Moral Fiber

Humans may be patients, poets, protestors, petermen and in the last resort politicians, but we are all self-reproducing bioreactors. We provide an environment for trillions of microbes, most of which cannot survive for long without the food, shelter and a place to breed that we provide. They inhabit us so thoroughly that not a single tissue in our body is sterile. They modify our health, personality, mood and behavior throughout life, they predict our death (1) and become our disassembly line after we die. The network of connections between our multicellular selves and our unicellular cousins is multi-dimensional and enormously complex, and the demarcation line where we stop and the bugs begin is rapidly disappearing. The microbiome is dead, long live the holobiome! (2)

But let’s stick to the old terms for a moment.

Our microbiome affects our development, character, mood and health, and we affect it via our diet, medications and mood states.

There is a vast and constantly shifting collaboration between us and our micro-passenger bacteria, between our bacteria, and between our bacteria and their micro-passengers, the phages. Everything is connected (ie 3, 4). 

One clinically important aspect of this is the growing realization that when we take non-antibiotic drugs, they modulate our microbiome (ie 5), which in turn influences those drugs’ positive and negative effects. This is the exciting new science of pharmacomicrobiomics (6-10).

But it goes much further than pharmaceuticals. Behind the gut/brain trope, it has become obvious that bacterial species not only affect who we are – they affect who we think we are, individually and socially.

Some of the early research in this area focused on the neurotransmitter serotonin. About 90% of serotonin is synthesised not in the brain but in the gut, where it plays a role in immunity.  In mice, Turicibacter and Clostridia bacteria send messages to the gut wall instructing it to boost serotonin synthesis by up to 50% (11); and there is some evidence that this serotonin may be transported to the brain via the vagus nerve (12).

This is not the whole story, however, and it may not necessarily be about serotonin at all, because vagal stimulation itself has anti-depressant effects (13); and so other scientists have focused on a different candidate gut / brain messenger, butyrate. This short chain fatty acid is produced by various gram-positive bacteria when they digest (or more properly ferment) prebiotic dietary fiber, and is a potent anti-inflammatory agent (ie 14-16).

Depression is strongly linked to inflammatory stress in the central nervous system (17, 18), and in clinical studies, higher numbers of butyrate-producing species in the gut are strongly linked to feelings of wellbeing (19).

As most butyrate is produced in the gut, it is not surprising that researchers find that the anti-inflammatory effects of butyrate are more pronounced in the gut than in other tissues (20). This is not, however, how things have to be – or how they used to be. Due to dietary change, and our increasing intakes of ultra-processed foods, our intakes of prebiotic fibers have fallen by an estimated 80-90% since the 19^th century (21, 22). Butyrate production in today’s gut is probably therefore an order of magnitude lower today than it was in the pre-industrial era. 

An outstanding team of scientists at Cork University believe that butyrate is indeed a potentially important element in the gut-brain axis (23): and others at the Universities of Beijing and Hiroshima have demonstrated significant anti-depressant activity in preclinical models with both butyrate itself (24) and the prebiotic fibers that produce butyrate in the gut (25).

Both serotonin and butyrate are part of the gut-brain connection, but there is more to it. Changes in the microbiome alter the synthesis of at least 8 gut peptides including neuropeptide Y, ghrelin and the ‘love hormone’ oxytocin, all of which act on multiple classes of receptors in the brain (26, 27). More on oxytocin later.

There may be multiple messengers, therefore, but the evidence linking neuroinflammation to depression is too pervasive to dismiss. And if you’re curious as to why inflammation should cause depression, it is probably because inflammation in the brain is linked, in evolutionary terms, to real or perceived danger signals (18, 28).

Neuroinflammation is a highly conserved response to adversity, and constitutes a survival strategy when an individual is faced with physical threat or injury. The perception of danger triggers the release of endogenous factors known as danger-associated molecular patterns (DAMP’s), which up-regulate pro-inflammatory cytokines in the brain. The cytokines then drive a suite of responses to injury or the threat of injury which include fatigue, psychomotor retardation, anhedonia and social and behavioral withdrawal. It is part of the fight/flight response, but geared towards flight; and like the fight/flight response, while it was pro-survival in primitive times, it can be triggered in the modern world by social defeat (29), or threats which may be merely symbolic or even imaginary. These all lead to brain inflammation, but now the responses of withdrawal, loss of resilience and depression may be largely or totally counter-productive. 

