. 50 μL of assay diluent 1 F (PBS with 2% Fetal Bovine Serum [FBS]) was added to each well. The plate was then incubated for 2 h at room temperature. Following the incubation, each well was washed three times with 400 μL of washed buffer. Excess wash buffer was removed and 200 μL of TNF-a conjugate was pipetted into the wells. The plate was then incubated for 1 h at room temperature.

After the second incubation the wells are washed again. A 200 μL of the substrate solution containing equal volumes of colored reagent A and B (kit component) was pipetted into the washed wells. The plate was then incubated for 20 min at room temperature. The reaction was stopped by adding 50 μL of stop solution. The absorbance of each well was read using an ELISA micro plate reader set at 450 nm.

Soluble TNF Receptor Type I (sTNF R1)

A dilution series for human sTNF RI standard was prepared as specified by the manufacturer's package insert (R&D Systems, Minneapolis MN). 200 μL of each standard and samples were pipetted into the designated wells of a 96-well microplate. 50 μL of assay diluent RD1M was added to each well. The plate was then incubated for 2 h at room temperature. Following the incubation each well was washed three times with 400 μL of washed buffer. Excess wash buffer was removed and 200 μL of sTNF RI conjugate was pipetted into the wells. The plate was then incubated for 2 h at room temperature. After the second incubation the wells are washed again. A 200 μL aliquot of the substrate solution containing equal volumes of colored reagent A and B was pipetted into the washed wells. The plate was then incubated for 20 min at room temperature. The reaction was stopped by adding 50 μL of stop solution. The absorbance of each well was read using an ELISA microplate reader set at 450 nm.

Total Mercaptans

A dilution series for Total Mercaptan assay standards was prepared as specified by the manufacturer's kit (Calbiochem-Novabiochem, San Diego CA) package insert. 100 μL of patient sample and standards was pipetted into spectrophotometer cuvettes. The total volume of the cuvettes containing the samples and standards was adjusted to 900 μL with buffer solution. 50 μL of solution R1 (1% trimethylbenzine [TMB]) was added to each cuvette mixed and the absorbance read at 356 nm using a spectrophotometer.

Glutathione

A dilution series for Glutathione assay standards was prepared as speci fied by the manufacturer's kit (Calbiochem-Novabiochem, San Diego CA) package insert. 100 μL of patient sample and standards was pipetted into spectrophotometer cuvettes. The total volume of the cuvettes containing the samples and standards was adjusted to 900 μL with buffer solution. 50 μL of solution R1 was added to each cuvette mixed and the absorbance read at 356 nm using a spectrophotometer.

Hemaglobin, Hematocrit, Chemistry Panels

Standard complete blood count and chemistry panels were performed in the clinical laboratory by established methods.

Statistics

Differences in laboratory parameters between baseline and 4 weeks or 6 months were determined by ANOVA. Differences in projected vs. average survival were evaluated by chi square testing.

Results

Twenty sequential late-stage cancer patients were that met inclusion criteria and were willing to participate were enrolled in order to avoid bias. By definition, the study inclusion criteria included an Oncologist projected survival of less than 6 months. Therefore, all patients technically should have been dead at 6 months. If one looks at the projected survival estimate (3.7 +/- 3.0), approximately 15 of the patients should be have been dead compared to only 4.

Immune parameters consistently and statistically improved over the 6 month trial. Natural Kliller function increased by over 400 % (Table 4).

Table IV. Baseline and 6 Month Natural Killer Cell Function Results (LU)

Four patients with very advanced cancer (mean estimated survival 1.5 months) died during the study. The patient with gastric cancer died after three weeks. The other three patients showed a partial response, dying between 4 and 6 months (bladder, non-small cell lung cancer and colon cancer). There was no correlation between type of tumor and response.

Many studies have verified the ability of Natural Killer cells to lyse cancer cells in vivo.^[15] Submitted data indicated that the synergistic, combination product Transfer Factor Plus alone could increase NK function by almost 250%. Combination nutraceuticals can be used to significantly boost Natural Killer function in the face of cancers that release toxins which inhibit NK function. The mean beseline NK function was 6.4 LU, which is significantly suppressed compared to normals, who have been determined to have over 20 LU activity.^[5]

TNF is a cytokine that is a potent anticancer agent. Serum recordings of TNF can be deceiving because end-stage patients often have high circulating levels as their immune system attempts, but ultimately fails, to control metastatic cancers. High serum TNF levels have been associated with cachexia in end stage cancer patients due to its patient's PBMC to release TNF alpha correlates with cancer killing and an ability to mount a response to the cancer.^[6] In the current study, PBMC-derived TNF alpha was increased by over 10,000% after 6 months on the regimen the subjects received (Table 5). Baseline levels were all less than 40 pg/mL, compared to an average person with approximately 300 to 600 pg/mL response under similar laboratory conditions.^[6]

Table V. Baseline and 6 Month Tumor Necrosis Factor Alpha Results (Adherent, Non stimulated PBMC Subpopulation, pg/mL)

TNF-alpha type I receptors are found in high numbers on tumor cells. The significant decrease (p<0.1; Table 6) found in this study could be considered a crude estimate of lowered tumor burden.

