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Archive for the ‘Autism & Alzheimer’s’ Category

The beauty of the complexity of our biological evolution with microbes and further evidence that supports “Acquiring Genomes: A Theory of the Origin of Species” by Lynn Margulis and Dorion Sagan is still occurring today. I so wish she were still alive because I would have immediately sent her this remarkable scientific finding. It is also further evidence of how perverted capitalism is towards undermining infant development and destroying their health that current hospital practices sabotage even mothers who choose to nurse their own babies. Formula is a Big Pharma product that has been sold to mothers all over the globe. They are not taught how the milk their own bodies produce are instrumental in calibrating the immune system and brain development of their infants.

I Contain Multitudes: The Microbes Within Us and a Grander View of Life by Ed Yong book excerpt.

“It is unclear why human breast milk stands out among that of other mammals. It has five times as many types of H.M.O.s as cow’s milk, and several hun­dred times the quantity. Even chimp milk is impoverished compared with ours. Mills suggests a couple of possible explanations for this difference. One involves our brains, which are famously large for a primate of our size, and which grow incredibly quickly during our first year of life. This fast growth partly depends on a nutrient called sialic acid, which also happens to be one of the chemicals that B. infantis releases while it eats H.M.O.s. It is possible that, by keeping this bacte­rium well fed, mothers can raise brainier babies. This might explain why, among monkeys and apes, social species have more milk oligo­saccharides than solitary ones, and a greater range of them to boot. Living in larger groups requires remembering more social ties, managing more friendships, and manipulating more rivals. Many scientists believe that these demands drove the evolution of primate intelligence; perhaps they also fueled the diversity of H.M.O.s.

An alternative idea involves diseases. In a group setting, pathogens can easily bounce from one host to another, so animals need better ways of pro­tecting themselves. H.M.O.s provide one such defense. When a pathogen infects our guts, it almost always begins by latching onto glycans—sugar molecules—on the surfaces of our intestinal cells. But H.M.O.s bear a striking resemblance to these glycans, so pathogens sometimes stick to them instead. They act as decoys, drawing fire away from a baby’s own cells. They can block a roll call of gut villains, including Salmonella; Listeria; Vibrio cholerae, the culprit behind cholera; Campylobacter jejuni, the most common cause of bacterial diarrhea; Entamoeba histolytica, a vora­cious amoeba that causes dysentery and kills a hundred thousand people every year; and many virulent strains of E. coli. H.M.O.s may even be able to obstruct H.I.V., which might explain why more than half of infants who suckle from infected mothers don’t get infected, despite drinking virus-loaded milk for months. Every time scientists have pitted a pathogen against cultured cells in the presence of H.M.O.s, the cells have come out smil­ing…

scientists have identified more than two hundred human milk oligosaccharides, or H.M.O.s. They are the third-most plentiful ingredient in human milk, after lactose and fats, and their structure ought to make them a rich source of energy for growing babies—but babies cannot digest them. When German first learned this, he was gobsmacked. Why would a mother expend so much energy manufacturing these complicated chemicals if they were apparently useless to her child? Why hasn’t natural selection put its foot down on such a wasteful practice? Here’s a clue: H.M.O.s pass through the stom­ach and the small intestine unharmed, landing in the large intestine, where most of our bacteria live. What if they aren’t food for babies at all? What if they are food for microbes?…

In 2006, the team found that the sugars selectively nourish one subspecies, Bifidobacterium longum infantis. As long as you provide B. infantis with H.M.O.s, it will outcompete any other gut bacterium. A closely related subspe­cies, B. longum longum, grows weakly on the same sugars, and the ironi­cally named B. lactis, a common fixture of probiotic yogurts, doesn’t grow at all. Another probiotic mainstay, B. bifidum, does slightly better, but is a fussy, messy eater. It breaks down a few H.M.O.s and takes in the pieces it likes. By contrast, B. infantis devours every last crumb using a cluster of thirty genes—a comprehensive cutlery set for eating H.M.O.s. No other Bif has this genetic cluster; it is unique to B. infantis. Human milk has evolved to nourish the microbe, and it, in turn, has evolved into a consummate H.M.O.vore. Unsurprisingly, it is often the dominant microbe in the guts of breast-fed infants. B. infantis earns its keep. As it digests H.M.O.s, it releases short-chain fatty acids, which feed an infant’s gut cells. Through direct contact, B. infantis also encourages gut cells to make adhesive proteins that seal the gaps between them, keeping microbes out of the bloodstream, and anti-inflam­matory molecules that calibrate the immune system. These changes only happen when B. infantis feeds on H.M.O.s; if it gets lactose instead, it survives but doesn’t engage in any repartee with the baby’s cells. In other words, the microbe’s full beneficial potential is unlocked only when it feeds on breast milk.” (Pages 92 – 96)

https://www.newyorker.com/tech/elements/breast-feeding-the-microbiome

https://www.goodreads.com/book/show/27213168-i-contain-multitudes

In other words, your baby’s immune system and brain will never fully develop because they never received your breast milk and they will be more prone to diseases throughout their lives. The same ruling class industrialists who created products off of their munitions technologies that have the precision of sniper fire on fetal development want women to feed their babies their formulas instead because it maximizes their
profits. (Should note that some of their products even destroy mammary development so some women are not even capable of nursing their babies. It’s a win win situation for them…)

“The study showed that the exclusively breastfed group had the fastest growth in myelinated white matter of the three groups, with the increase in white matter volume becoming substantial by age 2. The group fed both breastmilk and formula had more growth than the exclusively formula-fed group, but less than the breastmilk-only group.”
“We’re finding the difference [in white matter growth] is on the order of 20 to 30 percent, comparing the breastfed and the non-breastfed kids,” said Deoni. “I think it’s astounding that you could have that much difference so early.”
https://www.sciencedaily.com/releases/2013/06/130606141048.htm

Perchlorate (rocket fuel) contaminates all formula brands too
CDC found detectable levels of perchlorate in all 2820 urine samples tested, indicating widespread human exposure to perchlorate.”We found significantly higher levels of urinary perchlorate in children compared with adolescents and adults.”

“Perchlorate is commonly found in the environment and can impair thyroid function at pharmacological doses. As a result of the potential for widespread human exposure to this biologically active chemical, we assessed perchlorate exposure in a nationally representative population”

Thank you Wayback machine… (You can read the CDC removed report here.)

https://web.archive.org/web/20091129164121/https://www.cdc.gov/exposurereport/perchlorate1.htm

The CDC tested all infant powdered formulas and found perchlorate contamination in all of the brands. Here’s the CDC study.

Perchlorate exposure from infant formula and comparisons with the perchlorate reference dose.

2010 May;20(3):281-7. doi: 10.1038/jes.2009.18. Epub 2009 Mar 18.

Schier JG1, Wolkin AF, Valentin-Blasini L, Belson MG, Kieszak SM, Rubin CS, Blount BC.

Abstract

Perchlorate exposure may be higher in infants compared with older persons, due to diet (infant formula) and body weight versus intake considerations. Our primary objective was to quantitatively assess perchlorate concentrations in commercially available powdered infant formulas (PIFs). Secondary objectives were: (1) to estimate exposure in infants under different dosing scenarios and compare them with the perchlorate reference dose (RfD); (2) estimate the perchlorate concentration in water used for preparing PIFs that would result in a dose exceeding the RfD; and (3) estimate iodine intakes from PIFs. We quantified perchlorate levels in three samples (different lot numbers) of reconstituted PIF (using perchlorate-free water) from commercial brands of PIF in each of the following categories: bovine milk-based with lactose, soy-based, bovine milk-based but lactose-free, and elemental (typically consisting of synthetic amino acids). Exposure modeling was conducted to determine whether the RfD might be exceeded in 48 dosing scenarios that were dependent on age, centile energy intake per unit of body weight, body weight percentile, and PIF perchlorate concentration. We obtained three different samples in each of the five brands of bovine- and soy-based PIF, three different samples in each of the three brands of lactose-free PIF, and three different samples in two brands of elemental PIF. The results were as follows: bovine milk-based with lactose (1.72 microg/l, range: 0.68-5.05); soy-based (0.21 microg/l, range: 0.10-0.44); lactose-free (0.27 microg/l, range: 0.03-0.93); and elemental (0.18 microg/l, range: 0.08-0.4). Bovine milk-based PIFs with lactose had a significantly higher concentration of perchlorate (P<0.05) compared with all. Perchlorate was a contaminant of all commercially available PIFs tested. Bovine milk-based PIFs with lactose had a significantly higher perchlorate concentration perchlorate than soy, lactose-free, and elemental PIFs. The perchlorate RfD may be exceeded when certain bovine milk-based PIFs are ingested and/or when PIFs are reconstituted with perchlorate-contaminated water.

 

https://www.ncbi.nlm.nih.gov/pubmed/19293845?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1

“Then the United States delegation, embracing the interests of infant formula manufacturers, upended the deliberations.

American officials sought to water down the resolution by removing language that called on governments to “protect, promote and support breast-feeding” and another passage that called on policymakers to restrict the promotion of food products that many experts say can have deleterious effects on young children.

When that failed, they turned to threats, according to diplomats and government officials who took part in the discussions. Ecuador, which had planned to introduce the measure, was the first to find itself in the cross hairs.

The Americans were blunt: If Ecuador refused to drop the resolution, Washington would unleash punishing trade measures and withdraw crucial military aid. The Ecuadorean government quickly acquiesced.”