There is evidence too that when faced with too many threats and/or defeats, the neuro-inflammatory machinery becomes permanently activated, as occurs in abused children (30); leading to damaged development and impaired cognition and data processing (31).

This has led researchers to investigate anti-inflammatory approaches to various brain disorders. One candidate compound, chosen on the basis of its low molecular weight and ability to enter the brain (and all physiological compartments), is molecular hydrogen. It is an intriguing new approach to depression, anxiety and PTSD (32), and has already been shown to increase resilience in animal models (33).

But there are other ways. The bidirectional relationships between diet, the gut microbiota, immunity and resilience, create the possibility of treating affective disorders with dietary tools (34). There is a body of evidence to support this. High intakes of fruit and vegetables have documented antidepressant activity (35) and have been shown to increase resilience (36-38). The omega 3 HUFA’s also have well documented anti-inflammatory and antidepressant effects.

Plant foods are complex combinations of chemical compounds, and the most interesting candidates in this context are polyphenols and prebiotic fibers. Scientists generally want to know which compound has which effect, but it may be that these two types of nutrient work conjointly. The polyphenols undoubtedly increase resilience (39), but we do not know whether they act directly in the brain or indirectly, at least in part, via the microbiome (40).

Based on experience with patients, I recommend both prebiotics and polyphenols as part of a treatment program for affective disorders, with omega 3 HUFA’s on the side. This is not new; in 19^th century England, the prison system emphasized use of a healthy diet (rich in polyphenols and prebiotic fibers) to improve prisoners’ health, behavior and rehabilitation (41).

The microbiome affects our thinking and our moods, but it goes deeper than that. It influences how we develop. It molds our personalities, our sociability, our responses to fear and pain and our proneness to brain disease; and may be as or more important in these respects than our genetic makeup (42-47).

For example, as we age we generally become more conservative and more risk-averse. Some of this is due to growing awareness of the impermanence of life and the painful accumulation of experience – with, perhaps, a little wisdom. But our single cell collaborators play a role here too. As the decades pass our microbiome changes in a way that creates a greater degree of neuroinflammation. This tends to make us more risk-averse because it amplifies responses to DAMP’s, and by reducing neuroplasticity, makes it more difficult to learn – so we become more fixed in our thinking (48). Fascinatingly, by changing the microbiome, these age-related changes in the brain and the associated changes in behavior can be reversed; in mice, at least (48).

So what is the take-home message?

There is pervasive evidence that the ageing process has accelerated within the last generation, due to dietary and lifestyle changes (49). A significant part of this is due to dysbiosis (50), which has become prevalent due to the removal of prebiotic fibers from today’s ultra-processed foods (51, 52). 

Hedge accordingly, but if you are experiencing accelerated ageing, have psychological problems or believe that you have dysbiosis, I do not recommend probiotics. There is clinical evidence that some probiotic species, if consumed regularly, reduce activity in the brain in areas related to emotion and pain and may have anxiolytic and anti-depressant activity (53). However, a more detailed analysis suggests that while probiotic supplements may improve cognitive functions associated with dysbiosis, they can be counter-productive when added to a normal microbiome (54) or a microbiome depleted by antibiotics (55).

Probiotics can also cause dysbiosis directly, with small bowel malcolonization and excess production of D-lactic acid leading to significant cognitive impairment. Previously known to occur in subjects with short bowel syndrome and/or carbohydrate malabsorption, this has now been documented in normal subjects also (56).

Prebiotics present better treatment options, because here one is not introducing novel species of microbes into the body but amplifying the probiotic species already in situ. Additionally, by adding prebiotics we are merely restoring what the multinational food companies have irresponsibly removed from our diet.

How will you feel? 

Personal experience may convince because of its intimacy, but has relatively little value because our personal experiences are so limited. Nonetheless, people often tell me they never felt depressed or anxious until they started having problems with their gut; and conversely, once their dysbiosis was resolved, they felt more stable. I have never been particularly depressed or anxious, but after starting to use a blended prebiotic I noticed significant changes. People who plank gain improved physical strength, balance, groundedness, posture and flexibility. I would describe what I experienced, personally, as the psychological equivalent. But the personal is the political, as second wave feminists never tired of telling us in the ‘70’s, and these gut feelings are very relevant to today’s incivility.

