Reply to: Alzheimer’s Disease and Copper Biochemistry
I would like to thank Professor Emeritus Leslie M. Klevay for having read the article carefully and for allowing an open debate on copper involvement in Alzheimer’s disease (AD).
Our approach to ceruloplasmin specific activity (eCp/iCp) was inspired from a study by Professor Brewer and colleagues  that described the eCp/iCp to be lower in a group of patients with respect to controls, while the percentage of free copper was higher in AD group than in controls. The ratio eCp/iCp was proposed by Milne and Johnson  as an indicator of copper status not affected by age, gender, or hormone use. In our previous study , we measured eCp/iCp in Wilson disease (WD), demonstrating that it was lower than in the control group. A previous study  on WD showed that ceruloplasmin values measured immunologically could give higher values than those measured enzymatically, as also suggested by Walshe . Merle et al.  proposed serum ceruloplasmin oxidase activity as a sensitive and highly specific diagnostic marker for WD. In fact, the commercial kits usually employed do not discriminate between apo-ceruloplasmin and the completely active form of the protein: the holo-ceruloplasmin. Therefore, in WD patients, an apparent normal value of ceruloplasmin concentration could camouflage an inactive ceruloplasmin. Our study  confirmed eCp/iCp as a good index of the real status of circulating ceruloplasmin in WD.
A lower activity of ceruloplasmin in WD derives from a defective ATP7B copper pump, which does not excrete the copper from the liver and does not load copper into the nascent apo-ceruloplasmin. This is the reason why, in WD, mutations of the ATP7B gene modify copper incorporation into the protein, producing apo-ceruloplasmin that is less active. Moreover, this dysfunction produces a higher fraction of copper not bound to ceruloplasmin (Non Cp-Cu), which is one typical altered parameter in WD .
The same concept can be applied to copper dismetabolism in AD. In the course of the last ten years, a number of studies demonstrated that increased concentrations of Non Cp-Cu and other copper abnormalities are also evident in AD (meta-analysis of copper in AD: [8-10]; meta-analysis of Non Cp-Cu in AD: ). Moreover, specific ATP7B gene variants have been reported to independently increase the risk of AD [12-17]. It is also important to note that a meta-analysis on brain metals in AD  demonstrated that copper was significantly depleted in AD. Thus, an in increase in copper in general circulation corresponds to a copper depletion in the brain.
In our opinion, the problem of copper dismetabolism is strictly related to ATP7B imbalance. It is clear that a low copper diet could be recommended only to patients with evidence of copper dismetabolism, both obtained from genetic evaluation of ATP7B gene or from biochemical copper panel measurements (copper, eCp/iCp, Non Cp-Cu). Copper, in fact, is an essential metal and, in normal metabolism, it is fundamental to intake the appropriate quantity from diet.
We agree that the eCp/iCp index measured in subjects without ATP7B impairment is a sensitive measurement of copper deficiency, because the lower the copper intake in the diet, the lower the biosynthesis of ceruloplasmin and the lower the eCp/iCp; even so, this is not the case in AD, as clearly demonstrated in our article, showing increased levels of Non Cp-Cu associated with eCp/iCp decrement.
So, finally, we do not agree with Prof. L.M. Klevay’s interpretation of our data, and strongly advise against taking copper supplements as stated in . In fact, supplements have been associated with an 18% increase in mortality  and cognitive decline  in large population studies: IOWA women (40,000 subjects) and CHAP study (~4,000 subjects follow up for 9 years), respectively.
Mariacristina Siotto and Rosanna Squitti
 Brewer GJ, Kanzer SH, Zimmerman EA, Celmins DF, Heckman SM, Dick R (2010) Copper and ceruloplasmin abnormalities in Alzheimer’s disease. Am J Alzheimers Dis Other Demen 25, 490-497.
 Milne DB, Johnson PE (1993) Assessment of copper status: Effect of age and gender on reference ranges in healthy adults. Clin Chem 39, 883–887.
 Siotto M, Pasqualetti P, Marano M, Squitti R (2014) Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson’s disease and in hepatic encephalopathy. J Neural Transm 121, 1281–1286.
 Macintyre G, Gutfreund KS, Martin WRW, Camicioli R, Cox DW (2004) Value of an enzymatic assay for the determination of serum ceruloplasmin. J Lab Clin Med 144, 294–301.
