Tratamiento GcMAF para autismo Marco Ruggiero MD, PhD Firenze, Italy

1. Tratamiento GcMAF para autismoMarco Ruggiero MD, PhDFirenze, Italy

2. The first paper on GcMAF (Gc-protein-derived Macrophage Activating Factor) published in a peer-reviewed scientific journal indexed in the National Library of Medicine of the USA is dated 1994.

3. About 20 years later the research on GcMAF is a solid scientific reality with scores of papers published on its effects in vitro and in vivo and with hundreds of presentations at international congresses describing its effects in a variety of experimental systems as well as in several pathologic conditions.

8. However … GcMAF has many more effects in addition to stimulating macrophages and fighting cancer. It is effective in neurological diseases and in AUTISM.

10. 5 Very considerable improvement 4 Considerable improvement 3 Moderate improvement 2 Slight improvement Clinical Global Impression of Improvement response to GcMAF after an average of 14 weekly subcutaneous injections.

11. 5 = Very considerable improvement. This response was demonstrated at school, during therapies, home and outside the home as substantial improvement to the point that many or most of the criteria of autism were no longer present!

12. Preliminary conclusions concerning GcMAF and autism The response to GcMAF was robust with regard to Nagalase reduction, as well as symptomatic improvements as shown by the Clinical Global Impression of Improvement scale. 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.

13. It is worth noticing that more and more data confirming these results are accumulating and there are now about 1.500 children under treatment.

14. Doctors reported the effects of GcMAF in patients with a variety of diseases. Scientists described its effects in human cancer cells, in human neurons and in cardiac function. There are so many different effects reported following GcMAF administration that a question spontaneously arises:

15. How can a single molecule, GcMAF, be effective in such a variety of conditions ranging from cancer, to autism, ME/CFS, chronic kidney disease, neurological diseases (such as Parkinson’s and Alzheimer’s diseases or Multiple Sclerosis) as well as conditions associated with environmental pollution ?

16. We recently elucidated the molecular mechanism of action that is responsible for such a variety of effects. Here I shall report original data demonstrating a direct effect of GcMAF on human neurons. These results help understanding the efficacy of GcMAF in treating autism and my help developing new molecules specifically designed to treat autism.

