A Look at the Work of Dr. Nobuto Yamamoto and GcMAF

DO NOT CONTACT DR. YAMAMOTO. HE CANNOT HELP YOU.

THIS INFORMATION IS FOR REFERENCE ONLY

"In fact weekly administration of 100 ng GcMAF to cancer patients showed curative effects on a variety of cancers. Types of cancer so far tested are prostate, breast, colon, stomach, liver, lung (including mesothelioma), kidney, bladder, uterus, ovarian, head/ neck, brain cancers, melanoma and fibrosarcoma."
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Oracle and Sandra posted on the chat room information about another promising but unpatentable cancer treatment, called GcMAF.(Pronounced "G C mauf"). We have assembled information for those interested. Like DCA, this looks to be another great therapy the FDA's patent-based new drug approval process will leave behind. Don't let it happen!

EASY SUMMARY: Cancer cells secrete a chemical that deactivates a protein fundamental to immune system response. This deactivated protein (Gc protein) cannot be converted to macrophage activating factor (MAF), meaning the cancer cells have turned off the body's macrophage response.

By incubating human Gc protein (derived from human blood) with some particular enzymes, a glycoprotein macrophage activating factor (GcMAF) is created. It has now been tested in humans and has shown a 100 percent cure rate over a time period of 4 to 7 years. It has no side effects.

HIV / AIDS

Newest Yamamoto paper: Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF). J Med Virol. 2009 Jan;81(1):16-26 Abstract
Link to full text.
"After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years."

Pathogenic Significance of α-N-Acetylgalactosaminidase Activity Found in the Envelope Glycoprotein gp160 of Human Immunodeficiency Virus Type 1 Nobuto Yamamoto. AIDS Research and Human Retroviruses. March 2006, 22(3): 262-271. doi:10.1089/aid.2006.22.262

Structural Modification of Serum Vitamin D3-Binding Protein and Immunosuppression in AIDS Patients. Nobuto Yamamoto, Venkateswara R. Naraparaju, Srinivasa M. Srinivasula. AIDS Research and Human Retroviruses. November 1995, 11(11): 1373-1378. doi:10.1089/aid.1995.11.1373.

See also FOCIS meeting abstract on HIV, 2009. Reports IV superior to IM. Link

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1. Original Sardi/Hubbell article
2. Yamamoto patents
3. Yamamoto's more relevant work
4. A look at his research dating back to 1957
5. Videos on GcMAF
6. Dr. Saharuddin Bin Mohamad and GcMAF
7. Related Work Supporting the use of MAF as a potent anti-cancer agent
8. GcMAF analogs
9. A biography of Dr. Nobuto Yamamoto
10. Definitions
11. Osteopetrosis, immunosuppression and cancer
12. April 2009 Italian paper showing Nagalase as causing impared immune function

13. Work by other researchers on macrophage activating factors (under development)

Gc Variations

The Yamamoto Patents:
Click to see a page listing details of Dr. Nobuto Yamamoto's patents

The Yamamoto Papers

The most recent papers are here.
For a look at his older works click here.

The 2009 FOCIS meeting abstracts states that IV is superior to IM. link.

Immunotherapy of prostate cancer with Gc protein-derived macrophage activating factor, GcMAF

Nobuto Yamamoto, Hirofumi Suyama and Hiroyuki Yamamoto
Year 2008, Volume 1, Issue 2

Abstract
Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no side effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 nanogram (ng) GcMAF. As the MAF precursor activity increased their serum Nagalase activity decreased. Since serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14-25 weekly administrations of GcMAF (100 ng/week), all sixteen patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor free. No recurrence occurred for seven years.
Entire report: http://www.transonc.com/pdf/manuscript/v01i02/neo08106.pdf

Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF)

abstract (Introduction to this paper)
International Journal of Cancer / Volume 122, Issue 2, Date: 15 January 2008, Pages: 461-467
Nobuto Yamamoto, Hirofumi Suyama, Nobuyuki Yamamoto, Naofumi Ushijim

Abstract
Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum -N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized -galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. © 2007 Wiley-Liss, Inc.