(Surprised that The New York Times does not capitalize on this headline and include a formula advertisement.)

https://mobile.nytimes.com/2018/07/08/health/world-health-breastfeeding-ecuador-trump.html

The “Council of Gods” have literally destroyed millions of years of evolutionary development. Hormones in breast milk also build infant brains. Microbes ride the vagus nerve of infants to help develop their brains. Your hippocampus is loaded with estrogen receptors and develops from the hormones in breast milk.

“Seung’s new book, Connectome: How the Brain’s Wiring Makes Us Who We Are, explains how mapping out our neural connections in our brains might be the key to understanding the basis of things like personality, memory, perception and ideas, as well as illnesses that happen in the brain, like autism and schizophrenia.

“These kinds of disorders have been a puzzle for a long time,” says Seung. “We can look at other brain diseases, like Alzheimer’s disease and Parkinson’s disease, and see clear evidence that there is something wrong in the brain.”

But with schizophrenia and autism, there’s no clear abnormality during autopsy dissections, says Seung.

“We believe these are brain disorders because of lots of indirect evidence, but we can’t look at the brain directly and see something is wrong,” he says. “So the hypothesis is that the neurons are healthy, but they are simply connected together or organized in an abnormal way.”

One current theory, says Seung, is that there’s a connection between the wiring that develops between neurons during early infancy and developmental disorders like schizophrenia and autism.

“In autism, the development of the brain is hypothesized to go awry sometime before age 2, maybe in the womb,” he says. “In schizophrenia, no one knows for sure when the development is going off course. We know that schizophrenia tends to emerge in early adulthood, so many people believe that something abnormal is happening during adolescence. Or it could be that something is happening much earlier and it’s not revealed until you become an adult.”

What scientists do know, he says, is that the wiring of the brain in the first three years is critical for development. Infants born with cataracts in poor countries that don’t have the resources to restore their eyesight remain blind even after surgery is performed on them later in life.

“No matter how much they practice seeing, they can never really see,” says Seung. “They recover some visual function, but they are still blind by comparison to you and me. And one hypothesis is that the brain didn’t wire up properly when they were babies, so by the time they become adults, there’s no way for the brain to learn how to see properly.”

At birth, he says, you are born with all of the neurons you will ever have in life, except for neurons that exist in two specific areas of the brain: the dentate gyrus of the hippocampus, which is thought to help new memories form, and the olfactory bulb, which is involved in your sense of smell.

“The obvious hypothesis [is] that these two areas need to be highly plastic and need to learn more than other regions, and that’s why new neurons have to be created — to give these regions more potential for learning,” says Seung. “But we don’t really have any proof of that hypothesis.”

But not everything is set in stone from birth. The complex synaptic connections that allow neurons to communicate with one another develop after babies have left the womb.

“As far as we know, this is happening throughout your life,” he says. “Part of the reason that we are lifelong learners — that no matter how old you get, you can still learn something new — may be due to the fact that synapse creation and elimination are both continuing into adulthood.”

https://www.goodreads.com/book/show/11346470-connectome
NARRATOR: But, in comparing the brain scans of identical twins discordant for autism, Kaufman finally saw the definitive data he was searching for: an area in the brain linked to learning, memory and emotions—called the hippocampus—was smaller in the twin with severe autism. But how could the same genes create different brain structures? Kaufmann asked Andy Feinberg at Johns Hopkins University.

ANDREW FEINBERG: And suddenly we were able to form an epigenetic hypothesis. And that hypothesis is that they have the same genome, but one of them maybe has an epigenetic change that’s leading to a difference in their brain that you don’t see in the other twin.

NARRATOR: Kaufmann and Feinberg are now searching for methyl marks in the DNA of identical twins discordant for autism. The work has just begun, but the hope is that by finding identical genes that differ in their expression, some causes of autism may emerge.

Epigenetic changes occur from external or environmental impacts.

Ghost in Your Genes
https://www.pbs.org/wgbh/nova/transcripts/3413_genes.html

Organochlorine munitions in bomb, pill, spray, injection, and chemical additive forms shrink the hippocampus. The article “U.S. Nerve Gas Hit Our Own Troops in Iraq” explains, “According to Dr. Linda Chao, a neurologist at the University of California Medical School in San Francisco, “Because part of their brains, the hippocampus, has shrunk, they’re at greater risk for Alzheimer’s and other degenerative diseases.”
A Czech chemical-weapons detection unit found “trace concentrations of sarin, a nerve-paralyzing substance” drifting into Saudi Arabia. French, British and U.S. intelligence units found similar evidence.

Tracy Elledge, a former combat engineer and one of the veterans I interviewed, said, “Alarms went off all the time.… Our officers told us they were false and to disconnect them.”

However, Elledge and others were breathing poison.
https://renchemista.wordpress.com/2017/04/21/u-s-nerve-gas-hit-our-own-troops-in-iraq-by-barbara-koeppel/

Remember the “A” in Sarin stands for Nazi Otto Ambros and he expanded all organochlorine munition markets after serving only 3 years for his mass murder slavery conviction. He was IG Farben’s director of chemical operations and Hitler’s Director of Chemical Weapons. The Council of Gods were fully aware of the biological impacts of their organochlorine munitions and that was why they rounded up their infant and children victims in Vienna and exterminated them. They had market expansion plans so they had to cover up the damage.

Asperger’s Children: The Origins of Autism in Nazi Vienna by Edith Sheffer
https://renchemista.wordpress.com/2018/11/15/important-book-excerpts-from-aspergers-children-the-origins-of-autism-in-nazi-vienna-by-edith-sheffer-2/

Silent Spring by Rachel Carson

Excerpt From Chapter 13: Through a Narrow Window 

Plants treated with benzene hexachloride (BHC) or lindane became monstrously deformed with tumorlike swellings on their roots. Their cells grew in size, being swollen with chromosomes which doubled in number. The doubling continued in future divisions until further cell division became mechanically impossible.

The herbicide 2,4-D has also produced tumor like swellings in treated plants. Chromosomes become short, thick, clumped together. Cell division is seriously retarded. The general effect is said to parallel closely that produced by X-rays.

Recent medical findings in the field of chromosome abnormalities are of extreme interest and significance. In 1959 several British and French research teams found their independent studies pointing to a common conclusion—that some of humanity’s ills are caused by a disturbance of the normal chromosome number. In certain diseases and abnormalities studied by these investigators the number differed from the normal. To illustrate: it is now known that all mongoloids have one extra chromosome. Occasionally this is attached to another so that the chromosome number remains the normal 46. As a rule, however, the extra is a separate chromosome, making the number 47. In such individuals, the original cause of the defect must have occurred in the generation preceding its appearance.

A different mechanism seems to operate in a number of patients, both in America and Great Britain, who are suffering from a chronic form of leukemia. These have been found to have a consistent chromosome abnormality in some of the blood cells. The abnormality consists of the loss of part of a chromosome. In these patients the skin cells have a normal complement of chromosomes. This indicates that the chromosome defect did not occur in the germ cells that gave rise to these individuals, but represents damage to particular cells (in this case, the precursors of blood cells) that occurred during the life of the individual. The loss of part of a chromosome has perhaps deprived these cells of their “instructions” for normal behavior.

The list of defects linked to chromosome disturbances has grown with surprising speed since the opening of this territory, hitherto beyond the boundaries of medical research. One, known only as Klinefelter’s syndrome, involves a duplication of one of the sex chromosomes. The resulting individual is a male, but because he carries two of the X chromosomes (becoming XXY instead of XY, the normal male complement) he is somewhat abnormal. Excessive height and mental defects often accompany the sterility caused by the condition. In contrast, an individual who receives only one sex chromosome (becoming XO instead of either XX or XY) is actually female but lacks many of the secondary sexual characteristics. The condition is accompanied by various physical (and sometimes mental) defects, for of course the X chromosome carries genes for a variety of characteristics. This is known as Turner’s syndrome…

An immense amount of work on the subject of chromosome abnormalities is being done by workers in many countries. A group at the University of Wisconsin, headed by Dr. Klaus Patau, has been concentrating on a variety of congenital abnormalities, usually including mental retardation, that seem to result from the duplication of only part of a chromosome, as if somewhere in the formation of one of the germ cells a chromosome had broken and the pieces had not been properly redistributed. Such a mishap is likely to interfere with the normal development of the embryo.

According to present knowledge, the occurrence of an entire extra body chromosome is usually lethal, preventing survival of the embryo. Only three such conditions are known to be viable; one of them, of course, is mongolism. The presence of an extra attached fragment, on the other hand, although seriously damaging is not necessarily fatal, and according to the Wisconsin investigators this situation may well account for a substantial part of the so far unexplained cases in which a child is born with multiple defects, usually including mental retardation.

This is so new a field of study that as yet scientists have been more concerned with identifying the chromosome abnormalities associated with disease and defective development than with speculating about the causes. It would be foolish to assume that any single agent is responsible for damaging the chromosomes or causing their erratic behavior during cell division. But can we afford to ignore the fact that we are now filling the environment with chemicals that have the power to strike directly at the chromosomes, affecting them in the precise ways that would cause such conditions? Is this not too high a price to pay for a sproutless potato or a mosquitoes patio?

We can, if we wish, reduce this threat to our genetic heritage, a possession that has come down to us through some two billion years of evolution and selection of living protoplasm, a possession that is ours for the moment only, until we must pass it on to the generations to come. We are doing little now to preserve its integrity. Although chemical manufacturers are required by law to test their materials for toxicity, they are not required to make the tests that would reliably demonstrate genetic effect, and they do not do so.