 Walshe J (2003) Wilson’s disease: the importance of measuring serum caeruloplasmin non-immunologically. Ann Clin Biochem 40, 115–121.
 Merle U, Eisenbach C, Weiss KH, Tuma S, Stremmel W (2009) Serum ceruloplasmin oxidase activity is a sensitive and highly specific diagnostic marker for Wilson’s disease. J Hepatol 51, 925–930.
 Easl (2012) EASL Clinical Practice Guidelines: Wilson’s disease. J Hepatol 56, 671–685.
 Ventriglia M, Bucossi S, Panetta V, Squitti R (2012) Copper in Alzheimer’s disease: a meta-analysis of serum, plasma, and cerebrospinal fluid studies. J Alzheimers Dis 30, 981–984.
 Wang ZX, Tan L, Wang HF, Ma J, Liu J, Tan MS, Sun JH, Zhu XC, Jiang T, Yu JT (2015) Serum iron, zinc, and copper levels in patients with Alzheimer’s disease: a replication study and meta-analyses. J Alzheimers Dis 47, 565–581.
 Schrag M, Mueller C, Oyoyo U, Smith MA, Kirsch WM (2011) Iron, zinc and copper in the Alzheimer’s disease brain: A quantitative meta-analysis. Some insight on the influence of citation bias on scientific opinion. Prog Neurobiol 94, 296–306.
 Squitti R, Simonelli I, Ventriglia M, Siotto M, Pasqualetti P, Rembach A, Doecke J, Bush AI (2014) Meta-analysis of serum non-ceruloplasmin copper in Alzheimer’s disease. J Alzheimers Dis 38, 809–822.
 Bucossi S, Mariani S, Ventriglia M, Polimanti R, Gennarelli M, Bonvicini C, Pasqualetti P, Scrascia F, Migliore S, Vernieri F, Rossini PM, Squitti R (2011) Association between the c. 2495 A>G ATP7B polymorphism and sporadic Alzheimer’s disease. Int J Alzheimers Dis 2011, 973692.
 Bucossi S, Polimanti R, Ventriglia M, Mariani S, Siotto M, Ursini F, Trotta L, Scrascia F, Callea A, Vernieri F, Squitti R (2013) Intronic rs2147363 variant in ATP7B transcription factor-binding site associated with Alzheimer’s disease. J Alzheimers Dis 37, 453–459.
 Squitti R, Polimanti R, Siotto M, Bucossi S, Ventriglia M, Mariani S, Vernieri F, Scrascia F, Trotta L, Rossini PM (2013) ATP7B variants as modulators of copper dyshomeostasis in Alzheimer’s disease. Neuromolecular Med 15, 515–522.
 Liu H, Lin W, Wang W, Tsai C, Wu W, Chiou M, Shen C, Wu B, Tsai F (2013) Genetic variability in copper-transporting P-type Triphosphatase (ATP7B) is associated with Alzheimer’s disease in a Chinese population. J Biol Regul Homeost Agents 27, 319–327.
 Squitti R, Ventriglia M, Gennarelli M, Colabufo NA, El Idrissi IG, Bucossi S, Mariani S, Rongioletti M, Zanetti O, Congiu C, Rossini PM, Bonvicini C (2017) Non-ceruloplasmin copper distincts subtypes in Alzheimer’s disease: a genetic study of ATP7B frequency. Mol Neurobiol 54, 671-681.
 Squitti R, Ventriglia M, Gennarelli M, Colabufo NA, El Idrissi IG, Bucossi S, Mariani S, Rongioletti M, Zanetti O, Congiu C, Rossini PM, Bonvicini C (2017) Erratum to: Non-ceruloplasmin copper distincts subtypes in Alzheimer’s disease: a genetic study of ATP7B Frequency. Mol Neurobiol 54, 682-683.
 Barnard ND, Bush AI, Ceccarelli A, Cooper J, de Jager CA, Erickson KI, Fraser G, Kesler S, Levin SM, Lucey B, Morris MC, Squitti R (2014) Dietary and lifestyle guidelines for the prevention of Alzheimer’s disease. Neurobiol Aging 35, 1–5.