Germ-free mice display increased depression / anxiety and autistic behavior, and are less sociable. Without microbes (and you could think of this as our intrinsic or default mode), we are more anti-social, less cooperative. One attractive theory suggests that our microbes found ways to make us more social so that they could spread more easily. In this sense they contributed to human social behaviors and the development and maintenance of human society (57). 

We are not germ-free, but our microbiomes have changed radically. Human diet (and shape) has shifted over the last century, with huge increases in ultra-processed foods, fats and oils, and obesity. Maternal obesity during pregnancy increases the risk of neurodevelopmental disorders, including autism spectrum disorders, in the offspring. More specifically, pre-clinical work has shown that a maternal high fat, low fiber diet changes the infant microbiome in ways that degrade the infants’ social behavior and ability to learn social cues. Even more specifically, these offspring had fewer oxytocinergic neurons in their hypothalamus, indicating that they were less able to love (58). Amazingly, however, restoring a healthy microbiome returned most brain functions to normal.

I believe that dietary shift has created a generation of humans less able to sustain or receive love. They suffer from reduced motivation, lower impulse control and are less informed by any sense of civic responsibility. At the same time they are more anxious, more depressed, more selfish and more polarized, and therefore more susceptible to the corrosive politics of identity. When combined with the corruption of crony capitalism, wide-spread environmental erosion and the slo-mo collapse of obsolescent 19^th century social institutions, this begins to look like the beginning of the collapse of Western civilization.

Some say the youth of today lack moral fiber, but it may be that what they really lack is prebiotic fiber. It is certainly easier to restore.

REFERENCES:

