Resumen y Antecedentes
Trastornos del espectro autista (TEA) son trastornos del desarrollo que afectan a 1:88 los niños, y que parecen estar asociados con una variedad de desregulación inmune compleja incluyendo autoinmunidad. La enzima, la alfa-N-acetilgalactosaminidasa (Nagalase) proteína sérica glicosilada Gc (vitamina D3 - proteína de unión) haciéndola incapaz de activar las defensas de los macrófagos. Aumento de la actividad Nagalase se ha asociado con una variedad de tumores malignos, trastornos inmunitarios, virales y las infecciones bacterianas.
Factor activador de macrófagos (GcMAF) ha sido publicado en varias ocasiones como una intervención para reducir la actividad Nagalase suero para una variedad de cáncer y pacientes con VIH. GcMAF es una proteína natural con seguridad bien establecido y beneficios terapéuticos apoyados por numerosos estudios en humanos.
Al principio, los padres de los 40 individuos con TEA buscaban pruebas de la actividad sérica Nagalase como parte de una evaluación de desregulación inmune. Nagalase medición de la actividad enzimática fue realizada por el Laboratorio Europeo de nutrientes (ELN), Bunnik, Países Bajos, usando un ensayo enzimático de punto final de un sustrato cromogénico. Algunos padres de pacientes con elevada actividad Nagalase optaron por inyecciones semanales de GcMAF. GcMAF es purificada a partir de suero humano obtenido de la Cruz Roja Americana mediante cromatografía de afinidad alta-D3 Sepharose 25-hidroxivitamina. La proteína se diluye después adicionalmente para obtener niveles terapéuticamente apropiadas para los pacientes en función de sus presentaciones clínicas.
Las personas con TEA (32 hombres y 8 mujeres, n = 40, edades: 1 año 4 meses - 21 años 2 meses) tuvieron evaluación inicial y posterior tratamiento de la actividad Nagalase. Dosificación de GcMAF fue recomendado basado en las curvas de respuesta se informó anteriormente, ajustada por el médico tratante para la edad, el peso y los niveles de Nagalase. El pretratamiento actividad media Nagalase del grupo de autismo era 1,93 nmol / min / mg de sustrato. Esto era muy superior a la de laboratorio reportó rango normal de, 0,95 nmol / min / mg. Para el grupo de ASD el nivel medio en el momento de la prueba fue de 1,03 segundos nmol / min / mg, lo que refleja una reducción media de 0,90 nmol / min / mg (P, 0,0001). Aparte de los posibles beneficios inmunológicos de la reducción de la actividad Nagalase de estos individuos, las observaciones no controladas de la terapia GcMAF indicaron mejoras sustanciales en el lenguaje, la socialización y la cognición. No se reportaron efectos secundarios significativos durante el curso de inyecciones.
En este primer informe de la actividad Nagalase en pacientes con autismo, parece que la mayoría de los individuos tienen niveles sustancialmente más altos que los rangos saludables esperados. Aunque Nagalase es un marcador inespecífico de desregulación inmune, los niveles observados en el autismo pueden tener tanto significado etiológico y terapéutico. Es importante destacar que este es también el primer informe de reducción de la actividad Nagalase en una población autismo con inyecciones de GcMAF.