 Mursu J, Robien K, Harnack LJ, Park K, Jacobs Jr. DR (2011) Dietary supplements and mortality rate in older women: the Iowa Women’s Health Study. Arch Intern Med 171, 1625–1633.
 Morris MC, Evans DA, Tangney CC, Bienias JL, Schneider JA, Wilson RS, Scherr PA (2006) Dietary copper and high saturated and trans fat intakes associated with cognitive decline. Arch Neurol 63, 1085–1088.
 Tecchio F, Vecchio F, Ventriglia M, Porcaro C, Miraglia F, Siotto M, Rossini PM, Rongioletti M, Squitti R (2016) Non-ceruloplasmin copper appears a distinct subtype of Alzheimer`s disease: a study of EEG-derived brain activity. Curr Alzheimer Res 13, 1374-1384.
Reply to: Is sporadic Alzheimer’s disease a form of diabetes?
Neither is necessarily a form of the other per se. Just as Alzheimer himself once said that an infectious process in the CNS can have the same cause and yet land on two different places in the brain creating two different brain disorders , it makes more sense if both Alzheimer’s disease and diabetes are the result of the same underlying ongoing amyloid-producing systemic pathology involving two different organ-systems in the body.
As far back as 2006, it became obvious that epidemiological studies were finding a link between diabetes, Alzheimer disease, and dementia . Shock waves from the Madrid Alzheimer's meeting sent scientists scurrying for an explanation for how a Seattle VA study showed that diabetes could lead to a nearly 70% increase in Alzheimer's disease. Why this posed a particularly challenging problem was that it left the possibility of the explosion of diabetes with approximately 20 million in the US already having the disease and 40 million prediabetics, closing ranks with the rapidly growing Alzheimer’s victims. If this were to occur, such a pool would vastly escalate the number of the 4.5 million American Alzheimer's sufferers at the time.
A Puget Sound Seattle VA study showed subtle improvement in Alzheimer's patients placed on insulin mist. As a result researchers began to reemphasize that diabetes not only attacks the body but the mind. Conclusion: These results supported longer trials of intranasal insulin therapy for patients with amnestic mild cognitive impairment and patients with AD .Yet another study concluded that among well-functioning older adults, DM and poor glucose control among those with DM are associated with worse cognitive function and greater decline. This suggested that the severity of DM might contribute to accelerated cognitive aging .
As studies continued to appear, the one thing that most agreed upon was that as blood sugar control in diabetes worsens, Alzheimer's risk climbed.
While some speculated that diabetic's insulin deficient or resistant cells led to a cut-off of vital blood sugar to brain neurons, disabling their ability to remove clumps of incapacitating Alzheimer's amyloid –others thought it was a one-two punch: Alzheimer's tendency to attack cell's mitochondria or energy factories, leading to a neuronal death facilitated by diabetes's own attack on the brain's neurons. Or perhaps, although it was a not yet proven – there was a link between diabetes and Alzheimer disease due to malfunctioning of a gene that is involved in the function of the insulin-degrading enzyme (IDE). There were even those who, as a result of such studies, now consider diabetes as a precursor for Alzheimer's .
But to pathologist Phillip Schwartz, lead research pathologist for the State Facility in Warren Pennsylvania it was none of these. After a 50-year-autospsy-driven study of cases ranging from age 16 through 87, Schwartz published a report of 331 autopsied cases of diabetes, finding amyloidosis of the pancreas in 224 out of the 331 autopsies with the same chronic mycobacterial infectious focus in the body, either active or quiescent, that he had documented for a similar amyloid-generating process going on in autopsy after autopsy on Alzheimer’s brains .
Schwartz, who was the originator of the Thioflavin-S dye used to detect amyloid, reported that in the case of diabetes, most of those diagnosed as diabetic prior to death showed intense islet cell amyloidosis. And Schwartz hypothesized that once amyloidosis of the pancreatic islet cells hit a critical mass, the result was diabetes mellitus. Thus most cases of pancreatic amyloidosis, as well as the inflammatory infiltrate of the islet cells characteristic of Juvenile diabetes, and Alzheimer's, ought to be considered one and the same immunopathy – from, in all but a few cases, a foci of chronic mycobacterial infection in the body, which he found in just about every case in which he did a thorough and exhaustive examination.