17. NH2 4 8 11 9 2 10 1 7 5 6 3 Asp Tyr Arg Asp Arg Leu Lys Glu Lys Glu Gly 12 Val 15 17 20 25 26 13 18 29 30 31 23 16 21 22 24 27 28 14 19 32 33 34 35 Tyr Lys Leu Lys Glu Phe Leu Ser Ser Arg Lys Phe Asp Glu Gly Leu Val Leu Ser His Thr Ser Cys 36 Phe 61 59 50 44 58 57 55 51 42 41 39 56 52 48 46 40 38 60 43 45 37 54 53 49 47 62 Leu Leu Lys Leu Ser Gln Ala Glu Val Thr Ser Pro Ala Thr Val Val Gln Gln Phe Gly Cys Glu Val Glu Cys Glu 63 76 67 69 70 75 68 71 72 73 78 79 74 80 64 65 66 77 82 84 85 81 83 86 87 Ala Ala Asn Phe Pro Asp Pro Asp Ala Cys Glu Ser Ser Pro Tyr Arg Thr Ser Lys Ser Leu Cys Asp Thr Ser 88 Val 97 93 99 96 94 92 91 89 90 98 95 100 Lys Cys Pro His Glu Thr Cys Glu Thr Gly Ala Gly Domain I 101 104 105 102 106 103 107 108 Leu Arg Met Glu Cys Lys Leu Ala 109 115 120 116 113 112 124 100 125 121 117 126 122 118 114 123 119 111 Ala 127 Thr Pro Pro Lys Asp Pro Glu Phe Asn Val Thr Tyr Glu Gln His Leu Gln Glu 134 142 148 149 150 131 130 132 144 146 129 133 135 140 143 145 147 141 139 137 128 136 138 151 Cys Glu Ala Phe Arg Lys Asp Pro Lys Glu Tyr Ala Asn Gln Phe Met Trp Glu Tyr Ser Thr Asn Ile Tyr 152 172 161 173 157 176 175 174 171 166 162 160 170 169 163 168 167 165 158 156 155 154 159 153 164 177 Glu Val Met Gly Ser Thr Cys Ser Leu Ser Lys Val Cys Ser Ser Thr Tyr Tyr Ser Leu Leu Leu Pro Ala Gln Ala 178 182 183 186 185 181 189 190 184 187 188 179 180 191 Ser Thr Pro Lys Leu Arg Leu Gln Leu Val Cys Phe Lys Glu 192 His 203 202 201 200 199 198 197 196 195 194 193 Leu Val Arg Asn Ser Leu Thr Thr Leu Leu Ser 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 Tyr Gly Glu Leu Ile Lys Leu Ala Gln Lys Cys Ser Gln Tyr Ala Ala Lys Lys Ser Arg Leu Ser Asn 227 Val 252 251 241 250 249 248 247 246 245 244 243 242 240 239 238 237 236 235 234 233 232 231 230 229 228 Ala Thr Pro Cys Cys Lys Ser Leu Ile Asn Thr Ile Glu Ala Leu Pro Leu Val Asp Glu Leu Asp Asp Ser Glu 253 Ala 255 259 271 254 272 261 266 256 262 270 274 276 257 258 263 265 267 268 269 273 275 260 264 Asp Asn Asp Gln Thr Ser Lys Gln Cys Met Ala Leu Cys Leu Thr Ser Lys Pro His Val Lys Leu Glu 277 Asp 288 287 286 285 284 283 282 281 280 279 278 Phe Lys Ser Thr Lys Glu Glu Cys Cys Asp Glu Domain II 289 Ala 290 291 292 293 294 295 296 297 298 299 Asp Val Phe Val Cys Thr Tyr Phe Met Met 300 316 315 314 313 312 310 309 308 307 306 304 303 301 311 305 302 Lys Asn Thr Pro Leu Arg Val Asp Pro Leu Glu Pro Leu Gln Ala Ala Pro 317 Asp 331 332 333 318 319 320 334 335 336 337 321 322 323 325 326 327 328 329 330 324 338 Pro Gly Asn Thr Lys Val Met Asp Lys Val Leu Ser Arg Arg Val Cys Asp Thr Phe Glu Thr 339 362 360 359 358 352 351 350 349 347 346 345 340 361 357 356 355 354 353 344 343 342 341 348 363 His Val Asp Cys Cys Glu Gly Lys Ser Lys Leu Thr Pro Glu Leu Val Lys Ser Leu Phe Val Glu Pro Leu Glu 364 365 366 367 368 369 371 372 374 375 376 370 373 377 Asp Ser Thr Thr Cys Phe Asp Ala Lys Gly Pro Leu Leu Lys 378 Lys 381 383 380 379 389 387 385 384 382 388 386 Ser Ser Leu Glu Gln Gly Lys Asp Ile Glu Phe 390 Leu 401 407 408 402 391 392 393 394 395 396 397 398 400 411 412 413 399 404 405 406 409 410 403 Domain III Lys Leu Arg Leu Asp Tyr Ser Asn Thr Phe Thr Lys Lys Ala Lys Cys Ala Glu Lys Tyr Ala Glu Glu 414 Leu 421 429 439 420 423 418 430 428 427 426 425 424 422 438 437 436 435 434 433 432 431 417 416 415 419 440 Ala Glu Pro Ser Cys Cys Asn Ser Ala Phe Asp Ser Lys Asn Val Leu Lys Leu Pro Ala Thr Thr Arg Ile Glu Asn 441 458 453 COOH 442 443 444 445 446 447 448 449 450 451 454 455 456 457 452 Ser Leu Asp Ala Leu Asn Ile Leu Tyr Cys Asp Ser Glu Ile Lys Pro Pro Glu Gc protein (known as Vitamin D3-binding protein) 420 thr

18. In inflammation and immune responses, Thr 420 is de-glycosylated and Gc protein transformed into GcMAF, one of the most powerful activators of the immune system discovered so far. The biochemical events occurring in vivo thanks to the enzymes produced by B- and T-lymphocytes, can be reproduced in the laboratory or may occur during fermentation of milk thanks to enzymes produced by lactic ferments.