(That’s because manufacturers were already well aware of the biological impacts of the technologies they were expanding)

 

 

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Asperger’s Children: The Origins of Autism in Nazi Vienna by Edith Sheffer

Chapter 1: Enter the Experts
“Lazar was particularly interested in “dissocial” youths who had fallen afoul with the law. He felt it important to differentiate what he called endogenous and exogenous causes—between children whose disociability stemmed from internal factors (physical or psychiatric) versus external factors (illness or milieu).In endogenous cases, Lazar boasted that his Curative Education Clinic was “the first attempt” to distinguish between “the mental and physical defects of the wayward and criminal.” Committed to physiological detail, for example, Lazar would examine the genitals of a child “right away,” and send boys whose testicles had not dropped to a surgeon….

For all his purported idealism, Lazar could be damning in his judgements. As his coworkers put it, “He was not sentimental,” and would pronounce children “morally” damaged, “degenerate,” or even “waste.”….

Chapter 5: Fatal Theories
Jekelius wrote to her parents that he had done them a service: “you and the child will be spared much suffering.” Because Paula had “Mongolian idiocy” (Down syndrome), she “would never have walked or learned to speak, and would have been a constant burden for you.”

However, the true intent of the child euthanasia program was not to make life easier for parents, but to purge the Reich of undesirable citizens. And child killers were holding very different conversations among themselves.

*(Should note that the author is not educated in endocrine disruption science nor fetal development or she would have understood the significance of an epidemic of boys born with undescended testicles and children born with Down Syndrome. Their munition technology causes have been known since the 1950s. Since IG Farben had market expansion plans it would have been in their best interest to destroy any evidence that their products caused harm.)

 

Chapter 6: Asperger and the Killing System
While Asperger effectively worked down the hall. Hamburger who supervised numerous medical experiments on children at the Children’s Hospital. One medical student exposed children and infants to extreme temperature changes and measured the effects. Elmar Turk, one of Asperger’s associates as a postdoctoral student under Hamburger, used premature infants to study the effect of vitamin D on rickets; knowing that premature babies were particularly susceptible to the condition, Turk withheld prophylaxis, allowing thirteen of the fifteen infants in his control group to develop rickets.

Hamburger took special interest in Turk’s lethal tuberculosis experiments on children. Turk selected babies as his test subjects who he considered to be “severely damaged from birth trauma, unviable, and idiotic.” He administered a tuberculosis vaccine, Calmette-Guerin (BCG), to two of them, and then infected all three babies with “virulent tuberculosis bacillus.” He sent them to Speigelgrund for observation and, eventually, autopsies. The two vaccinated babies died within a month—not of tuberculosis, but reportedly of pneumonia, the main official cause of death at Spiegelgrund. The unvaccinated child overcame the tuberculosis after a painful four-month ordeal but still died.

Turk repeated his tuberculosis experiment one year later on an “idiotic, syphilitic” three-and-a-half-year-old, Adolf Guttmann, whom he did not. As he transferred little Adolf to Spiegelgrund for observation, Turk sent a macabre so-called “wish list” to the director for the boy’s death and postmortem study: “I would request that you inform me in the event of the child’s death so that I may be present at the autopsy, as I intend to conduct various histological examinations.” While the child still lived, Spiegelgrund staff were to take specialized notes on Adolf’s condition and conduct x-rays at regular intervals. Turk added, “I hope you are not burdened by this.” After Adolf arrived at Spiegelgrund, staff reported that the boy “was quiet and peaceful, laughs occasionally when one strokes him on the cheek.” Adolf was killed two and a half months after his arrival…

Hamburger’s postdoctoral student Heribert Goll, with whom Asperger had copublished in 1939, also conducted experiments on babies at the Children’s Hospital. Overseen by Hamburger, Goll explained that he selected “only infants unfit to live.” For his 1941 publication in the Munich Medical Weekly, Goll deprived babies of Vitamin A in order to measure the vitamin’s effects on the development of keratomalacia, a common cause of blindness. The condition dried the cornea and membrane that covers the white of the eye, resulting potentially, over time, in frothy patches called Bitot spots, ulceration, infection, and rupture of the eye. After Goll, withheld vitamin A from the infants for months, a number of them did, indeed, develop preliminary keratomalacia. Then, in a second experiment, Goll sought to infect babies with keratomalacia by placing secretions from the eye of one girl with the disease onto the eyes of four healthy children. When the method failed, he tried again by localizing the bacteria, which again failed.

Goll raised the stakes of his research in his 1942 publication for the Munich Medical Weekly, depriving twenty babies of fats and vitamin A in periods of up to three hundred days. After the infants died—perhaps forcibly killed, perhaps perishing from their maltreatment—Goll examined their livers from autopsies. Six-month-old Anna Mick was selected for the study; her health had been “robust” despite her hydrocephalus and bed sores on her head. She wasted away on Goll’s diet, lying in the Children’s Hospital while staff prodded her eyes and body for fluid and tissue samples. In less than four months, Anna died from “increasing feebleness.”

Asperger worked in the midst of his colleagues’ human experiments at the Children’s Hospital and would have known about their deadly methods, which they touted in prominent journals. He walked past babies in daily life who were injected, infected, and starved… Asperger—in cofounding the Vienna Society for Curative Education with Franz Hamburger, Erwin Jekelius, and Max Gundel in 1941—was collaborating with three top perpetrators of child killing in Vienna…

Asperger then brought up “eugenic issues” before the Vienna Society, pointing out that “proper assessment” of children was “already a good portion of their ‘treatment.’” Again, one could take these words at face value, as advocating careful care of children. But “treatment,” or Behandlung, was a euphemism that euthanasia personnel used for killing a child. It is curious that Asperger used quotation marks around the word, which suggests he might, indeed, be signaling a veiled meaning for “‘treatment’” —especially on the heels of recommending “prolonged and stationary observation” at Spiegelgrund. After all, the Vienna Society was run by notorious Spiegelgrund leaders, and Jekelius’s inaugural address had already set the stage with his reference to children unworthy of care. Given widespread knowledge of the euthanasia program in Vienna, it is likely that many in the audience were aware of the potential consequences of sending “difficult cases” to Spiegelgrund, as well as the regime’s desire to eliminate children deemed to be defective…..

Heirich Gross, one of the most notorious figures, came to work at Spiegelgrund in mid-November 1940 as child killing were gaining momentum. He was barely twenty-six, having graduated from the University of Vienna Medical School just the year before and worked for a short time at the psychiatric institution Ybbs an der Donau. At Spiegelgrund, Gross served under medical director Jekelius, ten years his senior. Like Jekelius, Gross had been a Nazi enthusiast when the party was still a fringe terrorist organization in Austria; he joined the Hitler Youth in 1932 and the SA in 1933, gaining successive promotions to SA squad leader by 1938.

Seven months after Gross came to work at Spiegelgrund, in June 1941, he went to Germany to train for six weeks under curative education proponent Hans Heinze, who taught killing methods to spiring euthanasia doctors, one of the top three figures in child euthanasia in the entire Reich.

When Gross returned to Vienna after his training with Heinze, the death rate at Spiegelgrund more than tripled… Gross also emulated Heinze in harvesting and preserving victims’ brains. Heinze was distributing many hundreds of adult and child brains to German physicians for research as Gross began his collection of children’s brains at Spiegelgrund for his own work. Children at Spiegelgrund called Gross, who practiced medicine in military uniform, “scythe” or the “Grim Reaper.” (The preservation and research of victim brains is evidence that they would have been able to examine and see the morphological changes to the brain from their organochlorine munition technologies.)

The second director of Spiegelgrund, Ernst Illing, also trained with Heinze. Like Heinze, Illing had spent his early career at the University of Leipzig, and Illing then followed Heinze to Gorden in 1935. Illing worked under Heinze for seven years, conducting some fo the Reich’s first child killings. Illing was highly trained, then, when Heinze and Vienna’s Public Health Office tapped him, at age thirty-eight, to succeed Jekelius as medical director of Spiegelgrund, where he served from July 1, 1942, until April 1945….

And child euthanasia came to pervade Vienna’s medical community at large, reaching far beyond just curative education, as many doctors acquiesced and even welcomed the measures. Illing described in his October 1945 deposition how Viennese physicians readily ridded their wards of children they deemed disabled. He singled out Hamburger and Asperger’s Children’s Hospital by name:

My clinic was always overcrowded since the other clinics, the Welfare Clinic, the Children’s Hospital Glanzing, and the University Children’s Hospital handed over, or wanted to hand over, these hopeless cases—obviously in the belief that euthanasia was legally possible at my clinic due to the aforementioned directive [euthanasia order], while they themselves were not allowed to conduct euthanasia. I am completely confident that the leaders of these institutions were in the know about euthanasia and the aforementioned directives.

Asperger had publicly encouraged his colleagues to transfer “difficult cases” of children to Spiegelgrund—and he followed his own recommendation…

Austrian scholar Herwig Czech has uncovered that Asperger’s panel reviewed the files of 210 children in a single day, slotting them into special schools supposedly appropriate to their level of disability. The commission deemed 35 of the 210 children, 9 girls and 26 boys, “incapable of educational and developmental engagement.” These youths were sent to Spiegelgrund, as the written committee instructions required, to be “dispatched for Jekelius Action.”