Schwartz once said this regarding infectious amyloidosis: “Our investigations have disclosed amyloidosis to be one of the most frequent diseases of the human species, and cerebral amyloidosis, because of its enormous incidence in the aged, to be the most important condition in neuropathology.” Yet the fact that it could hit the pancreas with its islet cells was to him also a given. One immunologic process of infectious origin, two diseases.
1. Alzheimer A (1911) On certain peculiar diseases of old age. Zeit fur die Ges Neur und Psych 4: 356-485.
2. Susman E. News From the International Conference on Alzheimer’s Disease and Related Disorders Neurology Today. 19 September 2006 6:18: 25-26.
3. Craft S, Baker LD, Montine TJ, Minoshima S, Watson GS, Claxton A, Arbuckle M, Callaghan M, Tsai E, Plymate SR, Green PS, Leverenz J, Cross D, Gerton B. Intranasal insulin therapy for Alzheimer disease and amnestic mild cognitive impairment: a pilot clinical trial. Arch Neurol. 2012 Jan; 69(1):29-38.
4. Yaffe K, Falvey C, Hamilton N, Schwartz AV, Simonsick EM, Satterfield S, Cauley JA, Rosano C, Launer LJ, Strotmeyer ES, Harris TB. Diabetes, glucose control, and 9-year cognitive decline among older adults without dementia. Arch Neurol. 2012 Sep;69(9):1170-5
5. P. Schwartz, Amyloidosis—Cause and Manifestation of Senile Deterioration (Springfield, Illinois: Charles C Thomas, 1970), 363pp
Reply to: Alzheimer’s Disease and Spirochetosis: A Causal Relationship
Dr. Judith Miklossy has certainly presented a valid and provocative presentation towards a conceivable causal relationship between an infectious agent and Alzheimer’s and is to be deeply commended for doing so.
With regards to Oskar Fischer’s suggestion that senile plaques are reminiscent of bacterial colonies and his inability to cultivate them - by 1907, Oskar Fischer went to the extent of saying that many of the senile plaques he found “resemble more closely the central cell-free part of a tubercle.” Thus, by 1911 Alzheimer wrote the following:
"Hitherto opinions about the nature of the plaques have been very divergent. Fischer pointed out their similarity to bacterial colonies and reported that he had undertaken cultivation experiments and complement-fixation tests which however produced negative results."
At the time Fischer undertook cultivation and compliment fixation tests to validate his germ, negative results were the rule, not the exception. Thus, the fact that Fischer’s cultivation experiments and complement fixations were negative did not rule out the existence of Fischer’s brain microbe, which to him resembled a Strepothrix. For example, in the same year that Alzheimer made his 1911 statement, Harbitz and Grondahl reported repeatedly negative attempts at running complement-fixation tests in all cases using the serum of patients with known Streptothrix (actinomycosis) patients. Before that, Woodhead, Director of Laboratories at the Royal College of Physicians, pointed out that any attempted failure to cultivate Streptothrix could easily occur when trying to cultivate the well-developed club-shaped form of the organism that Fischer repeatedly documented. Woodhead wrote this about such experiments:
“It is interesting to note that most of the experiments that have been made on the cultivation of this organism [Streptothrix] have been attended with complete failure—a failure that in some measure, at any rate, appears to be due to the fact that almost all experimenters have used for their inoculating material only those colonies in which the club-shaped [Streptothrix] organisms have become well developed. The first attempt that was at all successful was made by Bostrom, who, throwing aside the club-like processes, took for his inoculating material the central network, selecting as far as possible young growing colonies for his seed material.”
But even Bostrom succeeded in getting only eleven positive growths out of several hundred planted.  And when German investigator Fritzsche  addressed this same topic three years before Alzheimer challenged Fischer’s microorganism, Fritzsche found not only a limited number of cases in which complement fixation for Streptothrix proved positive, but, in addition, there were frequent cross-reactions in his meager positive test samples for Streptothrix with tuberculosis. He added that Streptothrix was further confused with TB because they both could stain with tubercular acid-fast dyes, and both could have filamentous as well as club-like forms. But Bolton  later pointed out that unlike TB, Fischer’s Streptothrix rarely involved the central nervous system. Furthermore, although the first case of Streptothrix involving the CNS was reported by Ponfick  in 1882, Harz never succeeded in cultivating the organism from there.  Most of this literature was readily available to the Alzheimer group. And, even in the case of using complement-fixation tests to detect the far, far more prevalent tuberculosis, Corper  reported as late as 1916 that such tests were positive in only 30 percent of already-proven cases of tuberculosis—whether active or inactive.