|Dr Bradstreet Initial Observatons of Elevated Nagalase Activity|
|Associated with Autism and Observed Reductions from GcMAF|
|Nagalase dataset for pre-post GcMAF therapy with iCGl response per subject.|
|Gender||Age||Pre Result||Post Result||Days Between||Difference||iCGl *|
|Male||6||0.90||0.47||76||-0.43||Considerable Improvement (4)|
|Male||21||1.00||0.44||79||-0.56||Very Considerable Improvement (5)|
|Male||7||1.30||0.72||143||-0.58||Moderate Improvement (3)|
|Male||10||2.20||1.30||200||-0.90||Slight Improvement (2)|
|Male||7||1.90||0.76||112||-1.14||Considerable Improvement (4)|
|Female||11||1.90||1.00||112||-0.90||Very Considerable Improvement (5)|
|Female||9||1.90||1.20||111||-0.70||Very Considerable Improvement (5)|
|Male||4||1.70||1.10||112||-0.60||Considerable Improvement (4)|
|Male||4||1.00||0.76||116||-0.24||Moderate Improvement (3)|
|Male||6||1.20||0.79||69||-0.41||Considerable Improvement (4)|
|Female||5||1.66||0.40||76||-1.26||Very Slight Deterioration (-1)|
|Male||12||1.69||0.47||88||-1.22||Very Slight Improvement (1)|
|Male||12||7.80||4.40||56||-3.40||Moderate Improvement (3)|
|Male||7||1.50||0.90||60||-0.60||Considerable Improvement (4)|
|Female||12||1.98||0.81||104||-1.17||Moderate Improvement (3)|
|Male||1||1.50||1.00||95||-0.50||Considerable Improvement (4)|
|Male||6||2.60||2.50||95||-0.10||Slight Improvement (2)|
|Male||1||2.80||1.80||111||-1.00||State Unchanged (0)|
|Male||18||1.30||0.92||69||-0.38||Moderate Improvement (3)|
|Male||4||3.00||1.00||87||-2.00||Very Considerable Improvement (5)|
|Male||17||1.20||0.80||128||-0.40||Considerable Improvement (4)|
|Male||4||1.60||1.10||53||-0.50||Moderate Improvement (3)|
|Female||5||0.92||0.62||103||-0.30||State Unchanged (0)|
|Male||11||1.00||0.90||88||-0.10||Moderate Improvement (3)|
|Male||5||1.40||0.81||105||-0.59||Moderate Improvement (3)|
|Female||5||3.90||1.60||126||-2.30||Very Considerable Improvement (5)|
|Female||4||1.10||0.61||89||-0.49||Slight Improvement (2)|
|Male||18||4.00||1.40||70||-2.60||Considerable Improvement (4)|
|Male||3||2.60||1.40||81||-1.20||Moderate Improvement (3)|
|Male||5||1.20||0.96||184||-0.24||Moderate Improvement (3)|
|Male||9||1.79||0.57||95||-1.22||Slight Improvement (2)|
|Female||18||1.90||1.20||159||-0.70||State Unchanged (0)|
|Male||16||1.82||0.62||77||-1.20||Slight Improvement (2)|
|Male||11||2.90||0.93||82||-0.97||Moderate Improvement (3)|
|Male||7||1.73||0.51||38||-1.22||Considerable Improvement (4)|
|Male||7||2.90||1.20||75||-1.70||Very Considerable Improvement (5)|
|Male||10||1.20||0.82||103||-0.38||Very Slight Improvement (1)|
|Male||8||1.10||0.68||103||-0.42||Slight Improvement (2)|
|Male||4||1.00||0.89||117||-0.11||Slight Improvement (2)|
|Male||4||1.20||0.91||138||-0.29||Very Considerable Improvement (5)|
Autism is a complex neurodevelopmental disorder which appears in the first three years of life. Once a rare disorder, it is now approaching epidemic, if not pandemic, proportions. A recent report from the US National Center on Birth Defects and Developmental Disabilities revealed a variable range of state by state prevalence from 4.8 to 21.2 per 1,000 for children aged 8 years (data from 2008).1 This data reflects a remarkable 23% increase over a mere two-year earlier evaluation. While no consensus exists, this trend in autism is at least suggestive of an infective pathogen. Within this context, various organisms have been postulated to be involved, including: gastrointestinal infections,2 Polyomaviruses,3 Chlamydophila,4 Bornaviruses,5 Paramyxoviruses,6 and Borrelia burgdorferi.7
While any of these may contribute to a small percentage of autism cases, it seems unlikely that any of them individually represents the origin of this epidemic. Despite this uncertainty, growing evidence supports significant immune dysfunction, including autoimmunity, in autism.8
Simultaneously, oxidative stress9 and mitochondrial dysfunction10 are common findings in this population. One possible explanation for the pattern of immune dysregulation and oxidative stress observed in autism spectrum disorders (ASD) could be persistence of active pathogens, perhaps from the perinatal or a subsequent period of child development.11
Viruses are also known to subvert intracellular calcium regulation to their own functional requirements and in that process to disrupt mitochondrial activity.12 Measurement of biomarkers related to immune dysregulation and putative infectious agents is a routine part of the author’s (JB) evaluation of ASD in a clinical setting. Recently, evaluation of the activity of alpha-N-acetylgalactosaminidase (Nagalase) has been made commercially available as a diagnostic laboratory measurement.