1. Salosensaari A and 21 other authors. Taxonomic Signatures of Long-Term Mortality Risk in Human Gut Microbiota. MedRxiv. doi: https://doi.org/10.1101/2019.12.30.19015842
2. Greer R, Dong X, Morgun A, Shulzhenko N. Investigating a holobiont: Microbiota perturbations and transkingdom networks. Gut Microbes. 2016; 7(2): 126–135.
3. Hsu BB, Gibson TE, Yeliseyev V, Liu Q, Lyon L, Bry L,  Silver PA, K Gerber G.K. Dynamic Modulation of the Gut Microbiota and Metabolome by Bacteriophages in a Mouse Model. Cell Host Microbe. 2019 Jun 12;25(6):803-814.e5.
4. Chang CS, Kao CY. Current understanding of the gut microbiota shaping mechanisms. J Biomed Sci. 2019 Aug 21;26(1):59.
5. Fung TC, Vuong HE, Luna CDG, Pronovost GN, Aleksandrova AA, Riley NG, Vavilina A, McGinn J,  Rendon T, L Forrest LR,  Hsiao EY. Intestinal serotonin and fluoxetine exposure modulate bacterial colonization in the gut. Nat Microbiol. 2019 Dec;4(12):2064-2073.
6. Xie Y, Hu F, Xiang D, Lu H, Li W, Zhao A, Huang L, Wang R. The Metabolic Effect of Gut Microbiota on Drugs. Drug Metab Rev. 2020 Feb;52(1):139-156.
7. Noh K, Kang YR, Nepal MR, Shakya R, Kang MJ, Kang W, Lee S, Jeong HG, Jeong TC. Impact of gut microbiota on drug metabolism: an update for safe and effective use of drugs. Arch Pharm Res. 2017 Dec;40(12):1345-1355.
8. Kim DH. Gut Microbiota-Mediated Drug-Antibiotic Interactions. Drug Metab Dispos. 2015 Oct;43(10):1581-9.
9. Wilson ID, Nicholson JK. Gut microbiome interactions with drug metabolism, efficacy, and toxicity. Transl Res. 2017 Jan;179:204-222.
10. Weersma RK, Zhernakova A, Fu J. Interaction between drugs and the gut microbiome. Gut. 2020 May 14:gutjnl-2019-320204. Online ahead of print.
11. Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, Nagler CR, Ismagilov RF, Mazmanian SK, Hsiao EY. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell. 2015 Apr 9;161(2):264-76.)
12. Neufeld KAM, Bienenstock J, Bharwani A, Champagne-Jorgensen K,  Mao Y, West C, Liu Y, Surette MG, Kunze W, Forsythe P. Oral selective serotonin reuptake inhibitors activate vagus nerve dependent gut-brain signaling. Sci Rep. 2019 Oct 3;9(1):14290.
13. Carreno FR, Frazer A. Vagal Nerve Stimulation for Treatment-Resistant Depression. Neurotherapeutics. 2017 Jul;14(3):716-727.
14. Hui W, Yu D, Cao Z, Zhao X. Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis. Int Immunopharmacol. 2019 Mar;68:226-233.
15. Sitkin S, Pokrotnieks J. Clinical Potential of Anti-inflammatory Effects of Faecalibacterium prausnitzii and Butyrate in Inflammatory Bowel Disease. Inflamm Bowel Dis. 2019 Mar 14;25(4):e40-e41.
16. Theiler A, Bärnthaler T, Platzer W, Richtig G, Peinhaupt M, Rittchen S, Kargl J, Ulven T, Marsh LM, Marsche G, Schuligoi R, Sturm EM, Heinemann A. Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival. J Allergy Clin Immunol. 2019 Sep;144(3):764-776.
17. Kiecolt-Glaser JK, Derry HM, Fagundes CP. Inflammation: depression fans the flames and feasts on the heat. Am J Psychiatry. 2015 Nov 1;172(11):1075-91.
18. Franklin TC, Xu C, Duman RS. Depression and sterile inflammation: Essential role of danger associated molecular patterns. Brain Behav Immun. 2018 Aug;72:2-13.
19. Valles-Colomer M, Falony G, Darzi Y, Tigchelaar EF, Wang J, Tito RY, Schiweck C, Kurilshikov A, Joossens M, Wijmenga C, Claes S, Van Oudenhove L, Zhernakova A, Vieira-Silva S, Raes J. The neuroactive potential of the human gut microbiota in quality of life and depression. Nat Microbiol. 2019 Apr;4(4):623-632.
20. Bach Knudsen KE, Lærke HN, Hedemann MS, Nielsen TS, Ingerslev AK, Gundelund Nielsen DS, Theil PK, Purup S, Hald S, Schioldan AG, Marco ML, Gregersen S, Hermansen K. Impact of Diet-Modulated Butyrate Production on Intestinal Barrier Function and Inflammation. Nutrients. 2018 Oct 13;10(10):1499. 
21. Clayton P, Rowbotham J. An unsuitable and degraded diet? Part two: realities of the mid-Victorian diet. J R Soc Med. 2008 Jul;101(7):350-7.