“Jekelius Action” was an instruction to kill. All of the 35 youths transferred by Asperger’s commission died. …

At Spiegelgrund, Herta’s photograph showed her crying, her dark hair shaved, and staring straight into the camera. Herta’s mother reportedly beseeched doctor Margarethe Hubsch, in tears: “If the child could not be helped, perhaps it would be better if she would die, as she would have nothing in this world anyway, she would be a laughing stock of the others.” Hubsch explained that, “as a mother of so many other children, she would not wish that on her, so it would be better if she died.” Herta’s mother conveyed at least some of her sentiments to Asperger, too, as he noted in his Spiegelgrund transfer order that “when at home, this child must present an unbearable burden to the mother.” On August 8, Jekelius sent Herta’s records to the Reich Committee in Berlin for authorization to kill the girl. Herta died soon thereafter, two months after Asperger’s transfer. Pneumonia was Herta’s official cause of death. (Herta was only two-years-old)

Chapter 7: The Daily Life of Death
Spiegelgrund’s second director, Ernst Illing, inflicted diagnostic practices on children that could be deadly. Pneumatic encephalography, for example, was an excruciating procedure that injected air into children’s brains after the removal of spinal fluid in order to conduct X-rays showing the cerebral ventricles. Spiegelgrund doctors also collected children’s body parts for research. Most notorious was Dr. Heinrich Gross, who preserved the brains of over four hundred children in jars meticulously stacked and labeled on shelves in the basement, which he used in his research through the 1980s. Indeed, the body parts of children killed at Spiegelgrund were disseminated among a number of research facilities, providing the basis of research long after the war….

Deaths also became part of the daily routine. Marianne Turk and Heinrich Gross even resided on the Spiegelgrund grounds, with Ernst Illing choosing to live with his family in Pavilion 15, the death ward. After the war, Turk reflected on just how habituated she had become to the life of murder, ordering overdoses of Luminal, Veronal, and morphine in injections and in pulverized tablets added to cocoa powder or other foods that children would gladly eat. “With the cases that we had by the dozens in the institution, putting an end to this human wretchedness was an automatic thought.” Turk outlined how the implementation of death orders was quotidian, too:

“The nurses—who undertook the actual execution since they added the sleeping pills to the food—had access to the medicine cabinet. They would be told by Dr. Illing or me that the decisions about child X or Y had arrived, and the nurses knew what they had to do.”…

The mother of Herta Gschwandtner, Luise, openly confronted Spiegelgrund staff about the killings. Herta was born “mongoloid,” and transferred to Spiegelgrund in 1943 at one-and-half years old. She died just eleven days after her transfer, ostensibly of pneumonia. Luise Gschwandnter was incredulous at the speed of her daughter’s death. She wrote to Ernst Illing and Spiegelgrund nurses, “I still can’t grasp why my dear little Herta had to leave me so fast, to die so quickly. […] We still can not believe that our child Herthi was not curable.” Gschwandtner went on, “I am completely heartbroken. I would gladly sacrifice my life for my child…. Please excuse me for my bad writing, which I wrote with very tearful eyes.” In her letter, Gschwandtner went so far as to suggest that Herta was murdered. “Now I have to bear twice the pain because people are saying straight to my face that she was simply poisoned, so to speak, eliminated.” Illing wrote back that nothing was amiss with her daughter’s death. He warned Gschwandtner that he would launch police action if the deaths at Spiegelgrund continued to be questioned: “I would also ask you to vigorously oppose rumors of that kind; if necessary, I will lodge a complaint against such rumormongers.”

But rumors about the killings were rife in Vienna, and exacerbated families’ anguish over the deaths of their children. The parents of two-month-old Hermann Dockl were devastated when their daughter, diagnosed as “mongoloid,” perished five weeks after her admission to Spiegelgrund, reportedly of pneumonia. The Dockl’s family doctor, Hans Geyer, asked Illing to give him a medical accounting of Hermine’s death in order to alleviate the relatives’ torment; he said a proper explanation would bring the family “peace and dispel all the hushed rumors and conjectures.” Hermine’s mother, Geyer warned, was “expressing suicidal intentions and cannot be left alone.” Illing replied simply that the infant had “severe weakness of life” and that the good doctor should surely know that “mongoloid” children had shorter life expectancies.”

The majority of parents’ letters in the case files of children killed at Spiegelgrund are heart wrenching. They voice sorrow, disbelief, anger—and frequently demand more information about how their children died. There was, nevertheless, a wide range of responses. A number of families expressed acceptance, even approval of their child’s untimely death. After all, many in the Reich even sought their child’s admission to killing wards in the hopes their child might perish. They might complain about the burden of caring for their child, perhaps while struggling to make ends meet, with other children at home or a husband away at war. Yet discussions of child-killing was not just limited to to the strains of the Third Reich. The idea of ending “life unworthy of life” had circulated long before the Nazis came to power. In 1925, Ewald Meltzer, the head of the asylum in Saxony, grew so concerned about the ethics of the issue that he asked the parents of children at his institution: “Would you agree to the painless curtailment of the life of your child if experts had established that it was suffering from incurable idiocy?” To his dismay, 73 percent of the parents who participated in the survey answered “yes.”

Spiegelgrund staff said that some parents held explicit conversations about death wishes for their children. The mother of a toddler who Asperger transferred to Spiegelgrund, Herta Schreiber, allegedly told doctor Margarethe Hubsch, “It would be better if she died.” Marianne Turk noted that one mother of a child with epilepsy “thought it would be a comfort and a reassurance to her if the child could close her eyes forever.” Both children were, indeed, killed…

Survivor Leopoldine Maier, contemplating her experience at Spiegelgrund, suggested that complicity in the cruelty—and in the Nazis system as a whole—was pervasive and inescapable. She said people’s potential for depravity would torment her throughout her life.

“Each person raises the question in me: Are you for me or against me? It was always a question of survival. And that question still lingers with me somehow when I meet somebody: With whom is he siding now and with whom was he siding then. And would he have helped you had he known or would he not have helped you at all. […] I am not angry with anybody for how can you be mad with somebody when the evil has no name, when the evil is just part of life, like it was the case there. But the evil belonged there, it was everyday life, and nobody questioned it.”

Chapter 9: In Service to the Volk
The mission of eliminated undesirable children mirrored the Reich’s ambition to eliminate undesirable populations. While Nazi psychiatrists killed youths at home, sequestered behind the walls of hospitals and sanatoria, the Reich wreaked Armageddon across the continent…

The Nazi state aimed to establish a new order in Europe. From 1939 until 1942, its goal appeared to be in sight. The Reich occupied territories, created satellite states, and forged alliances across eastern and western Europe, from Bulgaria to Estonia to Norway to France. Germany even had aspirations in North Africa, too, waging war in Morocco, Algeria, Tunisia, Libya, and Egypt.

In Vienna, most people, including Asperger, supported the Reich. Many were pleased that the Nazi state invested in rebuilding the Austrian economy; Germany coordinated Austria into its war machine, unemployment dropped, large firms boomed, commerce modernized….. The former nation became seven Reich districts, or Reichsgaue, that were collectively called the Ostmark and, after 1942, the “Danubian and Alpine Districts.” The new boundaries tripled the size of greater Vienna, making it the second-largest city in the Reich—yet the regime reduced Vienna’s power from a capital to a provincial city and kept Austria peripheral to the Reich overall…

For Asperger and his associates in Vienna, quality of life remained relatively high throughout the war. Reich citizens fared well compared to the populations they subjugated, typically better fed, housed, and spared the ravages of the battlefield.

*(This author clearly never read the book, The Last Train From Berlin, by Howard K. Smith, 1942. Independent journalists tell a far different story about the state of “Reich citizens.” )

Vienna was also spared the worst of Allied bombing. The Allies unleashed explosives on sixty-one Reich cities during the war, destroying one in five homes and killing around six hundred thousand civilians; the fire-bombing of Hamburg and Dresden raised street temperatures over 1,500 degrees Celsius that turned people into ash in seconds. But Vienna, known as the “Reich’s air raid shelter,” was out of reach until the Allies established bombing flotillas in Italy in the spring of 1944. Missions then aimed for tactical targets in the city rather than carpet bombing, and killed twenty-four thousand civilians.

As Europe collapsed, Germans and Austrians continued to sustain the Reich. Asperger and his colleagues worked through the destruction, publishing, debating, and delivering lectures to one another. Nazi child psychiatry was only a small pocket of activity within the regime’s efforts to remake Europe. But practitioners took their task seriously, and continued to work in earnest even as the continent descended into mass slaughter.
The Third Reich harnessed a curious juxtaposition of efforts, of total war versus erudite debates, of genocide versus journal articles. But the task to mold the mind mattered, as psychiatrists discussed the finer points of Nazi philosophy while carnage raged around them…

Later in life, Asperger emphasized what he had gained from his time in Yugoslavia, highlighting his fortitude and heroism. Echoing longstanding masculine ideals of proving one’s mettle in battle, Asperger recalled, “I was in Croatia, deployed in the partisan war. I wouldn’t want to have missed any of these experiences. It is good to know how you behave when facing danger, as the bullets whistle by—it is also a place where you are tested.”

Apparently Asperger found fulfillment amid the carnage and the Wehrmacht’s notoriously vicious conduct in Yugoslavia. Reich reprisal policy, for example, meant killing one hundred citizens for every German soldier killed….