Nagalase has been published as a biomarker associated with various types of cancer,22–24,29,30 systemic lupus erythematosus SLE),13 influenza,14 and human immunodeficiency virus infection (HIV).31 It is enzymatically distinct from hepatic galactosaminidase and appears to be far more biologically active.
Nagalase is therefore a nonspecific biomarker, which appears to be an important indicator of secondary immune dysregulation. Nagalase is a component of viral emagglutinin and is released by the action of trypsin on hemagglutinin.14
Since hemagglutinin is a common glycan-binding lectin of many viruses (including influenza, paramyxoviruses and polyomaviruses), several viruses may individually or jointly contribute to hemagglutininderived Nagalase activity in the blood.15 In the absence of recent viral infection or malignancies, elevated Nagalase activity likely represents a marker of viral production of hemagglutinin protein being acted upon by inflammatory cell mediated trypsin activity; as such it may represent viral persistence, active transcription, and inflammation.
Viral protein transcription is one potential mechanism of autoimmunity.16,17 Beyond this, Nagalase is an enzyme that deglycosylates the Gc protein also known as vitamin D binding protein (VDBP), rendering it incapable of conversion to active GcMAF (Gc protein derived Macrophage Activating Factor) and thereby preventing its regulation of macrophage activation.18 It is noteworthy that vitamin D deficiency, either in pregnancy or during postnatal development, is an apparent risk factor for autism.19
The impact of Nagalase on VDBP transportation of vitamin D is not known. However, vitamin D deficiency is a known risk factor for autoimmunity.20 In light of the possible involvement of immune abnormalities, autoimmunity, vitamin D deficiency, and potential viral persistence, Nagalase screening was added by JB and other physicians to the biomarker profile21 of children presenting for biomedical evaluation of autism-related disorders and co-morbidities.
A retrospective chart review for analysis of routine Nagalase testing was accomplished on the initial cohort of patients tested by the clinician (JB). All records were reviewed by JB for confirmation of test results, confirmed diagnosis of autism, time intervals between testing, dosing of subsequent GcMAF used, and the observed clinical/parental/therapist/teacher responses. All patients met the criteria for autism (299.00 DSM-IV revised) and were diagnosed by either a child neurologist or developmental psychologist, in addition to receiving the evaluation of the clinician. No significant changes were made to the participants’ treatments apart from the introduction of GcMAF during the time frame reported in this study.
Additional pre-screening assessments
In addition to meeting DSM-IV revised criteria for autism and having independent determination of autistic severity performed by non-affiliated practitioners, the clinician in this study used an in-house severity scoring system. This consisted of 20 questions (Table 4) designed to evaluate relative severity in the standard domains required for the diagnosis of autism (ie, language, socialization, and stereotypies), as well as other meaningful determinants. Within each category the clinician and parents agreed on a score as follows:
1 = normal or near normal
2 = mild
3 = moderate
4 = severe
All of the participants assessed in this initial evaluation were in the 3–4 range for question 1 (General Impression of Autism Severity or Delayed Development). This was also true for the core domains of autism (questions 2, 4, and 10). Additionally, all participants scored a 4 (severe) for question 9, indicating a lack of imaginative or age-appropriate play. Significant variability was observed for most of the other domains in the questionnaire, especially in the areas related to motor (both fine and gross), where the greatest initial variability was observed. Specific assessments within each domain were beyond the limited scope of this initial retrospective analysis and would instead be appropriate for a future prospective investigation.
The parents provided written informed consent for phlebotomy and evaluation of potential medical comorbidities occurring in their children with autism. Specifically, Nagalase was discussed with the parents as a potential marker of immune dysregulation. Upon agreement by the parents, sufficient venous blood was withdrawn to fill a 9 ml EDTA tube, which was then immediately inverted at least 5 times. The tube was then centrifuged for 10 minutes at 3000 rpm to separate the plasma. After separation, approximately 3 ml of the clear plasma was transferred to the plasma collecting tube, which was then immediately frozen to −20 °C for at least 24 hours. The specimen was then shipped frozen overnight to an intermediary laboratory in New Jersey, United States. Collected specimens were kept frozen during further shipping to ELN. The cohort in this initial study consisted of 40 patients whose records included both pre- and post-treatment Nagalase blood test results.