Received: 29 March 2007; Accepted: 27 July 2007

Immunotherapy of metastatic colorectal cancer with vitamin D-binding protein-derived macrophage-activating factor, GcMAF.

Link. 6 December 2007. Alternate link. Yamamoto N, Suyama H, Nakazato H, Yamamoto N, Koga Y.

Abstract  Serum vitamin D binding protein (Gc protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of colorectal cancer patients was lost or reduced because Gc protein is deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Deglycosylated Gc protein cannot be converted to MAF, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent macrophage-activating factor (GcMAF) ever discovered, but it produces no side effect in humans. Macrophages treated with GcMAF (100 pg/ml) develop an enormous variation of receptors and are highly tumoricidal to a variety of cancers indiscriminately. Administration of 100 nanogram (ng)/human maximally activates systemic macrophages that can kill cancerous cells. Since the half-life of the activated macrophages is approximately 6 days, 100 ng GcMAF was administered weekly to eight nonanemic colorectal cancer patients who had previously received tumor-resection but still carried significant amounts of metastatic tumor cells. As GcMAF therapy progressed, the MAF precursor activities of all patients increased and conversely their serum Nagalase activities decreased. Since serum Nagalase is proportional to tumor burden, serum Nagalase activity was used as a prognostic index for time course analysis of GcMAF therapy. After 32–50 weekly administrations of 100 ng GcMAF, all colorectal cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of metastatic tumor cells. During 7 years after the completion of GcMAF therapy, their serum Nagalase activity did not increase, indicating no recurrence of cancer, which was also supported by the annual CT scans of these patients.

Immunotherapy of HIV-Infected Patients With Gc Protein-Derived Macrophage Activating Factor (GcMAF)

Nobuto Yamamoto, Naofumi Ushijima, and Yoshihiko Koga. Journal of Medical Virology 81:16–26 (2009)

Abstract: Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by a-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized b-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CDþcell counts were maintained for 7 years. J. Med. Virol. 81:16–26, 2009.
2008 Wiley-Liss, Inc.

Cancer cell-killing by macrophages treated with Gc protein-derived macrophage activating factor (GcMAF)

Proc Amer Assoc Cancer Res, Volume 45, 2004. Experimental and Molecular Therapeutics 11: Specific Immune Mechanisms and Cancer Vaccines: Clinical Studies Abstract #1255 Nobuto Yamamoto and Masumi Ueda

Abstract: Macrophages, when highly activated via inflammation (e.g. intratumor BCG administration), can eradicate cancerous cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum Gc protein (vitamin D-binding protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with ß-galactosidase of inflammation-primed B cells and sialidase of T cells yields a potent macrophage activating factor (MAF), a Gc protein with N-acetylgalactosamine as the remaining sugar. Thus, Gc protein is the precursor for MAF. Stepwise treatment of purified Gc protein with immobilized ß-galactosidase and sialidase generates the most potent macrophage activating factor (GcMAF) ever discovered which produces no side effect in humans and animals. Macrophages activated by GcMAF (100 pg every 4 days to Ehrlich ascites tumor bearing mice) eradicate the tumor cells. After more than 25 weekly administrations of 100 ng GcMAF to cancer patients, the majority of prostate and breast cancer patients, excluding extremely advanced, exhibited healthy control levels of the tumor markers (e.g., -N-acetylgalactosaminidase), indicating eradication of tumors confirmed by CAT-scan and MRI. We demonstrated rapid in vitro cancer cell-killing using mouse and human macrophages activated by GcMAF. Since activation of macrophages leads to enhanced phagocytosis and antigen presentation to lymphocytes for development of humoral and cellular immunity, GcMAF therapy of Ehrlich ascites tumor-bearing mice and prostate and breast cancer patients appeared to develop antibodies against their respective cancerous cells. When in vitro cancer cell-killing study with mouse and human macrophages activated by GcMAF was performed using Ehrlich ascites tumor, and breast and prostate cell lines in the presence of serum (or IgG) fraction of GcMAF-treated Ehrlich tumor bearing mice, prostate or breast cancer patients, greatly accelerated cell-killings were observed, indicating that sera of GcMAF treated tumor-bearing mice and cancer patients contain IgG antibodies against these cancer cells because inflammation (or GcMAF)-primed activation of macrophages is known to develop Fc-receptor mediated cell-killing and phagocytosis of targets preferentially