19. sialidase Thr Thr Thr Thr Thr Thr of T cells GalNAc SA GalNAc (a) b-galacto- sidase of B cells* GalNAc Gal GalNAc SA SA Gc protein Macrophage activating Events occurring in vivo factor (MAF) (b) immobilized immobilized b-galacto- sidase GalNAc Gal sialidase GalNAc SA Purified Macrophage activating Gc protein factor (GcMAF) Events reproduced in the laboratory or during milk product fermentation

20. A VDR GcMAF Vit D Vit D cytoplasm GcMAF Oleic acid Plane of the membrane

21. B Vit D Plane of the membrane VDR cytoplasm GcMAF Oleic acid Oleic acid

23. GcMAF by interacting with the VDR modifies the expression of a number of genes in cells expressing the VDR and elicits a series of responses that go well beyond its capability of activating macrophages. BAG gene has been implicated in age related neurodegenerative diseases as Alzheimer's

25. Here I shall focus on The effects of GcMAF on human neuronal viability and metabolic activity. These effects, in addition to the immuno-stimulating properties of GcMAF, help explaining its therapeutic efficacy in autism. I shall present original data on human neurons in culture as well as on experimental rats.

26. It is well ascertained that significant neuroanatomical changes occur in autism, and they are consistent with the neuropathologic symptoms that are characteristic of this condition. It has been hypothesized that these anatomical changes are associated with defects in connectivity. Atypical brain connectivity in autism is associated with reduction of grey matter volume in different areas of the brain. Therefore, it can be hypothesized that factors stimulating neuronal viability and metabolism might counteract such alterations and improve clinical symptoms in ASD patients.

27. GcMAF and energy production at the cellular (mitochondrial) level.

29. The following figure shows that GcMAF (8-800 pM) significantly increased neuronal cell viability and metabolic activity in a dose-dependent manner with significant effects even at the lowest pM concentration.

31. The following figure shows that increased viability and metabolic activity following GcMAF stimulation were consistent with the morphological changes induced by GcMAF in human neurons. In the absence of GcMAF, neuronal cells, stained with haematoxylin-eosin, appeared as small, undifferentiated cells with large nuclei.

33. After 24-72 h stimulation with 8 pMGcMAF, cells showed a significant change in morphology that was consistent with the induction of neuronal differentiation. The cytoplasm was enlarged and several cytoplasmic elongations resembling neuritis and dendrites could be observed. These changes indicate that, after stimulation with GcMAF, neurons increase their metabolic activity and their level of connectivity.

35. Taken together these results indicate that GcMAF, at pMconcentration, increases neuronal cell viability, metabolic activity and differentiation, with the first effects being observed at 24h. It is worth noticing that the assay that we used to determine cell viability measures mitochondrial activity, and mitochondrial dysfunction is known to be hallmark of ASD. Therefore, the effects of GcMAF described here might directly counteract one of the basic alterations of neuronal function at the sub-cellular level.

37. Having observed a significant stimulatory effects of GcMAF on mitochondrial activity in human neurons, we decided to study its effects in a well-established model of neuropathy that is the osteoarthritic pain induced by monoiodoacetate. This model is used in neuropharmacology to evaluate the effects of neuro-protective drugs and to avoid any type of placebo effects.

42. GcMAF, 15 minutes after i.p. administration, increased the withdrawal threshold and was still effective after 24h. The weight tolerated on the ipsilateral paw was 55+6 g, that is almost identical to the weight tolerated on the controlateral paw and in control animals. It is worth noticing that GcMAF did not modify the weight tolerated in the controlateral healthy paw, that remained 60+5 g. This demonstrates that the effects of GcMAF were not to be attributed to non-specific analgesia.

43. Future directions GcMAF is now available in different forms that can be combined to obtain the best individualized response. Injectable pure GcMAF. Sublingual GcMAF. Food-based GcMAF. GcMAF for topical use as a mouth-wash or as an ointment.

44. Why food-based MAF? We were inspired by Nature’s way of conferring powerful immunity to mammal newborns and we hypothesized that GcMAF could be produced in one’s own kitchen in a fully natural way.

46. Certain strains of bacteria produce the very same enzymes, that synthesize GcMAFin vivo and in vitro. We hypothesized that GcMAF could be obtained through a natural milk fermentation process that would convert mammal milk Gc-protein into GcMAF.

47. Milk-derived Gc-MAF would activate the MALT in the walls of the entire gastrointestinal tract and, in the Waldeyer's-Pirogov tonsillar ring.

50. Desideravo informarla che Saverio ora assume circa 60 ml di Bravo Probiotic al dì. Attualmente, sono circa tre settimane che ha iniziato la cura, noto una notevole diminuizione della sua stereotipia vocale (mugugnava sempre) ed una maggiore attenzione a ciò che accade attorno a lui, straordinariamente migliorata è inoltre ogni capacità inerente alle autonomie personali. Esce persino da casa per andare da solo ad acquistare gelati o merende prendendo i soldi senza dirmi nulla.