22. Moshfegh AJ, Friday JE, Goldman JP, Ahuja JK. Presence of inulin and oligofructose in the diets of Americans. J Nutr. 1999 Jul;129(7 Suppl):1407S-11S.
23. Stilling RM, van de Wouw M, Clarke G, Stanton C, Dinan TG, Cryan JF. The neuropharmacology of butyrate: The bread and butter of the microbiota-gut-brain axis? Neurochem Int. 2016 Oct;99:110-132.
24. Yamawaki Y, Yoshioka N, Nozaki K, Ito H, Oda K, Harada K, Shirawachi S, Asano S, Aizawa H, Yamawaki S, Kanematsu T, Akagi H. Sodium butyrate abolishes lipopolysaccharide-induced depression-like behaviors and hippocampal microglial activation in mice. Brain Res. 2018 Feb 1;1680:13-38.
25. Yang XD, Wang LK, Wu HY, Jiao L. Effects of prebiotic galacto-oligosaccharide on postoperative cognitive dysfunction and neuroinflammation through targeting of the gut-brain axis. BMC Anesthesiol. 2018 Nov 30;18(1):177. 
26. Lach G, Schellekens H, Dinan TG, Cryan JF. Anxiety, Depression, and the Microbiome: A Role for Gut Peptides. Neurotherapeutics. 2018 Jan;15(1):36-59.
27. Clarke G, Stilling RM, Kennedy PJ, Stanton C, Cryan JF, Dinan TG. Minireview: Gut microbiota: the neglected endocrine organ. Mol Endocrinol. 2014 Aug;28(8):1221-38. 
28. Slavich GM, Irwin MR. From Stress to Inflammation and Major Depressive Disorder: A Social Signal Transduction Theory of Depression Psychol Bull. 2014 May; 140(3): 774–815.
29. Weber MD, Godbout JP, Sheridan JF. Repeated Social Defeat, Neuroinflammation, and Behavior: Monocytes Carry the Signal. Neuropsychopharmacology. 2017 Jan; 42(1): 46–61.
30. Danese A., McEwen B.S. Adverse childhood experiences, allostasis, allostatic load, and age-related disease. Physiol. Behav. 2012;106(1):29–39.
31. Balter LJT, Bosch JA, Aldred S, Drayson MT, Veldhuijzen van Zanten JJCS, Higgs S, Raymond JE, Mazaheri A. Selective effects of acute low-grade inflammation on human visual attention. NeuroImage, 2019; 202: 116098 
32.  Ichihara M, Sobue S, Ito M, Ito M, Hirayama M, Ohno K. Beneficial biological effects and the underlying mechanisms of molecular hydrogen – comprehensive review of 321 original articles. Med Gas Res. 2015; (5):12.
33. Qiang Gao, Han Song, Xiao-ting Wang, Ying Liang, Yan-jie Xi, Yuan Gao, Qing-jun Guo, Tyler LeBaron, Yi-xiao Luo, Shuang-cheng Li, Xi Yin, Hai-shui Shi, Yu-xia Ma. Molecular hydrogen increases resilience to stress in mice. Sci Rep. 2017; 7: 9625.
34. Dantzer R, Cohen S, Russo SJ, Dinan TG. Resilience and immunity. Brain Behav Immun. 2018 Nov;74:28-42. 
35. Saghafian F, Malmir H, Saneei P, Milajerdi A, Larijani B, Esmaillzadeh A. Fruit and vegetable consumption and risk of depression: accumulative evidence from an updated systematic review and meta-analysis of epidemiological studies. Br J Nutr. 2018 May;119(10):1087-1101.
36. Whatnall MC, Patterson AJ, Siew YY, Kay-Lambkin F, Hutchesson MJ. Are Psychological Distress and Resilience Associated with Dietary Intake Among Australian University Students? Int J Environ Res Public Health. 2019 Oct 24;16(21):4099.
37. Bonaccio M, Di Castelnuovo A, Costanzo S, Pounis G, Persichillo M, Cerletti C, Donati MB, de Gaetano G, Iacoviello L. Mediterranean-type diet is associated with higher psychological resilience in a general adult population: findings from the Moli-sani study. Eur J Clin Nutr. 2018 Jan;72(1):154-160.
38. Greene GW, Lofgren I, Paulin C, Greaney ML, Clark PG. Differences in Psychosocial and Behavioral Variables by Dietary Screening Tool Risk Category in Older Adults. J Acad Nutr Diet. 2018 Jan;118(1):110-117.
39. Aubry AV, Khandaker H, Ravenelle R, Grunfeld IS, Bonnefil V, Chan KL, Cathomas F, Liu J, Schafe GE, Burghardt NS. A diet enriched with curcumin promotes resilience to chronic social defeat stress. Neuropsychopharmacology. 2019 Mar;44(4):733-742.