Therapeutic Efficacy of the Most Potent Macrophage Activating Factor (GcMAF)
for Prostate and Breast Cancers

Nobuto Yamamoto, Director, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, Masumi Ueda, Chief, Microbiology Department, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, Charles Benson, Head, Infectious Diseases, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA

Inflammation-primed macrophage activation is the principal macrophage activation process that requires serum vitamin D-binding protein (known as Gc protein) and participation of B and T lymphocytes. A trisaccharide composed of N-acetylgalactosamine with dibranched galactose and sialic acid termini at 420 threonine residue of Gc protein is hydrolyzed by the β-galactosidase (Bgl) of inflammation-primed B cells and the Neu-1 sialidase of T cells to yield the macrophage activating factor (MAF). Thus, Gc protein is the precursor for the principal MAF.

However, the MAF precursor activity of serum Gc protein of cancer patients was lost or reduced because Gc protein is deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Serum Nagalase activity is proportional to tumor burden and serves as a prognostic index. Stepwise treatment of highly purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent macrophage activating factor (GcMAF) that produces no side effect in humans.

When human macrophages were treated in vitro with GcMAF (100 pg/ml) for 3 h, the macrophages developed enormous variation of receptors that recognize a variety of cellular abnormalities, i.e., tumor associated antigens, in cancerous cell surface. Thus, the activated macrophages were highly tumoricidal to a variety of cancerous cells.

Administration of 100 ng GcMAF/human results in the maximal activation of systemic macrophages. When nonanemic prostate and breast cancer patients (total of 36 patients) were treated with 14–25 weekly administrations of GcMAF (100 ng/week), all cancer patients exhibited healthy control levels of the serum Nagalase activity, indicating eradication of tumors. doi:10.1016/j.clim.2007.03.512

Treatment of HIV-infected Patients with Vitamin D-binding Protein Derived Macrophage Activating Factor (GcMAF) Eradicates HIV-Infection

Nobuto Yamamoto, Director, Socrates Institute for Therapeutic Immunology, Philadelphia, PA, Masumi Ueda, Immunology, Chief, Microbiology, Philadelphia, PA, Charles Benson, Head, Infectious Diseases, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, PA

Serum vitamin D-binding protein (known as Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Since Nagalase is the intrinsic component of gp120, serum Nagalase activity is the sum of enzyme activities expressed in both HIV virions and envelope proteins released from HIVinfected cells.

Because of Nagalase being an HIV viral protein and immunogenic, serum Nagalase was already complexed with anti-HIV immunoglobulin G (IgG) in patient blood stream. These antibodies, however, were largely not neutralizing antibodies. The IgG-bound viral proteins retained Nagalase activity that deglycosylates Gc protein. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effect in humans. Macrophages activated by administration of GcMAF (100 ng/patient) develop a large amount of Fc receptors as well as enormous variation of receptors that recognize IgG bound and unbound HIV virions.