Chapter 10: Reckoning.
Spiegelgrund survivors continued to suffer after their release. For Franz Pulkert, life did not improve much. “The violence, that was commonplace at the time, I mean, with my parents this wasn’t different either, because my mother wasn’t any better.” Friedrich Zawrel recalled, “My father continued drinking. At home, it was as bad as before.” Karl Hamedler, who was in his mid-teens, was bitter. “At that age you just don’t know what to do with yourself. There you are in the world, and nobody gives a shit about you, to put it bluntly. Even Leopoldine Maier, whose mother had journeyed to Spiegelgrund every week and rescued her, was troubled. She confessed, “I also very often ran away from my mother. I always had my bag with leftover food so I would not starve.”….

For Leopoldine Maier, legal recognition was cold comfort. She was still “crippled by these childhood memories,” she explained. “When I do not watch myself, I always pull my neck in as if I were in constant fear of being hit in the neck with a stick or something else. […] Whenever I wake up in the morning, I tell myself that I am old and it is over and that to me it will never happen again. This is my ritual each and every morning. I tell myself it is over and I survived it.”

Maier dedicated her life to sustaining life. She became a nurse in Vienna and, she said, “I would have loved to have a child just to spare the child what I had to go through.” But Maier found her fallopian tubes were blocked. Although there is no record of it in her files, she suspects she was sterilized during the Third Reich. Haunted by Spiegelgrund’s physical and mental abuse, Maier confided, “The term ‘unworthy of life’ is still ringing in my ears. There is still a sign above my life that says: strictly speaking, you have no right to live.”….

Even top Reich-level euthanasia figures, Hans Heinze and Werner Villinger, had flourishing postwar careers as Germany’s leading psychiatrists. Franz Hamburger, who had become emeritus in 1944, never faced trial. The enormous role Hamburger’s Children Hospital played in the killing system also went unrecognized. Hamburger’s student, Elmar Turk, who conducted tuberculosis experiments on children with Hamburger, practiced through the 1990s and drew on his human experiments from the Third Reich. The body parts of children killed at Spiegelgrund continued to circulate among Vienna’s research laboratories, the basis of its physicians’ publications for decades.

Spiegelgrund doctor Heinrich Gross published thirty-eight articles over twenty-five years—several based on the preserved brains of over four hundred children that he had harvested at Spiegelgrund during the Third Reich, collaborating with associates (such as Andreas Rest, who named Rett syndrome). Gross became a preeminent physical in Austria and was awarded the government’s Honorary Cross for Science and Art in 1975. Despite court proceedings against him in 1948 and 1981, Gross managed to evade conviction for murder. A rock solid case against Gross finally headed to court in 2000, but Gross was deemed unfit to stand trial due to advanced dementia—a condition many observers disputed. Gross died in 2005 at age ninety….

In 2002, the remains of Spiegelgrund’s victims were buried and memorialized—including the brains Gross had collected, which were discovered in Spiegelgrund’s basement, in neatly stacked glass jars. *(Thus they buried the evidence of the morphological and structural damages from IG Farben’s munition technologies branched from their ethylene tree)

Asperger was cleared of wrongdoing after the war… Asperger benefited from the vacuum and was appointed interim director of the University of Vienna Children’s Hospital from 1946 to 1949… He enjoyed a long career. In 1957, Asperger was named director of the University of Innsbruck Children’s Hospital; in 1962, Asperger followed in Hamburger’s footsteps as permanent director of the University of Vienna Children’s Hospital. Asperger wrote a textbook, Heilpadagogik, which found success in several editions—and his field of “curative education” expanded and shifted toward mainstream “special education.”

The Curative Education Clinic sent children to Spiegelgrund’s successor institution, Wilhelminenberg…. Anna Theresa Kimmel, seen in Asperger’s clinic, later described her encounter with Asperger. “I stood facing a tall, tall man in a white coat. Light-haired. The size difference was enormous. And I only know that he greeted my mother, and then looked at me and punched me in the stomach with full force. Yes? My reaction was: no howling, nothing, but I probably looked at him angrily. And so he told me, he told my mother that I had aggression.” Kimmel said she was institutionalized, held in a cage bed for a month. Afterward, Kimmel reflected, “I never heard from Asperger again. I don’t know, was I a test subject? Was I a person? Was I a piece of wood? A guinea pig? I have no idea.”…

Asperger held that the souls of the terminally ill youths “were always very different from the ‘normal’” As he put it, “Their fine spiritual differentiation results from a weakening in their primitive vitality through the existing disease—a consequence of the disease. In other words, illness changed children’s souls and permanently aged them, completing their development. It was appropriate that they perished earlier than others. In his 1975 article, “The Dying Child,” Asperger invoked scripture to conclude the point, citing the Wisdom of Solomon: children who die young “live a long life in a short span of time.”…

In Asperger’s view, the doctor’s role was to guide the child and his or her parents, particularly the grieving mother, down the path of death—to “fulfill his noble duty as guide into the realm of the natural.”

In his 1975 article, “The Dying Child,” Asperger also wrote that the doctor should “serve in death.” It is unclear exactly what Asperger meant by the phrase, but juxtaposed it with what he called “active euthanasia.” – Asperger’s Children: The Origins of Autism in Nazi Vienna by Edith Sheffer

The IG Farben employees, Vienna doctors, destroyed the evidence of the harm of their employer’s munition technology products. Theo Colborn called their benzene technology products, womb terrorists. They didn’t destroy the technologies causing all the harm. They expanded their ethylene tree based munition markets and exterminated the infants and children destroyed by them.
The Last Train From Berlin by Howard K. Smith
“Other vegetables came to count as luxuries. Tomatoes were rationed too for a while, then disappeared altogether to canning factories where they could be preserved and sent to the Eastern front. Two-vegetable meals became virtually extinct. Scarcities were made more severe by the prudence of the food ministry which, having its palms slapped once, began to play it safe by preparing more and more canned goods for the troops in the event that the war should last through the winter….

Ersatz foods flourished. Icing for the few remaining pastries tasted like a mixture of saccharine, sand and cheap perfume. White bread was issued after the third month of the campaign only on the ration cards formerly for pastry. A red coloured paste called Lachs Galantine, resembling salmon in color and soggy sawdust in taste, appeared in restaurants on meatless days. Several strange bottled sauces made of incredible combinations of acid-tasting chemicals made their appearance in shops to answer the public’s growing demand for something to put a taste of some kind in their unattractive and scanty meals…

Cigarettes suffered the most rapid decline in quality… My tobacconist told me “Johnnies” were made of the same dry, powdery, inferior tobacco as other cigarettes, but the leaves were sprayed with a chemical to give them a distinctive flavour and kill their original one. The chemical, he said, was severely damaging to the lungs, which I can believe…

It caused visible pain to the old bar-tender to answer an order for a cocktail saying he was dreadfully sorry but today, precisely today, he had run out of all the ingredients. But perhaps tomorrow. Actually, all he had was some raw liquor the management had been able to squeeze out of a farm-house outside Berlin, Himbeergeist, or a fake Vodka that took the roof off your mouth, or wood alcohol with perfume in it which was served under the name of Sclibovitz, two fingers to a customer and no more…

Civilian hospitals are overcrowded and doctors overworked. Environment, which has a great deal to do with mental health and well-being has grown seedy and ugly. Hours are longer and real wages immeasurably lower than they were before the Russian war. Families are losing their youngest and strongest members, or seeing them some home legless and armless. The horizon of the average German is desolate….

Today, also after two years of war, there are only two meat dishes on the menu, one of which is struck through with a pencil mark along the strategy of the Kaiserhof Hotel. The other is generally two little sausages of uncertain contents, each about the size of a cigar butt. Before the meat they give you a chalky, red, warm liquid called tomato soup, but which a good-natured waiter-friend of mine always called: Ee-gay Farben Nummer zwei nulleex! all of which means, “Dye trust formula number 20-X.” With the meat you get four or five yellow potatoes with black blotches on them…

Hitler’s solution to the crisis was not by making useful goods but by producing the greatest aggregation of arms, which nobody can eat, or wear, in all history…”
Portions from pages 120 – 149)*(You can actually consume munitions, Americans consume so much splenda that it’s the number 1 artificial sweetener. It’s an organochlorine and you can guarantee that it destroys your brain like all the rest of those sulfur chlorine carbon creations…)

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U.S. Nerve Gas Hit Our Own Troops in Iraq

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Effects of decabrominated diphenyl ether (PBDE 209) exposure at different developmental periods on synaptic plasticity in the dentate gyrus of adult rats In vivo.

Xing T1, Chen L, Tao Y, Wang M, Chen J, Ruan DY.
Author information

Abstract
Polybromininated diphenyl ethers (PBDEs) are widely used as flame-retardant additives. Previous studies have demonstrated that PBDEs exposure can lead to neurotoxicity. However, little is known about the effects of PBDE 209 on synaptic plasticity. This study investigated the effect of decabrominated diphenyl ether (PBDE 209), a major PBDEs product, on synaptic plasticity in the dentate gyrus of rats at different developmental periods. We examined the input/output functions, paired-pulse reactions, and the long-term potentiation of the field excitatory postsynaptic potential slope and the population spike amplitude in vivo. Rats were exposed to PBDE 209 during five different developmental periods: pregnancy, lactation via mother’s milk, lactation via intragastric administration, after weaning, and prenatal to life. We found that exposed to PBDE 209 during different developmental periods could impair the synaptic plasticity of adult rats in different degrees. The results also showed that PBDE 209 might cause more serious effects on the postsynaptic cell excitability in synaptic plasticity, and the lactation period was the most sensitive time of development towards PBDE 209.

http://www.ncbi.nlm.nih.gov/pubmed/19535737

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Inhibition of progenitor cell proliferation in the dentate gyrus of rats following post-weaning lead exposure.

