Reply to: The Relationship Between Infections and Alzheimer’s Disease Is Modified by Vitamin D Status
We thank Dr. Grant for the interest in our study  and for the opportunity to clarify several points. Our study observed a small (5%) increase in the risk of Alzheimer’s disease (AD) associated with infectious disease burden, with the risk increasing gradually with longer time since first infection and peaking after 12-30 years (11%). Dr. Grant suggests that the underlying etiology for the observed association could be vitamin D status rather than infectious disease per se, arguing that vitamin D supplementation has been shown to reduce the risk of infection and also the risk of AD. Dr. Grant provides several references in support of the link between vitamin D status and the risk of specific infections, for which our study allegedly found an association with an increased risk of AD in infection-specific analyses. The references include ‘observational studies’, ‘clinical trials’, and ‘Mendelian randomization studies’. Moreover, Dr. Grant groups the infections included in our study based on etiology into ‘viral’ and ‘bacterial’.
First, we must clarify that our study was not designed to assess the potential role of vitamin D in the association between infectious disease burden and the risk of AD. One reason was that we did not have access to patients’ vitamin D levels, which precluded the possibility of vitamin D specific analyses.
Second, and although we are not experts in the field of vitamin D, we would like to mention that a brief review of the provided references does not necessarily support the ‘causal’ language used in Dr. Grant’s letter regarding the potential link between vitamin D status and the risk of AD. For example, the studies listed under “clinical trials” include a preclinical study conducted in diabetic mice (reference 4) and one one-arm intervention study comparing the rates of eradication treatment failure for Helicobacter pylori infection between patients with vitamin D deficiency and those without (reference 6). Of note, the analyses in the latter study were unadjusted, which probably introduced important confounding.
Third, we would like to clarify that the etiology-based classification proposed by Dr. Grant was not part of our study. Indeed, we specifically decided against separate analyses for ‘viral’ and ‘bacterial’ infections during the planning stage of the study, given the difficulty in estimating the causal pathogen for several of the infections of interest (e.g., pneumonia). The reason is that we did not have access to patient’s laboratory data.
Fourth, we must mention that the risk estimates shown by Dr. Grant refer to our infection-specific analyses for the outcome of overall dementia and not for the outcome AD. Of note, our infection-specific analyses on the risk of AD showed a positive association only for gastritis.
Overall, and while the potential role of vitamin D in the association between infection and AD is an interesting hypothesis that could be addressed by future research, our study was not designed to address this issue. Thus, no conclusions can be drawn in this regard.
Antonios Douros, MD, PhD, and Paul Brassard, MD, MSc
 Douros A, Santella C, Dell'Aniello S, Azoulay L, Renous C, Suissa S, Brassard P (2021) Infectious disease burden and the risk of Alzheimer’s disease: A population-based study. J Alzheimers Dis, doi: 10.3233/JAD-201534
Reply to: The Role of Combined Treatment with Citicoline in Alzheimer’s Disease
The answer is not very elegant, because it is in turn the harsh and resentful comment to a different opinion, without leaving room for a serene dialectical and scientific comment.
It is quite astonishing that eminent researchers do not take into account the remarkable role of experimental studies, which are crucial and somewhat propaedeutic for understanding the possible clinical role. Few clinical studies were conducted on citicoline, but the starting point was just its relevant role at a biochemical and cellular level.
The roles on SIRT 1, glutamate, synaptogenesis, cell membranes, mitochondria are crucial for understanding why it works! Missing these aspects means misunderstanding why citicoline should be used instead of other cholinergic precursors marketed up to now. Furthermore, it does not sound respectful towards all the past studies that opened the route towards possible clinical outcomes [1 - 9].
Last point, the poster you have mentioned on the role of the triple therapy (citicoline + memantine + AchEIs - Alzheimer’s and Dementia, 2020 ) was submitted more than one year ago and presented online at the AAIC first, on July 24, 2020, and at the Italian Congress of Psychogeriatrics on mid-- September. Maybe it will not be published, but the idea of the triple association was conceived some years ago. That’s all for those who want to understand!
 Secades JJ, Frontera G (1995) CDP-choline: a pharmacological and clinical review. Methods Find Exp Clin Pharmacol 17(suppl B), 1–54.
 Hurtado O, Lizasoain I, Moro MÁ (2011) Neuroprotection and recovery: recent data at the bench on citicoline. Stroke 42(suppl1), S33–S35.
 Hurtado O, Moro MA, Cardenas A, Sánchez V, Fernández-Tomé P, Leza JC, Lorenzo P, Secades JJ, Lozano R, Dávalos A, Castillo J, Lizasoain I (2005) Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: Effects on glutamate transport. Neurobiol Dis 18, 336–45.