Just ten years before Oskar Fischer found Actinomycosis-like Streptothrix in Alzheimer’s cerebral plaque, Babèş and immunologist Levaditi reported in On the Actinomycotic Shape of the Tuberculous Bacilli that typical Actinomyces-like clusters [Drüsen] with clubs appeared in the tissue of rabbits inoculated with tubercle bacilli beneath the dura mater of their brains. Once introduced into the brain this way, reported Babes, TB bacilli not only branched out like the Actinomycosis such as Streptothrix, but showed filamentous fungal forms – and as they developed rosettes that were identical to the "drüsen" that Oskar Fischer spotted in Alzheimer’s plaque. 
Also, with regard to the Mawanda and Wallace’s Can Infections Cause Alzheimer’s Disease mentioned here, Mawanda and Wallace’s12 2013 review gave seven annotated references as to why HSV-1 “remains questionable” as a cause for Alzheimer’s; nine studies referenced as to why there was “no evidence to suggest an association between Chlamydia pneumoniae infection and AD pathogenesis”; and six “rigorous studies which found no evidence to suggest that spirochetal B. Burgdorferi, is “causally linked to AD.” Wallace also mentioned that although Riviere et al. found oral spirochetal Treponema, including T. denticola, T. pectinovorum, T. vincentii, T. amylovorum, T. maltophilum, T. medium, and T. socranskii in a significantly higher proportion of postmortem brain specimens from AD cases than controls,13 that these results have, however, not been replicated. Also, Oskar Fischer, the discoverer of Alzheimer’s plaque, failed to observe Alzheimer’s neuritic plaque in the brains of 45 cases with neurosyphilis.14
What Mawanda and Wallace did maintain however was the emerging evidence that supported an infectious pathogen and what to them were two prime suspects for Amyloid beta deposition to the extent that it was going on in Alzheimer’s. They said this:
“In addition, amyloidopathy—a condition characterized by elevated levels of serum amyloid and by amyloid deposition and aggregation in tissues—is a frequent occurrence in several acute and chronic systemic inflammatory conditions, especially chronic infections like tuberculosis and leprosy.” IBID p.162
Mawanda and Wallace seemed to have no dearth of referenced studies to substantiate this mycobacterial assertion. [15-21] Streptothrix was of the Actinobacteria and its colonies form fungus-like branched networks of hyphae. The aspect of these colonies initially led to the incorrect assumption that the organism was a fungus and to the name Actinomyces, "ray fungus" (from Greek actis, ray, beam and mykes, fungus).
So it becomes all but obvious that Fischer’s 1907 portrayal of Alzheimer plaque as often appearing like bacterial “Streptotriches” had to be weighed within the context that when Fischer found that nearly all brain plaque in the 60 to 80μ microscopic range had an appearance reminiscent of “glandular” actinomyces, that American bacteriologist Davis mentioned that Fischer’s “glandular” actinomycosis were among the most difficult to differentiate from tuberculosis: “the two are often all but indistinguishable.” 
 Fischer O. Miliary Necrosis with Nodular Proliferation of the Neurofibrils, a Common Change of the Cerebral Cortex in Senile Dementia, Monatsschrift fuer Psychiatrie und Neurologie, vol. XXII, edited by Th. Ziehen (Berlin: S. Karger, 1907), 361–72.
 Alzheimer A. H. Forstl, and R. Levy, an English translation of Alzheimer’s 1911 paper Uber Eigenartige Krankheitsfalle des Spateren Alters (“On Certain Peculiar Diseases of Old Age”), History of Psychiatry 2 (1991): 71–101.
 Harbitz F, Grondahl NB, Actinomycosis in Norway: Studies in the Etiology, Modes of Infection, and Treatment, American Journal of Medical Science 142 (1911): 386–95.