Schneider JS1, Anderson DW, Wade TV, Smith MG, Leibrandt P, Zuck L, Lidsky TI.
Author information

Abstract
Although lead is a potent developmental neurotoxin, the effects of postnatal lead exposure on progenitor cell proliferation in the hippocampus has not been examined. Postnatal day 25 rats were fed a lead containing diet (1500 ppm lead acetate) for 30-35 days and administered bromodeoxyuridine (BrdU, 50 mg/kg, i.p.) during the last 5 days of lead exposure. Animals were killed 24 h after the last BrdU injection. Proliferation of new cells in the subgranular zone and dentate gyrus was significantly decreased in lead-exposed rats compared to control animals that ate a similar diet devoid of lead. These results suggest that postnatal lead exposure can have significant deleterious effects on progenitor cell proliferation and thus the structure and function of the hippocampus.

http://www.ncbi.nlm.nih.gov/pubmed/15527882

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MisLEAD: America’s Secret Epidemic Documentary on Lead Poisoning in the US

 

 

The question on many minds today: what is the source of the sudden, alarming rise in the number of American children with ADD, ADHD, Autism Spectrum symptoms and similar neurological disorders—expensive impairments/disabilities that create challenges for families and cost our society more than $50,000,000,000 annually?

MisLEAD: America’s Secret Epidemic is the first documentary film that undertakes an intellectually rigorous, emotionally compelling and illuminating inquiry into a hidden epidemic that impacts one in three American children today. Tamara Rubin, an Oregon mother whose children were poisoned, travels the country talking with parents and top experts across many fields—uncovering surprising answers.

To access the film’s website click on the link below
misLEAD the movie

“Ammunition that contains lead-based primer or lead bullets will create smoke that contains lead. This lead can be inhaled or can settle on the floor, counters, doorknobs, and other surfaces in the range. Shooters and instructors can ingest lead if they touch contaminated surfaces and then eat, drink, or smoke without washing their hands. Range operators are at higher risk of lead exposure than recreational shooters – while periodic cleaning of the range is recommended, this activity can stir up settled dust and persons conducting the cleaning can be overexposed if they are not protected.” Firearm use should be added to potential lead exposures for children.

Taken from The Commonwealth of Massachusetts “Shooting Range Lead Safety Precautions”

Click to access olr-shooting-range-bulletin.pdf

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House Oversight and Government Reform Committee Investigation into Johnson and Johnson’s Recall of Children’s Tylenol and Other Children’s Medicines

Statement of
Joshua M. Sharfstein, M.D.
Principal Deputy Commissioner
U.S. Food and Drug Administration
Department of Health and Human Services

Before the
Committee on Oversight and Government Reform
U.S. House of Repersentatives

May 27, 2010
Introduction
Mr. Chairman and Members of the Committee, I am Joshua M. Sharfstein, M.D., Principal Deputy Commissioner, U.S. Food and Drug Administration (FDA or the Agency), which is an Agency of the Department of Health and Human Services. Thank you for the opportunity to discuss the Agency’s regulation of drug manufacturing, our oversight of McNeil Consumer Healthcare, LLC (McNeil), and lessons learned from the ongoing investigation into quality concerns at McNeil.

FDA Oversight of Drug Manufacturing
Under the Federal Food, Drug, and Cosmetic Act, FDA is charged with, among other things, ensuring that drugs marketed in the United States are safe and effective, and are manufactured in accordance with current Good Manufacturing Practice (cGMP).

The cGMP regulations for drugs contain minimum requirements for the methods, facilities, and controls used in manufacturing, processing, and packing of a drug product. The regulations are intended to ensure purity, potency, and quality of drug products, and to prevent unsafe products from reaching consumers.

Under the cGMP regulations, each manufacturer sets specifications for its own products for such factors as potency, stability and purity, and puts in place a quality system that ensures those specifications are met. Critical to the cGMP process is that a company must meet its own standards.

A violation of cGMP does not necessarily mean that a product is hazardous to the public. It does indicate, however, a breakdown in a manufacturer’s quality system and is an indication that a company needs to take effective steps to fix the problem promptly.

FDA inspects facilities to ensure compliance with cGMP standards. These inspections occur on average for domestic facilities every two to three years. We increase the frequency of inspections for facilities when warranted by past problems or by products that are difficult to manufacture or are especially high risk.

When on site, FDA inspectors identify gaps in manufacturing standards and discuss with companies how they can fix them. Firms may choose to recall products when there are cGMP violations, especially when those violations have a significant impact on product quality or safety.

For drugs, patterns of non-compliance or non-compliance that put the public’s health at risk leads to appropriate enforcement action by the Agency, including warning letters, seizures, injunctions and criminal prosecution.

Oversight of McNeil Consumer Healthcare, LLC (McNeil)
McNeil makes a variety of over-the-counter (OTC) products for the U.S. market from four manufacturing facilities in the United States and Canada. Over the last several years, FDA has had growing concerns about the quality of the company’s manufacturing process. These concerns have led to a number of unsatisfactory inspections and consumer recalls. FDA has inspected the company’s facilities with an increased frequency, and in February 2010, the Agency took the extraordinary step of convening a meeting with the management of the parent company, Johnson & Johnson, to express concern about a pattern of non-compliance.

Prior to 2009. Before 2009, FDA investigators identified several problems with cGMP compliance at facilities run by McNeil. These problems included laboratory controls, equipment cleaning processes, and a failure to investigate identified problems. The company generally fixed the specific problems, and the Agency inspected the firm regularly.

Spring/Summer 2009. At its Fort Washington facility, McNeil makes a wide variety of OTC products, including a large number of OTC liquid products for children.

In May and June 2009, FDA identified several cGMP violations, including McNeil’s failure to meet its own standard for quality in one of the ingredients in OTC liquids.

McNeil’s standard for this ingredient, known as microcrystalline cellulose, required that there be no gram negative bacteria. McNeil purchased the cellulose in partial lots that had not tested positive for this objectionable bacteria. The vendor tested other partial lots from the same large master lot and found a certain gram negative bacteria called B. cepacia. According to cGMP standards, McNeil should not have used any partial lots from this master lot.

In reviewing the situation, FDA scientists concluded that the risk to the public was remote. All of the drums used tested negative for the bacteria B. cepacia, all of the final product tested negative, and FDA agreed with the company’s assessment that this bacteria would be very unlikely to grow in the final product.

Yet, because the company had not kept to its standard, it represented a cGMP violation, and the company initiated a recall of almost eight million bottles of finished product in August 2009.

Fall 2009. At its Las Piedras, Puerto Rico, facility, McNeil makes a large number of OTC pills for the U.S. market.

In the fall of last year, FDA became aware that McNeil had received reports of products from this facility having a musty odor. Yet, McNeil had not fully investigated these reports for about a year and did not notify FDA despite the requirement that such reports be referred to the Agency within three days.

FDA inspectors urged McNeil to conduct a complete investigation, which eventually identified the source of the odor to be a chemical, called 2,4,6-Tribromoanisole or TBA, which was in the air because of a pesticide used on the wood of the pallets used to store empty medication bottles. McNeil initiated a series of recalls as the scope of the problem became clear.

The risk posed to the public by this problem included potential temporary, non-serious gastrointestinal reactions – including nausea, stomach pain, vomiting, or diarrhea. Very little is known about the chemical TBA, but in the small quantities transferred to the products, it is not thought to pose a serious risk for long-term health problems.

On January 15, 2010, FDA issued a warning letter to McNeil expressing serious concerns about the company’s control over the quality of its drugs and the company’s failure to aggressively investigate and correct quality problems. This letter identified significant violations of the cGMP regulations. FDA noted that neither upper management at Johnson & Johnson nor at McNeil assured timely investigation and resolution of the issues.

January and February 2010. In early 2010, FDA conducted focused inspections of McNeil at both the Las Piedras and Fort Washington facilities to follow up on a reported problem. The report identified a 6-year-old child who died. Prior to his death, the child had been given several products manufactured by McNeil at these facilities. FDA tested the products the child had taken for potential contamination, and all results were negative. Based on the results of the testing and the results of the inspection, FDA did not find evidence to link the products to the child’s death.

February 2010. On February 19, 2010, senior compliance staff from FDA’s Center for Drug Evaluation and Research and from FDA’s field organization met with senior officials from McNeil and its parent company, Johnson & Johnson. Attendees included the President of McNeil, the Company Group Chairman for OTC at Johnson & Johnson, as well as a number of Quality Assurance executives from both companies.

This was an extraordinary meeting. FDA requested that senior officials from Johnson & Johnson attend the meeting so they would be on notice regarding FDA’s rising concerns about whether McNeil’s corporate culture supported a robust quality system to ensure the purity, potency and safety of its products. FDA also raised concerns about Johnson & Johnson’s oversight of McNeil due to recent multiple recalls of McNeil products and recent warning letters FDA had issued to both McNeil and its parent company, Johnson & Johnson. Based on the Fort Washington and Las Piedras inspections in 2009 as well as the firm’s recent compliance history, FDA expressed its significant concern that there was a pattern of conduct including failure to report material information to FDA in a timely manner, miscalculating and/or misstating risks and benefits of their products, and reactive vs. proactive approaches to product quality problems. FDA told the company’s leadership that significant, immediate steps were needed to address issues of compliance and quality, especially in investigating product quality issues so that the company could take preventive action to avoid problems.