 Gutierrez-Fernandez M, Rodriguez-Frutos B, Fuentes B, Vallejo-Cremades, Alvarez-Grech, , (2012) CDP-choline treatment induces brain plasticity markers expression in experimental animal stroke. Neurochem Int 60, 310–7.
 Secades JJ. Citicoline: pharmacological and clinical review, 2010 update (2011) Rev Neurol 52 (suppl 2), S1–S62.
 Hurtado O, Hernández-Jiménez M, Zarruk JG, Cuartero, Ballesteros, , Moraga, Pradillo, Moro, (2013) Citicoline (CDPcholine) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke. J Neurochem 126, 816–819.
 Krupinski J, Slevin M, Badimon L (2005) Citicoline inhibits MAP kinase signaling pathways after focal cerebral ischemia. Neurochem Res 30 (8), 1067–1073.
 Álvarez-Sabín J and Román GC (2013) The Role of Citicoline in Neuroprotection and Neurorepair in Ischemic Stroke. Brain Sci 3, 1395-1414; doi:10.3390/brainsci3031395
Pietro Gareri, MD, PhD - Center for Cognitive Disorders and Dementia – Catanzaro Lido, ASP Catanzaro; Catanzaro, Italy; Antonino Maria Cotroneo, MD - Director Department of Geriatrics – Maria Vittoria Hospital ASL City of Turin, Italy.
Reply to: The Role of Combined Treatment with Citicoline in Alzheimer’s Disease
We thank Dr. Gareri and Dr. Cotroneo for their interest in our retrospective study, recently published in JAD .
It is a fact that several retrospective studies focused the role of citicoline, alone and in combined treatment, in older patients affected with cognitive impairment. As Gareri and Cotroneo well know, two of the authors (AC and RL) of our study participated in the vast majority of the studies they mentioned in the initial part of their letter [2-5]. We are therefore wondering what is the usefulness for the reader and the logic of such clarifications.
Additionally, what is the usefulness of listing other, and in some cases only experimental, mechanisms of action of citicoline , if the final result does not change in a clinical practice? Indeed, there is no doubt that successful use of citicoline depends on its chronic administration, as our study also confirmed. Moreover, the information the authors cited in their letter is well known to our research group, and already discussed in our article. Are repetitions useful for the reader?
We acknowledge that the statements related to the Citicholinage and the CitiMem studies were not correctly reported in our article. This was due to a printing error. We are grateful to the authors for pointing this out. Nevertheless, we are convinced that this did not distract the reader from the central theme and results of our article.
Finally, we want to clearly address the part of the letter in which the two authors wrote that “the first evidence of efficacy of the triple therapy (citicoline 1 g plus memantine and AChEIs at the maximum tolerated dosage) was shown some months ago…This was the CITIDEMAGE study…”. This is not correct. On December 7, 2020, Alzheimer’s and Dementia published in a supplement a poster entitled “The CITIDEMAGE Study: Stressing the cholinergic hypothesis for the best outcomes in dementia patients” . As reported, the enrolled patients were visited between 2016 and 2018. On the other hand, our study enrolled patients since January 1, 2014 (please see Materials and Methods, line 102). On December 1, 2020 our study was published (in early view) as a research article, after a long review process involving three reviewers and a handling associate editor (Prof. Patrizia Mecocci).
We read with great attention and interest the poster presented by Gareri et al., and we are grateful to them for this contribution. Their preliminary report confirms efficacy and safety of a combined therapy with citicoline, memantine, and an AChEI in older patients with AD, and reinforces scientific soundness of our article. Obviously, we wait to read the full-text article, if and when published after the review process.
Alberto Castagna1, Andrea Fabbo2, Ciro Manzo3, Roberto Lacava1, Carmen Ruberto1, Giovanni Ruotolo4
1Azienda Sanitaria Provinciale Catanzaro, Primary Care Departiment, Center for Cognitive Disorders and Dementia, Catanzaro, Italy; E-mail: firstname.lastname@example.org
2Health Authority and Services (AUSL) of Modena, Geriatric Service, Cognitive Disorders and Dementia Unit, Modena, Italy
3 Azienda Sanitaria Locale Napoli 3 sud, Internal and Geriatric Medicine department, Center for Cognitive Disorders and Dementia, health district no.51, Pomigliano d’Arco, Naples, Italy
4Azienda Ospedaliera Pugliese-Ciaccio di Catanzaro, Medical Department, Geriatric Unit, Catanzaro, Italy
 Castagna A, Fabbo A, Manzo C, Lacava R, Ruberto C, Ruotolo G (2021) A retrospective study on the benefits of combined citicoline, memantine, and acetylcholinesterase inhibitor treatments in older patients affected with Alzheimer's disease. J Alzheimers Dis 79, 1509-1515.