40. Cardona F., Andres-Lacueva C., Tulipani S., Tinahones F. J., Queipo-Ortuno M. I. (2013). Benefits of polyphenols on gut microbiota and implications in human health. J. Nutr. Biochem. 24 1415–1422.
41. Judith Rowbotham, SOLON; personal communication
42. Desbonnet L, Clarke G, Shanahan F, Dinan TG, Cryan JF. Microbiota is essential for social development in the mouse. Mol Psychiatry. 2014 Feb;19(2):146-8. 
43. Clarke G, Grenham S, Scully P, Fitzgerald P, Moloney RD, Shanahan F, Dinan TG, Cryan JF. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. Mol Psychiatry. 2013 Jun;18(6):666-73. 
44. Hoban AE, Stilling RM, Moloney G, Shanahan F, Dinan TG, Clarke G, Cryan JF. The microbiome regulates amygdala-dependent fear recall. Mol Psychiatry. 2018 May;23(5):1134-1144. 
45. Lauzon-Guillain Bd, Wijndaele K, Clark M, Acerini CL, Hughes IA, Dunger DB, Wells JC, Ong KK. Breastfeeding and infant temperament at age three months. PLoS One. 2012;7(1):e29326.
46. Mohamadi-Jorjafki E, Abbasnejad M, Kooshki R, Esmaeili-Mahani S, Raoof M. Mode of delivery alters dental pulp nociception and pain-induced changes in cognitive performance in adults’ male rats. Can J Physiol Pharmacol. 2020 Jun 9. doi: 10.1139/cjpp-2019-0370. Online ahead of print.
47. Borre YE, O’Keeffe GW, Clarke G, Stanton C, Dinan TG, Cryan JF. Microbiota and neurodevelopmental windows: implications for brain disorders. Trends Mol Med. 2014 Sep;20(9):509-18. 
48. Boehme M, van de Wouw M, Bastiaanssen TFS, Olavarría-Ramírez L, Lyons K, Fouhy F, Golubeva AV, Moloney GM, Minuto C, Sandhu KV, Scott KA, Clarke G, Stanton C, Dinan TG, Schellekens H, Cryan JF. Mid-life microbiota crises: middle age is associated with pervasive neuroimmune alterations that are reversed by targeting the gut microbiome. Mol Psychiatry. 2019 May 16. May 16. doi: 10.1038/s41380-019-0425-1. Online ahead of print.
49. Hulsegge G, Picavet HS, Blokstra A, Nooyens AC, Spijkerman AM, van der Schouw YT, Smit HA, Verschuren W. Today’s adult generations are less healthy than their predecessors: generation shifts in metabolic risk factors: the Doetinchem Cohort Study. Eur J Prev Cardiol. 2014 Sep;21(9):1134-44.
50. Reynolds A, Mann J, Cummings J, Winter N, Mete E, Te Morenga L. Carbohydrate quality and human health: a series of systematic reviews and meta-analyses. Lancet. 2019 Feb 2;393(10170):434-445.
51. Bibbò S, Ianiro G, Giorgio V, Scaldaferri F, Masucci L, Gasbarrini A, Cammarota G. The role of diet on gut microbiota composition. Eur Rev Med Pharmacol Sci. 2016 Nov;20(22):4742-4749.
52. Zinöcker MK, Lindseth IA. The Western Diet-Microbiome-Host Interaction and Its Role in Metabolic Disease. Nutrients. 2018 Mar 17;10(3):365.
53. Tillisch K, Labus J, Kilpatrick L, Jiang Z, Stains J, Ebrat B, Guyonnet D, Legrain-Raspaud S, Trotin B, Naliboff B, Mayer EA. Consumption of fermented milk product with probiotic modulates brain activity. Gastroenterology. 2013 Jun;144(7):1394-401, 1401.e1-4.
54. Suez J, Zmora N, Zilberman-Scapira G, Halpern Z, Segal E, Elinav E. Post-Antibiotic Gut Mucosal Microbiome Reconstitution Is Impaired by Prebiotics and Improved by Autologous FMT. Cell (174)6;1406-1423, 2018
55. Beilharz JE, Kaakoush NO, Maniam J, Morris MJ. Cafeteria Diet and Probiotic Therapy: Cross Talk Among Memory, Neuroplasticity, Serotonin Receptors and Gut Microbiota in the Rat. Mol Psychiatry. 2018 Feb;23(2):351-361.
56. Rao SSC, Rehman A, Yu S, de Andino NM. Brain fogginess, gas and bloating: a link between SIBO, probiotics and metabolic acidosis. Clinical and Translational Gastroenterology, 2018 Jun 19;9(6):162.
57. Stillin RM, Bordenstein SR, DinanTG, Cryan JF. Friends With Social Benefits: Host-Microbe Interactions as a Driver of Brain Evolution and Development? Front Cell Infect Microbiol. 2014 Oct 29;4:147.
58. Buffington SA, Di Prisco GV, Auchtung TA, Ajami NJ, Petrosino JF, Costa-Mattioli M. Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring. Cell. 2016 Jun 16;165(7):1762-1775.