Thus, macrophages activated by GcMAF preferentially phagocytize IgG-bound HIV virions via Fc receptor mediation. Cells latently infected with HIV are unstable and spontaneously release the virions at a high rate. After less than 18 weekly administrations of 100 ng GcMAF for twenty-one nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV infection.

http://www.4marks.com/articles/details.html?article_id=2457

Effects of Vitamin D3-Binding Protein-Derived Macrophage Activating Factor (GcMAF) on Angiogenesis. Shigeru Kanda, Yasushi Mochizuki, Yasuyoshi Miyata, Hiroshi Kanetake, Nobuto Yamamoto. Journal of the National Cancer Institute, Vol. 94, No. 17, 1311-1319, September 4, 2002

Conclusions: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Deglycosylation of Serum Vitamin D3-Binding Protein by α-N-Acetylgalactosaminidase Detected in the Plasma of Patients with Systemic Lupus Erythematosus

Nobuto Yamamoto, Venkateswara R. Naraparaju, Mary Moore and Lawrence H. Brent

Abstract
A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by β-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized β-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by α-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma α-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macrophage-activating factor. The resulting defect in macrophage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma α-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macrophage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Immunotherapy of BALB/c Mice Bearing Ehrlich Ascites Tumor with Vitamin D-binding Protein-derived Macrophage Activating Factor (full pdf)
Nobuto Yamamoto and Venkateswara R. Naraparaju / Cancer Res 1997 57: 2187-2192

Abstract: "Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized ß-galactosidase or treatment of human Gc protein with immobilized ß-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum -N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 105 cells) showed a mean survival time of 35 ± 4 days, whereas tumor-bearing controls had a mean survival time of 16 ± 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 105 tumor cells, survived up to 32 ± 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 105 cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 105 tumor cells, also survived up to 32 ± 4 days, while control mice that received the 5 x 105 ascites tumor cells only survived for 14 ± 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 105 Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90." (Note that the more injections, the longer the survival time)

F.128. Macrophages Activated By GCMAF Develop Enormous Variation of Receptors That Recognize and Eradicate Adenocarcinomas. Nobuto Yamamoto, Masahiro Urade, Yoshihiko Koga, Nobuyuki Yamamoto, Theodor Sery and Masumi Ueda. 2006. (no abstract given)

Intratumor induced inflammation generates maximally activated macrophages that can eradicate cancerous cells.
Nobuto Yamamoto*. Socrates Institute For Therapeutic Immunology, Philadelphia, PA. 2006 Source

Photodynamic action on mammalian tissues immediately results in severe inflammation, leading to macrophage activation. Intratumor PDT-induced inflammation generates maximally activated macrophages that can eradicate local as well as metastasized cancerous cells. Inflammation in mammalian tissues activates phospholipase A2 to releases lysophospholipids that efficiently activate macrophages. Because cancerous tissues contain alkylphospholipids, PDT-induced inflammation of cancerous tissue produces alkyl-lysophospholipids and alkylglycerols that activate macrophages with approximately 400 times more efficiency than lysophospholipids. These results imply that highly activated macrophages can kill cancerous cells. Inflammation-primed macrophage activation process is the principal macrophage activation cascade that requires serum vitamin D3-binding protein (known as Gc protein) and participation of B and T lymphocytes. Stepwise hydrolysis of Gc protein with the inducible membranous beta-galactosidase of inflammation-primed B cells and the membranous Neu-1 sialidase of T cells yields a potent macrophage activating factor (MAF), the protein with N-acetylgalactosamine as the remaining sugar. Thus, Gc protein is the precursor for the principal MAF. Stepwise treatment of highly purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF) ever discovered which produces no side effect in humans. Administration of 100 ng GcMAF per human and 100 pg GcMAF per mouse results in the maximal activation of macrophages, which develop enormous variation of receptors. When human macrophages were treated in vitro with GcMAF (100 pg/ml) for 3 hrs, the macrophages were highly activated. The activated macrophages can recognize and kill a variety of cancerous cells indiscriminately. When prostate and breast, cancer patients were treated with less than 25 weekly administrations of 100 ng GcMAF, the majority of cancer patients, excluding anemic patients, exhibited healthy control levels of the serum prognostic index, alpha-N-acetylgalactosaminidase, indicating eradication of the tumors. GcMAF therapy also develops antibodies against the tumors.