 Cotroneo AM, Castagna A, Putignano S, Lacava R, Fantò F, Monteleone F, Rocca F, Malara A, Gareri P (2013) Effectiveness and safety of citicoline in mild vascular cognitive impairment: the IDEALE study. Clin Interv Aging 8, 131–137.
 Putignano S, Gareri P, Castagna A, Cerqua G, Cervera P, Cotroneo AM, Fiorillo F, Grella R, Lacava R, Maddonni A, Marino S, Pluderi A, Putignano D, Rocca F (2012) Retrospective and observational study to assess the efficacy of citicoline in elderly patients suffering from stupor related to complex geriatric syndrome. Clin Interv Aging 7, 113-118.
 Castagna A, Cotroneo AM, Ruotolo G, Gareri P (2016) The CITIRIVAD Study: CITIcoline plus RIVAstigmine in elderly patients affected with Dementia Study. Clin Drug Invest 36, 1059-1065.
 Gareri P, Castagna A, Cotroneo AM, Putignano D, Conforti R, Santamaria F, Marino S, Putignano S (2017) The Citicholinage Study: citicoline plus cholinesterase inhibitors in aged patients affected with Alzheimer's disease study. J Alzheimers Dis 56, 557–565.
 Hurtado O, Hernandez-Jimenez M, Zarruk JG, Cuartero MI, Ballesteros I, Camarero G, Moraga A, Pradillo JM, Moro MA, Lizasoain I (2013) Citicoline (CDPcholine) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke. J Neurochem 126, 816-819.
 Gareri P, Cotroneo AM, Orsitto G, Putignano S (2020) The CITIDEMAGE study: stressing the cholinergic hypothesis for the best outcomes in dementia patients. Alzheimers Dement 16, e038178.
Reply to: A Role for Mycobacterium in Alzheimer’s Disease?
We are grateful to Dr. Broxmeyer’s Letter to the Editor regarding the article by Loupy et al. . The idea that Alzheimer’s disease might have, in some cases, a mycobacterial etiology merits further study. Tuberculosis infection remains one of the most common infections worldwide, with an estimated 2 billion people infected with M. tuberculosis . In the United States, a total of 9,287 new cases of tuberculosis were reported in 2016 . However, based on tuberculin skin tests (TST) in 2011-2012, it was estimated that 4.7% of the US population was TST positive (compared to the point estimate in foreign-born persons of 20.5%) . Also of concern are nontuberculous mycobacterial (NTM) infections, chronic infections that appear to be increasing in the United States, particularly among older age groups . This is of potential interest given the similar patterns of geographic variation of NTM infection  and Alzheimer’s disease , which could reflect shared environmental risk factors, such as the abundance of NTM, including potential pathogenic M. mucogenicum/phocaicum, M. avium complex, M. fortuitum complex, and M. abscessus complex, in municipal water sources . Of these, M. avium was recently classified as belonging to an emended genus, Mycobacterium (“Tuberculosis-Simiae” clade), whereas M. abscessus was classified as Mycobacteroides gen. nov. (“Abscessus-Chelonae” clade), and M. vaccae, M. mucogenicum, and M. fortuitum were classified as Mycolicibacterium gen. nov. (“Fortuitum-Vaccae” clade), highlighting the phylogenetic diversity of mycobacteria, and foreshadowing potential differences in their impacts on the human host.
Mycobacteria are intracellular parasites, and, thus, may utilize a well-documented “Trojan horse” mechanism to enter the brain, wherein mycobacteria enter the central nervous system following infection of host immune cells [8-12]. The extent to which NTM can enter the central nervous system in this manner and the effects on host neurophysiology remain to be determined.
It is also possible that chronic mycobacterial infection increases risk of Alzheimer’s disease through signaling of peripheral inflammation from the periphery to the central nervous system though afferent signaling pathways. We have shown that intratracheal administration of a heat-killed preparation of M. vaccae NCTC 11659 (coupled to nitrocellulose beads in order to localize the immune activation to the airways) in mice acutely activates a subset of serotonergic neurons located within the interfascicular part of the dorsal raphe nucleus (DRI) . This subset of serotonergic neurons projects to the hippocampus and prefrontal cortex and we have suggested previously that it modulates affective, cognitive, and stress resilience functions . Importantly, we have also shown that infection of mice with live, virulent M. tuberculosis (H37Rv) activates DRI serotonergic neurons 3 days and 7 days following infection, but this activation is absent 14, 21, and 28 days after infection, suggesting adaptation of the DRI serotonergic system following chronic infection . In convergence with Dr. Broxmeyer’s Letter to the Editor, we have shown an essential role for microRNA 135 (miR135a) for chronic stress resiliency, antidepressant efficacy, and serotonergic activity . Dysregulation of an miR135a-serotonin signaling pathway following chronic mycobacterial infection could conceivably impact the etiology and pathophysiology of Alzheimer’s disease.