Table VI. Baseline and 6 Month Tumor Necrosis Factor Alpha Type I Receptor Results (Optical Density)

Likewise, circulating mercaptans are associated with damaged DNA, including cacinogenic genes. A significant decrease (p<.01; Table 7) in the current study could be viewed as a similar marker for lessened DNA damage, and perhaps lower concentrations of oncogenes.

Table VII. Baseline and 6 Month Serum Mercaptans Results (Optical Density)

Glutathione is a potent intracellular antioxidant that improves immune function and was shown to be significantly improved in the study. Of the supplements used, ImuPlus has been shown to be the greatest inducer of glutathione, and may have been primarily responsible for the systemic increaase in the face of cancer, which typically lowers this protein. ^[13]

Table VIII. Plasma Glutathione and Hematocrit Levels (OD)

Discussion

Adequate levels of vitamins, minerals, orthomolecular compounds such as vitamin C, and nutrition from food are essential for health, and is especially important during times of physical and emotional stress. Cancer is stressful to the body in many ways. Numerous biological pathways and enzymes require vitamins for proper functioning. The immune system is no exception. Critical nutrients on which the immune system depends include vitamin C, folic acid, beta-carotene, and the minerals manganese, selenium, and zinc, among many others.

The February 25th, 1994 issue of Science magazine reported that scientists had been able to extend the lifespan of fruit flies by 40% with supplementation with antioxidant vitamins such as C, E, A, beta-carotene and selenium. It is postulated that vitamins protect cells from cancer in a nunmber of ways including strengthening the immune system (vitamins C, E, A, and beta-carotene and the minerals selenium, zinc and manganese), neutralizing carcinogens (C and E) and preventing DNA and cellular damage (vitamins A and E, beta carotene and the minerals selenium, zinc and maganese).

Ascorbic acid, when given at sufficiently high desages, has demonstrated preferential cytotoxicity to tumor cells in vitro and in vivo. Levels required to be cytotoxic in vivo to tumor cells are not attainable via oral administration. Cytotoxic effect requires intravenous administration of 50 Gms or more. ^[21]

It is hypothesized that ascorbic acid exhibits cytotoxic activity via a prooxidant effect. There is a 10 to 100 fold greater content of catalase in normal cells than in tumor cells. Due to this, cancer cells reach high levels of intracellular hydrogen peroxide leading to their destruction, while normal cells are protected.^{[22, 23]}

Andro graphis Paniculate is an herb commonly used in Ayurvedic medicine which possesses a number of pharmacological properties. Of particular interest to us is the observation that the herb is involved in restoration of cell cycle dynamics in cancer cells, a phenomenon often referred to as dedifferentiation.^[24]

The medicinal mushroom Agaricus Blazei Murill, like other medicinal mushrooms, has been observed to possess significant ability to stimulate macrophages, increase natural killer cells, and have other immunomodulatory effects. It is believed that the various fractions of beta glucans in medicinal mushrooms are responsible for this effect, but it appears that different fractions possess different immunomodulatory properties.^{[25, 26]} More recently Takaku identified a substance (ergosterol) in Agaricus possessing anti-angiogenic activity.^[26]

Glutathione is a potent intracellular tripeptide which vigorously binds damaging free radical molecules that would other wise harm the cell bey several mechanisms. Non-denatured milk whey protein (ImuPlus) has been shown to be a potent inducer of glutathione, thereby reducing cellular damage and improving intracellular function. Interestingly, a study by Kennedy^[17] showed that non-denatured milk whey protein increased glutathione in cancer cells.

Most patients with cancer usually have decreased hemaglobin and hematocrit levels. however, in unpublished, preliminary data, ImuPlus has been shown to increase these levels, improving oxygen carrying capaccity to tissues, improving their resistance to cancer.

It is beyond the scope of the current publication to discuss the actions and ramifications of all the constituents of the regimen used by the 20 study subjects, but the combination accomplished the following: immune function was significantly increased; markers of tumor load such as total circulating mercaptans were decreased; life span was increased; quality of life was improved; and glutathione and hemoglobin were increase. Combination approaches such as that used in the study may be necessary to improve the dismal statistics in stage III and IV cancers by acting at several sites in cancer growth, gene expression, biochemistry and mechanisms of metastases

Acknowledgments

We are grateful for the technical assistance of Dr. Romin Roshan. We are appreciative of the free product and partial funding of the study provided by Swiss Bioceuticals, and a portion of Transfer Factor Plus provided free of charge by 4-life International.

Natural Killer Cells in Human Cancer

Immune System & Diseases

Transfer Factor & Immune Function that affect Cancer

References

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^[6] Zhang M., Tracey K. Tumor Necrosis Factor, The Cytokine Handbook, 3, Academic press Ltd., 1998, pp. 517-548
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Transfer Factor can strengthen your immune cells (NK cells) by educating them to recognise harmful invasion to your body, remember the past invasion and respond accordingly with the best possible way.  NK cells are known for the important roll in the fight against cancer.