Macrophage-directed immunotherapy as adjuvant to photodynamic therapy of cancer
Korbelik M, Naraparaju VR, Yamamato N. / BRITISH JOURNAL OF CANCER Volume: 75 Issue: 2 Pages: 202-207 Published: 1997 Link to full pdf

Abstract: The effect of Photofrin-based photodynamic therapy (PDT) and adjuvant treatment with serum vitamin D-3-binding protein-dervived macrophage-activating factor (DBPMAF) was examined using a mouse SCCVII tumour model (squamous cell carcinoma). The results show that DBPMAF can markedly enhance the curative effect of PDT. The most effective DBPMAF therapy consisted of a combination of intraperitoneal and peritumoral injections (50 and 0.5 ng kg(-1) respectively) administered on days 0, 4, 8 and 12 after PDT. Used with a PDT treatment curative to 25% of the treated tumours, this DBPMAF regimen boosted the cures to 100%. The DBPMAF therapy alone showed no notable effect on the growth of SCCVII tumour. The PDT-induced immunosuppression, assessed by the evaluation of delayed-type contact hypersensitivity response in treated mice, was greatly reduced with the combined DBPMAF treatment. These observations suggest that the activation of macrophages in PDT-treated mice by adjuvant immunotherapy has a synergistic effect on tumour cures. As PDT not only reduces tumour burden but also induces inflammation, it is proposed that recruitment of the activated macrophages to the inflamed tumour lesions is the major factor for the complete eradication of tumours.

Some additional papers on the combination of Immunotherapy and photodynamic therapy:

• The pilot experience of immunotherapy-combined photodynamic therapy for advanced gastric cancer in elderly patients
• Combination Immunotherapy and Photodynamic Therapy for Cancer
• Induction of Tumor Immunity by Photodynamic Therapy

Prognostic Utility of Serum a-N-Acetylgalactosaminidase and Immunosuppression Resulted from Deglycosylation of Serum Gc Protein in Oral Cancer Patients. (full pdf) Nobuto Yamamoto, Venkateswara R. Naraparaju and Masahiro Urade. Cancer Research 57, 295-299, January 15, 1997.

Abstract: Vitamin D3-binding protein (Gc protein), a serum glycoprotein, is the precursor for the macrophage activating factor. Cancer patient sera contain -N-acetylgalactosaminidase that deglycosylates Gc protein. Deglycosylated Gc protein cannot be converted to macrophage activating factor, leading to immunosuppression. Of 46 oral cancer patients with squamous cell carcinoma, approximately 22% had greatly reduced precursor activities. The precursor activity of approximately 61% of these patients was moderately reduced. The remaining patients (17%) had precursor activities equivalent to those of healthy humans. Patients with low precursor activity of serum Gc protein had high serum -N-acetylgalactosaminidase activity. In contrast, patients with high precursor activity had low serum -N-acetylgalactosaminidase activity. Thus, levels of serum -N-acetylgalactosaminidase of individual patients have an inverse correlation with precursor activities of their serum Gc protein. Surgical removal of tumors resulted in a subtle decrease in serum -N-acetylgalactosaminidase activity with concomitant increase in the precursor activity of serum Gc protein. Serum enzyme analysis of nude mice transplanted with a human oral squamous carcinoma cell line revealed that serum -N-acetylgalactosaminidase activity is directly proportional to tumor burden. Thus, -N-acetylgalactosaminidase activity in patient blood-stream can serve as a diagnostic/prognostic index.

Antitumor Effect of Vitamin D-Binding Protein-Derived Macrophage Activating Factor on Ehrlich Ascites Tumor-Bearing Mice. Yoshihiko Koga, Venkateswara R. Naraparaju & Nobuto Yamamoto. Proceedings of the Society for Experimental Biology and Medicine Volume 220 Issue 1 Page 20-26, January 1999.