 Woodhead GS. Bacteria and Their Products (London and New York: Walter Scott, Ltd./Charles Scribner’s Sons, 1895), 258
 Cope VZ. Actinomycosis: The Actinomyces and Some Common Errors about the actinomyces and actinomycosis. Postgraduate Medical Journal 28 (1952): 572–4
 E. Fritzsche, “Experimentelle Untersuchungen Tiber Biologische Beziehungen des Tuberkelbazillus zu Einigen Anderen Saurefesten Mikroorganismen und Aktinomyzeten,” Archive for Hygiene 5 (1908): 181–220.
 Bolton CF, Ashenhurst EM. Review Article: Actinomycosis of the Brain. Canadian Medical Association Journal 90 (April 11, 1964): 922–8.
 Ponfick, Die Actinomykose des Menschen, Eine Neue Infectionskrankheit auf Vergleichend-Pathologischer und Experimenteller Grundlage Geschildert. Berlin: A. Hirschwald, 1882.
 Harz B, “Actinomyces Bovis, ein Neuer Schimmel in den Geweben des Rindes: Deutsche Zeitschr. f. their,” Med. und Vergl. Path. (1870): 125, Zweites Supplementheft.
 Corper HJ, “Complement-Fixation in Tuberculosis,” The Journal of Infectious Diseases 19, no. 3 (September 1916): 315–21.
 Babes V, Levaditi C. On the Actinomycotic Shape of the Tuberculosis Bacilli (Sur la Forme Actinomycosique du Bacilli de la Tuberculosis), In Arch. of Med. Exp. et D’anat, part 2, 9, no. 6 (1897): 1041–8.
 Mawanda F, Wallace R. Can infections cause Alzheimer's disease? Epidemiol Rev. 2013; 35:161-80.
 Riviere GR, Riviere KH, Smith KS. Molecular and immunological evidence of oral Treponema in the human brain and their association with Alzheimer’s disease. Oral Microbiol Immunol. 2002;17(2):113–118.
 Goedert M. Oskar Fischer and the study of dementia. Brain. 2009 Apr; 132(4): 1102–1111.
 De Beer FC, Nel AE, Gie RP, et al. Serum amyloid A protein and C-reactive protein levels in pulmonary tuberculosis: relationship to amyloidosis. Thorax. 1984; 39(3):196–200.
 Looi LM, Jayalakshim P, Lim KJ, et al. An immunohistochemical and morphological study of amyloidosis complicating leprosy in Malaysian patients. Ann Acad Med Singapore. 1988; 17(4):573–578.
 Looi LM. The pattern of amyloidosis in Malaysia. Malays J Pathol. 1994; 16(1):11–13.
 Röcken C, Radun D, Glasbrenner B, et al. Generalized AA-amyloidosis in a 58-year-old Caucasian woman with an 18-month history of gastrointestinal tuberculosis. Virchows Arch. 1999; 434(1):95–100.
 Wangel AG, Wegelius O, Dyrting AE. A family study of leprosy: subcutaneous amyloid deposits and humoral immune responses. Int J Lepr Other Mycobact Dis. 1982; 50(1):47–55.
 Tank SJ, Chima RS, Shah V, et al. Renal amyloidosis following tuberculosis. Indian J Pediatr. 2000; 67(9):679–681.
 Urban BA, Fishman EK, Goldman SM, et al. CT evaluation of amyloidosis: spectrum of disease. Radiographics.1993; 13(6):1295–1308.
 Davis DJ, Some Observations on Streptothrix Infections and Their Relation to Tuberculosis in the National Association for the Study and Prevention of Tuberculosis, Transactions of the Eleventh Annual Meeting, Seattle, Washington, June 14–16, 1915. Baltimore: Williams & Wilkins Company, 1935, 255–61
Reply to: Early Cognitive Deficits in Type 2 Diabetes: A Population-Based Study.
This paper has been selected as my top vote ie 1 out of 3
Reply to: Cerebrospinal Fluid Alzheimer's Disease Biomarkers Across the Spectrum of Lewy Body Diseases: Results from a Large Multicenter Cohort.
The resuts partly explains about the pathological differences of PDD and DLB.
Reply to: Insulin Resistance is Associated with Increased Levels of Cerebrospinal Fluid Biomarkers of Alzheimer's Disease and Reduced Memory Function in At-Risk Healthy Middle-Aged Adults.
This article confirmed suggestion that Alzheimer's disease is type 3 diabetes mellitus.