Since this was a preliminary and retrospective evaluation of both Nagalase activity and the response to GcMAF, these initial data and observations reflect only the period between the first and second Nagalase testing for patients. Of the 40 subjects, there were 32 males (ages at the point of first testing ranged from 1 year and 4 months to 21 years) and 8 females (ages ranged from 4 years 7 months to 18 years, median = 6.93 ± SD 5.08 years)
Following the procedure published by Yamamoto et al22,23, Nagalase activity was determined by using an endpoint enzymatic assay using a chromogenic substrate. ELN established a reference range of 0.32–0.95 nM/min/mg of substrate based on serum collected from healthy volunteers, a range nearly superimposable to that previously reported which was between 0.35 and 0.65 nM/min/mg.24
GcMAF preparation & application
Patients obtained commercially available GcMAF. Briefly, Gc protein was isolated from purified human serum obtained from the American Red Cross, using either 25-hydroxyvitamin D3-Sepharose high affinity chromatography or actinagarose affinity chromatography. The bound material was eluted and then further processed by incubation with three immobilized enzymes. The resulting GcMAF was filter sterilized. The protein content and concentration was assayed using standard Bradford protein assay methods.25 At the end of the production process, the GcMAF was checked for sterility in-house and externally by the UK Health Protection Agency and independent laboratories.26
After appropriate written informed consent by the parents, GcMAF was injected subcutaneously on a weekly basis using a 31 gauge insulin syringe. In adults with HIV or several different types of cancer, the typical dose described for response given either intramuscular or intravenous administration has been a minimum of 100 ng/wk. Despite levels of Nagalase activity well into the range of many cancer or HIV patients, the clinician elected not to exceed 100 ng/wk so as to prevent putative over-stimulation of macrophages. All patients started on low doses that were increased gradually over the course of treatment.
The doses ranged from 4 to 100 ng per wk and were adjusted based on clinical response, age, body weight, and the initial level of Nagalase activity. To further assess the clinical responses, all parents were interviewed at intervals of no longer than a month using the improved Clinical Global Impression Scale (iCGI) defined by Kadouri et al and described below.29
Improved response format for the clinical global impression of improvement scale
The improved response format for the Clinical Global Impression of Improvement scale is as follows:
5 = Very considerable improvement
4 = Considerable improvement
3 = Moderate improvement
2 = Slight improvement
1 = Very slight improvement
0 = State unchanged
−1 = Very slight deterioration
−2 = Slight deterioration
−3 = Moderate deterioration
−4 = Considerable deterioration
−5 = Very considerable deterioration
−6 = Maximum deterioration
Due to the relatively small population overall and only 8 females in this sample, the group was evaluated as a whole and not segregated based on gender or age.
Statistical comparison between pre and post treatment levels of Nagalase was performed by two-tailed, paired difference t-test and by using standard formulas in Microsoft Excel® 2010. Since one subject had a Nagalase result significantly higher than the mean, consideration for skewing artifact was made. Adjusting for the skew effect changed the median of the group from 1.71 to 1.68, which was not statistically significant.
The average pre-GcMAF treatment Nagalase activity was 1.93nM/min per mg, with a median of 1.68 nM/min per mg (SD ± 1.21 nM/min per mg), and with a range of 0.90 nM/min per mg to 7.80 nM/min per mg (Table 1). At the point of time of subsequent testing (average interval 100 days, ±SD 32 days), the average Nagalase activity during GcMAF treatment was 1.03 nM/min per mg, with a median of 0.90 nM/min per mg (±0.67 nM/min per mg), and with a range of 0.44 to 4.40 nM/min per mg. This reflects an average reduction of 0.90 nM/min per mg (P , 0.0001). Of this original cohort only 2 of 40 (5%) were observed to be initially within the laboratory reference range (0.90 and 0.92 nM/min per mg).
Because of the standard laboratory turnaround time and the necessary time to discuss treatment options with the parents, the actual number of weekly injections was substantially less than the number of weeks in the interval of retesting. The average number of weekly injections was 14 (±4 weeks SD). At the time of retesting, the Nagalase levels of 24 of the 40 patients (60%) had decreased to within the laboratory reference range of ,0.95 nM/min per mg. In view of these results, a minimum of 16 patients (40%) would be considered not to have received adequate therapeutic effect and would therefore be candidates for continued intervention.
Only 1 of 40 (2.5%) failed to respond with significant reduction of Nagalase activity (pre/post Nagalase difference of only 0.10 nM/min per mg). The families of the two patients whose initial Nagalase levels were within the upper part of the laboratory reference range both elected to initiate GcMAF therapy. Both patients experienced significant reductions in Nagalase activity, one with a considerable response (iCGI = 4), while the other was rated as a non-responder.