Abstract: Cancerous cells secrete α-N-acetylgalactosaminidase (NaGalase) into the blood stream, resulting in deglycosylation of serum vitamin D3-binding protein (known as Gc protein), which is a precursor for macrophage activating factor (MAF). Incubation of Gc protein with immobilized β-galactosidase and sialidase generates the most potent macrophage activating factor (designated GcMAF). Administration of GcMAF to cancer-bearing hosts can bypass the inactivated MAF precursor and act directly on macrophages for efficient activation. Therapeutic effects of GcMAF on Ehrlich ascites tumor-bearing mice were assessed by survival time and serum NaGalase activity, because serum NaGalase activity was proportional to tumor burden. A single administration of GcMAF (100 pg/mouse) to eight mice on the same day after transplantation of the tumor (5 × 105 cells) showed a mean survival time of 21 ± 3 days for seven mice, with one mouse surviving more than 60 days, whereas tumor-bearing controls had a mean survival time of 13 ± 2 days. Six of the eight mice that received two GcMAF administrations, at Day 0 and Day 4 after transplantation, survived up to 31 ± 4 days whereas, the remaining two mice survived for more than 60 days. Further, six of the eight mice that received three GcMAF administrations with 4-day intervals showed an extended survival of at least 60 days, and serum NaGalase levels were as low as those of control mice throughout the survival period. The cure with subthreshold GcMAF-treatments (administered once or twice) of tumor-bearing mice appeared to be a consequence of sustained macrophage activation by inflammation resulting from the macrophage-mediated tumoricidal process. Therefore, a protracted macrophage activation induced by a few administrations of minute amounts of GcMAF eradicated the murine ascites tumor.

Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

Special Feature. Immunology & Cell Biology. 76(3):237-244, June 1998.
YAMAMOTO, NOBUTO 1,2; NARAPARAJU, VENKATESWARA R 1

Summary: Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized [beta]-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice

A report on this work was given at the International Society for Biological Therapy of Cancer 2007 Meeting. http://www.isbtc.org/meetings/am06/program.pdf
Paper No.118 TREATMENT OF CANCER PATIENTS WITH VITAMIN D-BINDING PROTEIN-DERIVED MACROPHAGE ACTIVATING FACTOR (GCMAF) RAPIDLY ERADICATES CANCEROUS CELLS. Nobuto Yamamoto, Masumi Ueda / Socrates Institute for Therapeutic Immunology, Philadelphia, PA

Here is a link to his presentations at earlier meetings:. http://www.isbtc.org/meetings/am04/presentations/yamamoto.pdf
and
2005 conference: Paper 86 POTENT TUMORICIDAL CAPACITY OF MACROPHAGES ACTIVATED BY GC PROTEIN- DERIVED MACROPHAGE ACTIVATING FACTOR (GCMAF) AND ITS THERAPEUTIC EFFICACY FOR PROSTATE, BREAST AND COLORECTAL CANCERS. Nobuto Yamamoto1, Masahiro

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Videos on GcMAF

GcMAF explained.

Dr. Saharuddin Bin Mohamad and GcMAF
(click to see a review of his research)

Related work concerning
MAF as a potent anti-cancer agent

Vitamin D Binding Protein-Macrophage Activating Factor (DBP-maf) Inhibits Angiogenesis and Tumor Growth in Mice. Full article is linked. Neoplasia. 2003 January; 5(1): 32–40..Oliver Kisker, Shinya Onizuka, Christian M Becker, Michael Fannon, Evelyn Flynn, Robert D'Amato, Bruce Zetter, Judah Folkman, Rahul Ray, Narasimha Swamy, and Steven Pirie-Shepherd

Abstract: We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

Guide to abbreviations in the Kisker paper:
BCE=Bovine capillary endothelial
BCS=Bovine calf serum
bFGF=basic fibroblast growth factor
BSA=Bovine serum albumin
CAM= Chick chorioallantaic membrane
FCS=fetal calf serum