The Agency learned that McNeil was taking several major steps to address these issues, including implementing management reporting structure changes, hiring new managers, and engaging a third party manufacturing consultant. FDA indicated that it would continue to monitor closely and consider further action, and that it was concerned about whether the company’s corporate culture was appropriately focused on product quality issues.

April 2010. In April, FDA inspectors returned to McNeil’s Fort Washington facility. This inspection was scheduled sooner than usual due to McNeil’s recent history of compliance problems, including numerous recalls and cGMP deficiencies discovered in the June 2009 Fort Washington inspection, which had a significant impact on the scheduling of the April 2010 inspection.

Days before the inspectors arrived, McNeil shut down manufacturing because of manufacturing issues, including particulates found in a number of liquid medications. These particulates included acetaminophen, cellulose, nickel, and chromium. FDA inspectors identified a range of cGMP violations. These included the company failing to meet its own specifications for bacteria and particulates and, for one Tylenol product, the possibility of higher than expected concentrations of Tylenol per dropper.

In reviewing the situation, FDA scientists concluded that the risk posed to the public by these problems was remote. FDA did not find evidence that McNeil used raw materials that its tests found to be positive for bacterial contamination and all lots of finished product were tested by McNeil and found negative for bacterial contamination. The particulates would be expected to pass through the gastrointestinal tract. While there was a potential for higher concentrations of Tylenol per dropper, none of the final products released for sale tested with high levels. In addition, the increase in potency would not be expected to cause adverse effects.

Although the public health risk from these quality problems is low, these problems should never have occurred, and the cGMP failures at the facility that caused them were unacceptable. Following cGMP requirements assures that products are consistent in their safety and effectiveness and failure to follow those procedures undermines consumer confidence. On April 30, 2010, McNeil announced a voluntary recall of over 136 million bottles of liquid infants’ and children’s products.

Next Steps in FDA Oversight of McNeil

Based on the pattern of concerns found at McNeil’s facilities, FDA is working with the company to address its systemic quality issues. The Agency is closely monitoring the implementation of a corrective action plan developed by McNeil that includes significant enhancements to its quality system, organizational changes, and senior management oversight.

FDA will continue to investigate issues related to the Fort Washington facility including oversight related to renewal of manufacturing operations at that facility, to evaluate the facility’s suppliers, and evaluate the compliance of all other McNeil facilities. FDA will also take steps to help ensure that when the facility begins manufacturing again it will be able to produce safe products. FDA is also considering additional enforcement actions against the company for its pattern of non-compliance which may include seizure, injunction or criminal penalties.

Adverse Event Evaluation
It is understandable that many Americans, hearing about these large recalls, would wonder whether or not their children were put at risk. In assessing this question, FDA considers two basic sources of information – first, our assessment of the manufacturing problems themselves, and second, adverse event reports to the Agency.

As I discussed earlier, FDA analyzed the various manufacturing problems. Based on the circumstances in each case, our experts believe the risk for any child in the United States was remote.

FDA has also looked at adverse events reported to the Agency. FDA receives these reports and often requests and reviews medical records, coroner’s reports, and other supplementary data sources.

When we have adequate information about a case, the Agency reviews these reports to determine what role, if any, the medication played in the development of an adverse event. We can find that the medication likely had no role in the adverse event, that the medication’s activity as a drug could have caused a serious side effect, or that a quality problem may have contributed to the outcome.

All drugs have side effects, and some of the McNeil reports may reflect the side effects of OTC medications. Other reports appear unrelated to the medications.

So far, FDA has no cases with evidence that a product quality issue contributed to a significant adverse health outcome. We are continuing to receive information about certain cases and we will update the public and the Committee should our assessment change.

Lessons Learned
Every investigation presents an opportunity for FDA to improve our effectiveness in protecting the public health. One lesson to be drawn from the McNeil story is that it is important for the Agency to even more fully consider the corporate structure when investigating and enforcing the law. FDA will be developing new procedures to use what we learn at one facility in guiding our inspections of other facilities run by the same company.

FDA is also using these events as part of an ongoing review of our recall process. FDA has already made significant changes to its approach to recalls when there are urgent, life-threatening product quality concerns. For example, in recent months, FDA has moved aggressively to support several urgent food recalls. FDA is now looking at our process for clear expectations and standards with respect to other types of recalls, such as those undertaken by McNeil.

We will continue to work with Congress to secure additional authorities that could assist us in assuring product quality and acting more quickly when product quality issues occur.
FDA will also be considering enforcement actions in this case as part of the Agency’s ongoing changes in enforcement. FDA Commissioner Dr. Margaret Hamburg has called for FDA’s enforcement to be “vigilant, strategic, quick, and visible.” A range of activities are underway at the Agency to bring this vision to reality, including strengthening our criminal enforcement of FDA’s laws.

As we continue these efforts, as well as our other regulatory work, we will focus on entire companies and their systems in addition to focusing on specific violations, individuals, and sites, much as we are doing in the McNeil situation.

Conclusion
Thank you for the opportunity to explain FDA’s oversight of drug manufacturing and our engagement with McNeil. I look forward to your questions.

“Gentle on little tummies.. When it comes to reducing fever or relieving pain in infants, INFANTS’ TYLENOL® has been the brand recommended most by pediatricians for the last 20 years. INFANTS’ TYLENOL® works differently than other pain and fever medicines. It also won’t upset little stomachs…. anhydrous citric acid, butylparaben, D&C red no. 33, FD&C Blue no.1, flavors, glycerin, high fructose corn syrup, microcrystalline cellulose and carboxymethylcellulose sodium, propylene glycol, purified water, sodium benzoate, sorbitol solution, SUCRALOSE, xanthan gum.”

The Search For Sweet by Burkhard Bilger for The New Yorker – May 22, 2006

“The substance in the flask seemed to have all the makings of an excellent insecticide. It was a fine crystaline powder and its molecules were full of chlorine atoms, like DDT. ..by taking an eye-dropper full of sulfuryl chloride – a highly toxic chemical – and adding it to a sugar solution, one drop at a time. In the violent reaction that followed, a wholly new compound was born: 1′, 4,6,6′-tetrachloro-1′,4,6,6′-tetra-deoxygalactosucrose. “It isn’t of any use as an insecticide,” Hough told me recently, “That was tested.” But it has proven useful as a food. In its pure form, it is known as sucralose. When mixed with fillers and sold in bright yellow sachets, it’s known as Splenda, the best-selling artificial sweetener in America.”

Sucralose was declared safe by the Food and Drug Administration in 1998, but most of the taste researchers I talked to won’t eat it. “I look at that structure and I have an irrational fear of it,” one of them said.”

http://archives.newyorker.com/?i=2006-05-22#folio=040

J Toxicol Environ Health A. 2008;71(21):1415-29. doi: 10.1080/15287390802328630.
Splenda alters gut microflora and increases intestinal p-glycoprotein and cytochrome p-450 in male rats.
Abou-Donia MB1, El-Masry EM, Abdel-Rahman AA, McLendon RE, Schiffman SS.
Author information

Abstract
Splenda is comprised of the high-potency artificial sweetener sucralose (1.1%) and the fillers maltodextrin and glucose. Splenda was administered by oral gavage at 100, 300, 500, or 1000 mg/kg to male Sprague-Dawley rats for 12-wk, during which fecal samples were collected weekly for bacterial analysis and measurement of fecal pH. After 12-wk, half of the animals from each treatment group were sacrificed to determine the intestinal expression of the membrane efflux transporter P-glycoprotein (P-gp) and the cytochrome P-450 (CYP) metabolism system by Western blot. The remaining animals were allowed to recover for an additional 12-wk, and further assessments of fecal microflora, fecal pH, and expression of P-gp and CYP were determined. At the end of the 12-wk treatment period, the numbers of total anaerobes, bifidobacteria, lactobacilli, Bacteroides, clostridia, and total aerobic bacteria were significantly decreased; however, there was no significant treatment effect on enterobacteria. Splenda also increased fecal pH and enhanced the expression of P-gp by 2.43-fold, CYP3A4 by 2.51-fold, and CYP2D1 by 3.49-fold. Following the 12-wk recovery period, only the total anaerobes and bifidobacteria remained significantly depressed, whereas pH values, P-gp, and CYP3A4 and CYP2D1 remained elevated. These changes occurred at Splenda dosages that contained sucralose at 1.1-11 mg/kg (the US FDA Acceptable Daily Intake for sucralose is 5 mg/kg). Evidence indicates that a 12-wk administration of Splenda exerted numerous adverse effects, including (1) reduction in beneficial fecal microflora, (2) increased fecal pH, and (3) enhanced expression levels of P-gp, CYP3A4, and CYP2D1, which are known to limit the bioavailability of orally administered drugs.

Splenda: The Artificial Sweetener that Explodes Internally
By: Shane Ellison, MS for The People’s Chemist

“Splenda contains the drug sucralose. This chemical is 600 times sweeter than sugar. To make sucralose, chlorine is used. Chlorine has a split personality. It can be harmless or it can be life threatening.
In combo with sodium, chlorine forms a harmless “ionic bond” to yield table salt. Sucralose makers often highlight this worthless fact to defend its’ safety. Apparently, they missed the second day of Chemistry 101 – the day they teach “covalent” bonds.
When used with carbon, the chlorine atom in sucralose forms a “covalent” bond. The end result is the historically deadly “organochlorine” or simply: a Really-Nasty Form of Chlorine (RNFOC).
Unlike ionic bonds, covalently bound chlorine atoms are a big no-no for the human body. They yield insecticides, pesticides, and herbicides – not something you want in the lunch box of your precious child. It’s therefore no surprise that the originators of sucralose, chemists Hough and Phadnis, were attempting to design new insecticides when they discovered it! It wasn’t until the young Phadnis accidentally tasted his new “insecticide” that he learned it was sweet. And because sugars are more profitable than insecticides, the whole insecticide idea got canned and a new sweetener called Splenda got packaged.
To hide its dirty origin, Splenda pushers assert that sucralose is “made from sugar so it tastes like sugar.” Sucralose is as close to sugar as Windex is to ocean water.”