The initial levels of Nagalase activity in the group of patients that we studied ranged from the upper range of normal to beyond levels typically observed in metastatic cancer patients30,31 and HIV-infected patients.32
Despite concerns about autoimmunity in autism, none of the patients observed in this study experienced significant side-effects, and none were required to suspend or drop out of treatment.
During the first few weeks of treatment, 3 of 40 patients (7.5%) experienced low to moderate rise in body temperature, typically occurring 24 to 48 hours after the GcMAF injection and lasting less than 24 hours. Parents were instructed to use ibuprofen only if the temperature exceeded 102°F (approximately 39°C), and two were treated during the first few weeks.
By the second month, no patients experienced significant febrile events. Interestingly, during the first 3 weeks, 6 of 40 patients (15%) were observed to have rashes compatible with viral exanthemas (generally on the trunk and in fine papules more commonly than maculae). Petechiae were not observed. These rashes could represent the manifestation of latent or persistent viral infections interacting with activated macrophages.
Since this is an open-label, non-controlled, retrospective analysis, caution must be employed when ascribing cause and effect to any treatment outcome. However, the response to GcMAF was robust with regard to Nagalase reduction, as well as symptomatic improvements as shown by the iCGI. Despite the short observational time period, the result that 67.5% of the group responded in the 3 to 5 CGI-I range was unexpectedly substantial (Tables 2 and 3). In this small population, it does not appear that an obvious association exists between the iCGI response and the change in the Nagalase activity (Table 3). Further statistical analysis was therefore not deemed appropriate.
The changes in Nagalase activity in response to GcMAF treatment in this ASD population reflected similar robust responses observed using GcMAF in the treatment of HIV infection and cancer. However, autism represents a developmental disorder with substantial delays in core domains of cognitive activity (language, socialization, and behavior) and is generally felt to be a life-long condition. Therefore, these initial observations give support to the notion that autism per se may be the consequence of treatable underlying pathophysiology. Given that ASD are now affecting more than 1% of US children, the observed response to GcMAF warrants urgent and further prospective evaluation.
Although Nagalase is a non-specific marker believed to be derived from viral hemagglutinin, it may be useful as a biomarker of therapeutic significance in ASD, and as such also warrants further investigation. Regardless of any immediate clinical improvement, the reduction of Nagalase to more desirable levels is of potential benefit to these patients, since Nagalase is known to impair immune defenses.
1. Baio J. Prevalence of Autism Spectrum Disorders—Autism and Developmental Disabilities Monitoring Network, 14 Sites, United States, 2008. MMWR Surveill Summ. Mar 30, 2012;61(3):1–19.
2. Finegold SM, Downes J, Summanen PH. Microbiology of regressive autism. Anaerobe. Apr 2012;18(2):260–2.
3. Lintas C, Altieri L, Lombardi F, Sacco R, Persico AM. Association of autism with polyomavirus infection in postmortem brains. J Neurovirol. Mar 2010; 16(2):141–9.
4. Contini C, Seraceni S, Cultrera R, Castellazzi M, Granieri E, Fainardi E. Chlamydophila pneumoniae Infection and Its Role in Neurological Disorders.Interdiscip Perspect Infect Dis. 2010;2010:273573. Epub Feb 21, 2010.
5. Pletnikov MV, Rubin SA, Vasudevan K, Moran TH, Carbone KM. Developmental brain injury associated with abnormal play behavior in neonatally Borna disease virus-infected Lewis rats: a model of autism. Behav Brain Res. Apr 1999;100(1–2):43–50.
6. Chess S. Autism in children with congenital rubella. J Autism Child Schizophr. Jan–Mar 1971;1(1):33–47.
7. Bransfield RC, Wulfman JS, Harvey WT, Usman AI. The association between tick-borne infections, Lyme borreliosis and autism spectrum disorders.Med Hypotheses. 2008;70(5):967–74.
8. Onore C, Careaga M, Ashwood P. The role of immune dysfunction in the pathophysiology of autism. Brain Behav Immun. Mar 2012;26(3):383–92.
9. Chauhan A, Audhya T, Chauhan V. Brain region-specific glutathione redox imbalance in autism. Neurochem Res. Aug 2012;37(8):1681–9.