Christopher A. Lowry, PhD, and
Kelsey M. Loupy, BA, MS
 Loupy KM, Lee T, Zambrano CA, Elsayed AI, D'Angelo HM, Fonken LK, Frank MG, Maier SF, Lowry CA (2020) Alzheimer’s disease: protective effects of Mycobacterium vaccae, a soil-derived mycobacterium with anti-inflammatory and anti-tubercular properties, on the proteomic profiles of plasma and cerebrospinal fluid in rats. J Alzheimers Dis, doi: 10.3233/JAD-200568.
 U.S.Department of Health and Human Services Centers for Disease Control and Prevention (2019) Epidemiology of Tuberculosis. Atlanta, Georgia.
 Schmit KM, Wansaula Z, Pratt R, Price SF, Langer AJ (2017) Tuberculosis - United States, 2016. MMWR Morb Mortal Wkly Rep 66, 289-294.
 Miramontes R, Hill AN, Yelk Woodruff RS, Lambert LA, Navin TR, Castro KG, LoBue PA (2015) Tuberculosis infection in the United States: Prevalence estimates from the National Health and Nutrition Examination Survey, 2011-2012. PLoS One 10, e0140881.
 Winthrop KL, Marras TK, Adjemian J, Zhang H, Wang P, Zhang Q (2020) Incidence and prevalence of nontuberculous mycobacterial lung disease in a large U.S. managed care health plan, 2008-2015. Ann Am Thorac Soc 17, 178-185.
 Kirson NY, Meadows ES, Desai U, Smith BP, Cheung HC, Zuckerman P, Matthews BR (2020) Temporal and geographic variation in the incidence of Alzheimer's disease diagnosis in the US between 2007 and 2014. J Am Geriatr Soc 68, 346-353.
 Gebert MJ, Delgado-Baquerizo M, Oliverio AM, Webster TM, Nichols LM, Honda JR, Chan ED, Adjemian J, Dunn RR, Fierer N (2018) Ecological analyses of mycobacteria in showerhead biofilms and their relevance to human health. MBio 9, e01614-18.
 van Leeuwen LM, Boot M, Kuijl C, Picavet DI, van Stempvoort G, van der Pol SMA, de Vries HE, van der Wel NN, van der Kuip M, van Furth AM, van der Sar AM, Bitter W (2018) Mycobacteria employ two different mechanisms to cross the blood-brain barrier. Cell Microbiol 20, e12858.
 Schwerk C, Tenenbaum T, Kim KS, Schroten H (2015) The choroid plexus-a multi-role player during infectious diseases of the CNS. Front Cell Neurosci 9, 80.
 Jain SK, Tobin DM, Tucker EW, Venketaraman V, Ordonez AA, Jayashankar L, Siddiqi OK, Hammoud DA, Prasadarao NV, Sandor M, Hafner R, Fabry Z; NIH Tuberculous Meningitis Writing Group (2018) Tuberculous meningitis: a roadmap for advancing basic and translational research. Nat Immunol 19, 521-525.
 Cain MD, Salimi H, Diamond MS, Klein RS (2019) Mechanisms of pathogen invasion into the central nervous system. Neuron 103, 771-783.
 Dando SJ, Mackay-Sim A, Norton R, Currie BJ, St John JA, Ekberg JA, Batzloff M, Ulett GC, Beacham IR (2014) Pathogens penetrating the central nervous system: infection pathways and the cellular and molecular mechanisms of invasion. Clin Microbiol Rev 27, 691-726.
 Lowry CA, Hollis JH, de Vries A, Pan B, Brunet LR, Hunt JR, Paton JF, van Kampen E, Knight DM, Evans AK, Rook GA, Lightman SL (2007) Identification of an immune-responsive mesolimbocortical serotonergic system: Potential role in regulation of emotional behavior. Neuroscience 146, 756-772.
 Hale MW, Lowry CA (2011) Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits. Psychopharmacology (Berl) 213, 243-264.