Patent related to the above publication: Method and Composition for the Treatment of Angiogenesis. Application number: 10/275,414. Filing date: Jun 13, 2003. (pdf from USPTO)

Treatment of Prostate Cancer with a DBP-MAF-Vitamin D Complex to Target Angiogenesis and Tumorigenesis Michael W. Fannon. Feb 2006. Full text

Abstract: The purpose of this study has been to determine the efficacy of combined therapy using vitamin D binding protein-macrophage activating factor (DBP-maf) and vitamin D as therapy for prostate cancer using a mouse model of human prostate tumors. In this phase of the study we have tested the ability of vitamin D and DBP-maf to inhibit the formation of endothelial cells into tubes. Both molecules were effective on their own, however, the combination of vitamin D and DBP-maf showed synergistic behavior. Because of its high affinity for actin, the possibility that DBP-maf acted by inhibiting microtubule formation was investigated using an anti-phalloidin antibody. No differences in phalloidin strength or localization were observed. Using immunoassays we were able to identify a 75 kDa protein in tumor cells that is phosphorylated by DBP-maf and a synthetic maf peptide. We are investigating the identity of that protein since the mechanism by which DBP-maf works remains unknown.

Grant info: http://www.childrenshospital.org/newsroom/Site1339/mainpageS1339P1sublevel88.html

Inhibition of angiogenesis by vitamin D-binding protein: Characterization of anti-endothelial activity of DBP-maf Satyan Kalkunte, Laurent Brard, Cornelius O. Granai and Narasimha Swamy. 2005. "These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting."

Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration Rajendra S. Apte*, Jennifer Richter, John Herndon, Thomas A. Ferguson. 2006. (added here to show support of the additional anti-cancer mechanism proposed in many of Dr. Yamamoto's papers of anti-angiogenesis.)

"Vitamin D Binding Protein-Macrophage Activating Factor as an Anti-Angiogenic Molecule"Invention Summary: Scientists in Dr. Folkman's laboratory at Children's Hospital Boston have identified a novel angiogenesis inhibitor, vitamin D binding protein-macrophage activating factor (DBP-maf). Murine tumor assays of DBP-maf's activity demonstrate that it inhibits solid tumor growth at doses as low as 4 ng/kg per day. At higher doses, DBP-maf causes regression of established tumors. Children's Hospital seeks an industrial partner who can diligently and aggressively pursue the preclinical and clinical development of this novel angiogenesis inhibitor. A patent link.

Macrophages and cancer Pat W. Whitworth, Charles C. Pak, Joseph Esgro,Eugenie S. Kleinerman and Isaiah J. Fidler. 2004. "Appropriately activated macrophages can fulfill these demanding criteria. Macrophages can be activated to become tumoricidal ..."

Upregulation of Vitamin D Binding Protein (Gc-Globulin) Binding Sites During Neutrophil Activation from a Latent Reservoir in Azurophil Granules. 2007. Stephen J. DiMartino, Glenda Trujillo, Lauren A. McVoy, Jianhua Zhang, and Richard R. Kew. Has some basic information on Gc Protein.

Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.
1: Comp Biochem Physiol A Mol Integr Physiol. 2002 May;132(1):1-8. Links
Mohamad SB, Nagasawa H, Uto Y, Hori H. / Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Tokushima 770-8506, Japan.

Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation.
PMID: 12062184 [PubMed - indexed for MEDLINE]

Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS.
Chad R. Borges, Jason W. Jarvis, Paul E. Oran, Stephen P. Rogers, and Randall W. Nelson.
Journal of Biomolecular Techniques 1 RF 9:167–176 © 2008
full text

Serum proteomic profile of cutaneous malignant melanoma and relation to cancer progression: Association to tumor derived alpha-N-acetylgalactosaminidase activity. Marilena Greco, Marianna De Mitri, Fernanda Chiriacò, Giuseppe Leo, Ettore Brienza, and Michele Maffia. Cancer Lett. 2009 Apr 24