10. Giulivi C, Zhang YF, Omanska-Klusek A, et al. Mitochondrial dysfunction in autism. JAMA. Dec 1, 2010;304(21):2389–96.
11. Fatemi SH, Cuadra AE, El-Fakahany EE, Sidwell RW, Thuras P. Prenatal viral infection causes alterations in nNOS expression in developing mouse brains. Neuroreport. May 15, 2000;11(7):1493–6.
12. Zhou Y, Frey TK, Yang JJ. Viral calciomics: interplays between Ca2+ and virus. Cell Calcium. Jul 2009;46(1):1–17. Epub Jun 16, 2009. Review.
13. Yamamoto N, Naraparaju VR, Moore M, Brent LH. Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus. Clin Immunol Immunopathol. Mar 1997;82(3):290–8.
14. Yamamoto N, Urade M. Pathogenic significance of alpha-N-acetylgalactosaminidase activity found in the hemagglutinin of influenza virus. Microbes Infect. Apr 2005;7(4):674–81.
15. Varki A, Cummings RD, Esko JD, et al. Essentials of Glycobiology. 2nd ed. Chapter 34. Microbial Lectins: Hemagglutinins, Adhesins, and Toxins. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009.
16. Khera TK, Dick AD, Nicholson LB. Mechanisms of TNFα regulation in uveitis: focus on RNA-binding proteins. Prog Retin Eye Res. Nov 2010;29(6):610–21. Epub Sep 8, 2010. Review. Erratum in: Prog Retin Eye Res. Mar 2011;30(2):147.
17. Giraudon P, Bernard A. Chronic viral infections of the central nervous system: Aspects specific to multiple sclerosis. Rev Neurol (Paris). Oct 2009; 165(10):789–95.
18. Yamamoto N, Naraparaju VR. Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor. Cancer Res. Jun 1, 1997;57(11):2187–92.
19. Kočovská E, Fernell E, Billstedt E, Minnis H, Gillberg C. Vitamin D and autism: Clinical review. Res Dev Disabil. Sep 2012;33(5):1541–450.
20. Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation. Annu Rev Nutr. 2003;23:117–45.
21. Bradstreet JJ, Smith S, Baral M, Rossignol DA. Biomarker-guided interventions of clinically relevant conditions associated with autism spectrum disorders and attention deficit hyperactivity disorder. Altern Med Rev. Apr 2010;15(1):15–32.
22. Yamamoto N, Naraparaju VR, Asbell SO. Deglycosylation of serum vitamin D-binding protein and immunosuppression in cancer patients. Cancer Res. 1996;56:2827–31.
23. Yamamoto N, Naraparaju VR, Urade M. Prognostic utility of serum α-N-acetylgalactosaminidase and immunosuppression resulted from deglycosylation of serum Gc protein in oral cancer patients. Cancer Res. 1997;57:295–9.
24. Yamamoto N, Suyama H, Yamamoto N. Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF. Transl Oncol. Jul 2008;1(2):65–72.
25. Bradford, MM. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976;7:248–54.
26. GcMAF assays. FIRST IMMUNE GCMAF. http://www.gcmaf.eu/info/ index.php?option=com_content&view=article&id=114&Itemid=53.
27. Pacini S, Morucci G, Punzi T, Gulisano M, Ruggiero M. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay. Cancer Immunol Immunother. Apr 2011; 60(4):479–85.
28. Pacini S, Punzi T, Morucci G, Gulisano M, Ruggiero M. Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells. Anticancer Res. Jan 2012;32(1):45–52.
29. Kadouri A, Corruble E, Falissard B. The improved Clinical Global Impression Scale (iCGI): development and validation in depression. BMC Psychiatry. Feb 6, 007;7:7.
30. Yamamoto N, Suyama H, Nakazato H, Yamamoto N, Koga Y. Immunotherapy of metastatic colorectal cancer with vitamin D-binding protein-derived macrophage-activating factor, GcMAF. Cancer Immunol Immunother. Jul 2008;57(7):1007–16.
31. Yamamoto N, Suyama H, Yamamoto N, Ushijima N. Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF). Int J Cancer. Jan 15, 2008; 122(2):461–7.
32. Yamamoto N, Ushijima N, Koga Y. Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF). J Med Virol. Jan 2009;81(1):16–26.
Full paper is available here