 Hollis JH, Goosen K, Orozco H, Wilkinson A, Lightman SL, Hernández-Pando R, Rook GA, Lowry CA (2009) A murine model of acute and chronic bronchopulmonary infection with Mycobacterium tuberculosis: Delayed and persistent activation of serotonergic neurons within the brainstem raphe complex. Brain Behav Immun 23 (Suppl 2), S40-S41.
 Issler O, Haramati S, Paul ED, Maeno H, Navon I, Zwang R, Gil S, Mayberg HS, Dunlop BW, Menke A, Awatramani R, Binder EB, Deneris ES, Lowry CA, Chen A (2014) MicroRNA 135 is essential for chronic stress resiliency, antidepressant efficacy, and intact serotonergic activity. Neuron 83, 344-360.
Reply to: Plasma amyloid-β oligomerization tendency levels are increased with age in healthy subjects
Blood-based biomarkers will be the holy grail in the field of Alzheimer’s disease (AD) research and practice, once the adequacy of their sensitivity and specificity is proven. In addition, an ideal diagnostic biomarker for AD should detect the fundamental features of the molecular pathogenesis of AD. Therefore, reliable blood-based biomarkers of AD should be derived from amyloid-β (Aβ).
According to recent studies, peripheral Aβ-related markers are suitable for the evaluation of cerebral amyloidopathy even at the preclinical stage of AD. There are three types of blood-based, amyloid-targeting AD markers. The first method involves measuring Aβ related molecules (Aβ1-42, Aβ1-40, or other APP fragments) and calculate their ratios. Highly sensitive ELISA techniques or specific mass spectrometry are being used for this. The second is measuring the ratio of α-form to β-form amyloid in the peripheral circulation. The last technique is observing increasing tendencies of oligomeric forms of Aβ in plasma after spiking synthetic Aβ peptides.
The comment written by Dr. Minn & Dr. Kim noted that this oligomerization tendency is shows a slight increase with age in healthy normal subjects in his study. This is a very important finding we should keep in mind. As glycated hemoglobin (HbA1C) for diabetes mellitus, this oligomerization tendency of Aβ in plasma measured by MDS-OAβ could be used as a monitoring marker for preclinical AD pharmacotherapy in the near future. In this situation, Dr. Minn & Dr. Kim’s findings are very important to establish the management guideline according to age.
There are two possible explanations for the increased oligomerization tendencies by age; one is the possibility that there were some preclinical AD patients in the older healthy normal subject group. Because cerebral amyloidopathy may begin 15~20 years before the onset of clinical symptoms, and amyloid PET studies also found some positive cases without clinical symptoms at older population group.
Another possibility is that there may be physiological increases of Aβ oligomerization tendencies according to age. We may explain this increasing tendency by increased production of Aβ or many factors related to oligomerization, or decreased clearance of Aβ or decreased inhibiting factors of oligomerization. However, at the moment, there isn’t enough evidence to definitively explain the mechanism; more time is needed for additional research regarding this.
In conclusion, this comment including good data showing the increased oligomerization tendency of Aβ in plasma by age is a good reference upon which to establish diagnosis or treatment guidelines using MDS-OAβ.
SangYun Kim1, Young Chul Youn2
1 Department of Neurology, Seoul National University College of Medicine, Seoul, Korea
Clinical Neuroscience Center of Seoul National University Bundang Hospital, Seongnam-si, Korea
2Department of Neurology, Chung-Ang University College of Medicine, Seoul, Republic of Korea
Reply to: Letter to the Editor: Comment on “Oral Monosodium Glutamate Administration Causes Early Onset of Alzheimer’s Disease-like Pathophysiology in APP/PS1 Mice"
Our article which was published by Plos ONE in 2010 presented that homocysteic acid (HCA) was a pathogen of 3xTg-AD model mice (1). And also we published that blood HCA was the pathogen of human AD (2). This HCA is the strong agonist of glutamate, which suggests MSG is the same glutamate.
1. Hasegawa T, Mikoda N, Kitazawa M, LaFerla FM (2010) Treatment of Alzheimer’s Disease with Anti-Homocysteic Acid Antibody in 3xTg-AD Male Mice. PLoS ONE 5(1): e8593. doi:10.1371/journal.pone.0008593
2. Tohru Hasegawa, Masayoshi Ichiba, Shin-ei Matsumoto, Koji Kasanuki, Taku Hatano, Hiroshige Fujishiro, Eizo Iseki, Nobutaka Hattori, Tatsuo Yamada, Takeshi Tabira (2012) Urinary Homocysteic Acid Levels Correlate with Mini-Mental State Examination Scores in Alzheimer’s Disease Patients. Journal of Alzheimer’s Disease 31, 59–64.