ABSTRACT: Currently clinical outcome in melanoma is not predictable by known serum biomarkers. The only reliable tool for the diagnosis of this tumor is the histopathological assay after surgical removing. We used a proteomic approach in order to identify novel non-invasive serum biomarkers of melanoma. Serum proteomic maps showed different patterns in relation to the presence and progression of the tumor in five regions of the map. Differently expressed spots were identified by MALDI-TOF mass spectrometry. Significant increases of expression were found for transthyretin (TTR) and angiotensinogen (AGT) while vitamin D binding protein (DBP) expression was decreased in presence of melanoma. Interestingly, protein expression came back to control values in stages I and II of the disease after 1month since surgical removal of suspected melanoma. We related the decrease of DBP spot to the impaired immune response of cancer patients. In fact cancer cells release the alpha-N-acetylgalactosaminidase that can deglycosylate DBP thus interfering with the immune cascade response in which DBP is involved, leading to immunosuppression in melanoma patients. Specific enzymatic activity of serum alpha-N-acetylgalactosaminidase was significantly increased in stage III melanoma patients, but not in early stages.

GcMAF Analogs

A Designed Glycoprotein Analogue of Gc-MAF Exhibits Native-like Phagocytic Activity.
Federica Bogani, Elizabeth McConnell, Lokesh Joshi, Yung Chang, and Giovanna Ghirlanda. Department of Chemistry and Biochemistry, Department of Bioengineering, School of Life Sciences, and The Biodesign Institute, Arizona State University, Tempe, Arizona 85287. J. Am. Chem. Soc., 128 (22), 7142 -7143, 2006. 10.1021/ja0604212 S0002-7863(06)00421-5
Web Release Date: May 16, 2006

A Designed Glycoprotein Analogue of Gc-MAF Exhibits Native-like Phagocytic Activity Justin Spiriti, Federica Bogani, Arjan van der Vaart and Giovanna Ghirlanda Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, United States Biophysical Chemistry Volume 134, Issue 3, May 2008, Pages 157-167

Abstract
The post-translational modification of proteins by the covalent attachment of carbohydrates to specific side chains, or glycosylation, is emerging as a crucial process in modulating the function of proteins. In particular, the dynamic processing of the oligosaccharide can correlate with a change in function. For example, a potent macrophage-activating factor, Gc-MAF, is obtained from serum vitamin D binding protein (VDBP) by stepwise processing of the oligosaccharide attached to Thr 420 to the core α-GalNAc moiety. In previous work we designed a miniprotein analog of Gc-MAF, MM1, by grafting the glycosylated loop of Gc-MAF on a stable scaffold. GalNAc-MM1 showed native-like activity on macrophages (Bogani 2006, J. Am. Chem. Soc. 128 7142–43). Here, we present data on the thermodynamic stability and conformational dynamics of the mono- and diglycosylated forms. We observed an unusual trend: each glycosylation event destabilized the protein by about 1 kcal/mol. This effect is matched by an increase in the mobility of the glycosylated forms, as evaluated by molecular dynamics simulations. An analysis of the solvent-accessible surface area shows that glycosylation causes the three-helix bundle to adopt conformations in which the hydrophobic residues are more solvent exposed. The number of hydrophobic contacts is also affected. These two factors, which are ultimately explained with a change in occupancy for conformers of specific side chains, may contribute to the observed destabilization

Additionally:

Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog. Spiriti J, Bogani F, van der Vaart A, Ghirlanda G. / Biophys Chem. 2008 May;134(3):157-67. Epub 2008 Feb 21

http://chemistry.asu.edu/faculty/g_giovanna.as

Biographical Information on Dr. Nobuto Yamamoto

Click here

Some definitions:

Macrophage activating factor, (from the very short Wikipedia entry): "A macrophage activating factor (MAF) is a lymphokine that primes macrophages to become cytotoxic to tumors. It also controls the expression of Ia antigens on the macrophage cell surface."

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