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Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits
Open – Molecular Psychiatry advance online publication 23 April 2013; doi: 10.1038/mp.2013.41

C C Y Wong1, E L Meaburn1,2, A Ronald1,2, T S Price1,3, A R Jeffries1, L C Schalkwyk1, R Plomin1 and J Mill1,4

Abstract

Autism spectrum disorder (ASD) defines a group of common, complex neurodevelopmental disorders. Although the aetiology of ASD has a strong genetic component, there is considerable monozygotic (MZ) twin discordance indicating a role for non-genetic factors. Because MZ twins share an identical DNA sequence, disease-discordant MZ twin pairs provide an ideal model for examining the contribution of environmentally driven epigenetic factors in disease. We performed a genome-wide analysis of DNA methylation in a sample of 50 MZ twin pairs (100 individuals) sampled from a representative population cohort that included twins discordant and concordant for ASD, ASD-associated traits and no autistic phenotype. Within-twin and between-group analyses identified numerous differentially methylated regions associated with ASD. In addition, we report significant correlations between DNA methylation and quantitatively measured autistic trait scores across our sample cohort. This study represents the first systematic epigenomic analyses of MZ twins discordant for ASD and implicates a role for altered DNA methylation in autism.

Introduction

Autism spectrum disorder (ASD) defines a collection of complex childhood neurodevelopmental disorders affecting ~1% of the population and conferring severe lifelong disability.1 ASD is characterized by a triad of impairments: (1) deficits in social interactions and understanding, (2) non-social impairments, such as repetitive behaviour and interests, and (3) impairments in language and communication development. Quantitative genetic studies indicate that ASD has a strong heritable component,2 which is supported by the recent identification of several susceptibility loci and an emerging literature implicating the relevance of de novo and inherited copy number variants (CNVs) in the disorder.3 Despite intense research effort during the past decade, however, no definitive biological or clinical markers for ASD have been identified. This can be partly explained by the highly heterogeneous nature of ASD, both clinically and aetiologically. The clinical manifestation of ASD displays considerable individual variability in the severity of impairments and quantitative genetic studies also report genetic heterogeneity between the three trait domains of ASD.4, 5, 6

Despite the high heritability estimates for ASD, there is notable discordance within monozygotic (MZ) twin pairs for diagnosed ASD, and often considerable symptom severity differences within ASD-concordant MZ twins,2 strongly implicating a role for non-genetic epigenetic factors in aetiology. Epigenetic mechanisms mediate reversible changes in gene expression independent of DNA sequence variation, principally through alterations in DNA methylation and chromatin structure.7 Epigenetic changes in the brain have been associated with a range of neurological and cognitive processes, including neurogenesis,8 brain development9 and drug addiction.10 Emerging evidence implicates epigenetic modifications in several neuropsychiatric disorders, including ASD.11, 12 In particular, epigenetic dysregulation underlies the symptoms of Rett syndrome and Fragile X syndrome, two disorders with considerable phenotypic overlap with ASD.11 Although few empirical studies have systematically examined the role of altered epigenetic processes in ASD, recent analyses provide evidence for altered DNA methylation and histone modifications in disease pathology.13, 14, 15

The use of disease-discordant MZ twins represents a powerful strategy in epigenetic epidemiology because identical twins are matched for genotype, age, sex, maternal environment, population cohort effects and exposure to many shared environmental factors.16, 17 Recent studies have uncovered considerable epigenetic variation between MZ twins,18, 19, 20 and DNA methylation differences have been associated with MZ twin discordance for several complex phenotypic traits, including psychosis21 and Type 1 diabetes.22 In ASD, Nguyen and co-workers23 recently examined lymphoblastoid cell lines derived from peripheral blood lymphocytes collected from three ASD-discordant MZ twin pairs, reporting several ASD-associated differentially methylated loci.23 Two loci (RORA and BCL2) reported as hypermethylated in ASD were found to be downregulated in RNA from post-mortem autism brains. These findings support a role for DNA methylation in ASD and highlight the successful use of peripherally derived DNA from discordant MZ twins to identify disease-associated epigenetic changes. Given the highly heterogeneous nature of ASD, however, more comprehensive genome-wide analyses across larger numbers of samples are warranted to investigate the extent to which ASD-associated epigenetic variation is individual- and symptom-specific.

Materials and methods

Samples for methylomic analysis

Participants were recruited from the Twins’ Early Development Study (TEDS), a United Kingdom-based study of twins contacted from birth records.24 For this study, a total of 50 MZ twin pairs were identified within TEDS using the Childhood Autism Symptom Test (CAST), which assesses dimensional ASD traits, at age 8 years. The CAST25 is a 31-item screening measurement for ASD, designed for parents and teachers to complete in non-clinical settings to assess behaviours characteristic of the autistic spectrum. Items within the CAST are scored additively and a score of 15 (that is, answering ‘yes’ on 15 items) is the cutoff for identifying children ‘at risk’ for ASD. On the basis of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria for autism, CAST items can be divided into three subscales: impairments in social symptoms (12 items); impairments in non-social symptoms (that is, restricted repetitive behaviours and interests (RRBIs); 7 items); and communication impairments (12 items).6 The CAST has been widely used in population-based studies of singletons25 as well as in twin studies.26 Within TEDS, the CAST has been shown to have good reliability and validity.27 Supplementary Figure 1 shows the distribution of total CAST and its three subscale scores within samples selected using parent ratings. Supplementary Table 1 provides a summary of the samples included in the analyses. Whole-blood samples were collected from subjects at age 15 years by a trained phlebotomist for DNA extraction and blood cell-count analysis. Blood cell counts were assessed for all collected samples and found to be within the normal range.

Genome-wide analysis of DNA methylation

For each individual, genomic DNA (500 ng) extracted from whole blood was treated with sodium bisulphite using the EZ 96-DNA methylation kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s standard protocol. The bisulphite conversion reaction was performed in duplicate for each sample to minimize potential bias caused by variable conversion efficiency, and pooled bisulphite-treated DNA was used for subsequent array analysis. Genome-wide DNA methylation was assessed using the Illumina Infinium HumanMethylation27 BeadChip (Illumina, San Diego, CA, USA), which interrogates the DNA methylation profile of 27 578 CpG sites located in 14 495 protein-coding gene promoters and 110 microRNA gene promoters, at single-nucleotide resolution.28 Illumina GenomeStudio software (Illumina, San Diego, CA, USA) was used to extract signal intensities for each probe and perform initial quality control checks, with all data sets (except two individuals) being considered to be of high quality and included in subsequent analyses. To ensure stringent data quality, probes with a detection P-value >0.05 in any of the samples were removed across all individuals (N=1161 probes) in addition to a set of probes (N=2923) that were reported as nonspecific and potentially unreliable in a recent survey of all probes on the microarray.29

Methylation microarray data processing

All computations and statistical analyses were performed within the R statistical analysis environment (http://www.r-project.org), and all analysis scripts are available on request from the authors. A customized pipeline was used for the analysis of Illumina 27K methylation data as described in a previous study of psychosis-discordant MZ twins.21 Briefly, signal intensities for each probe were normalized using quantile normalization to reduce unwanted interarray variation. The relative methylation level of each interrogated CpG site was calculated as the ratio of the normalized signal from the methylated probe to the sum of the normalized signals of the methylated and unmethylated probes. This gave an average DNA methylation value, described as average ‘β-value’ for each CpG site, ranging from 0 (unmethylated) to 1 (fully methylated). A density plot of β-values for every sample revealed that, as expected given the known distribution of probes on the array, the data followed a bimodal distribution (Supplementary Figure 2). An empirical variance stabilizing transformation was used to adjust for the bimodal distribution of the data.21 Raw microarray data are available for download from http://epigenetics.iop.kcl.ac.uk/ASDTwins/.

Identification of ASD-associated DMRs

Two major analysis strategies were used to identify DMRs associated with ASD and related traits. First, DNA methylation differences within pairs of MZ twins were examined in MZ twin pairs discordant for ASD and ASD-related traits. Second, case–control comparisons of DNA methylation were performed between groups of individuals scoring high and low for ASD traits. With the aim of identifying real, biologically relevant within-twin and between-group DNA methylation differences, we used an analytic approach that incorporates both the significance (that is, t-test statistic) and magnitude (that is, absolute delta-β (Δβ)) of any observed differences to produce a ranked list of DMRs.21 A summary of the analysis strategy is presented in Supplementary Figure 3. This combined approach, where data are interpreted based on the combination of fold change and statistical significance, is routinely used in genome-wide gene expression studies and has been shown to produce gene lists of higher reproducibility and biological relevance.30 We recently used a similar approach successfully to identify disease-associated epigenetic changes in a psychosis-discordant MZ twin study.21 Given the known phenotypic and aetiologic heterogeneity, we also screened for large Δβ-values within each discordant MZ twin pair to examine the possibility that disease-associated epigenetic changes are potentially private and not consistent across all families. Finally, we examined whether quantitative CAST scores are correlated with DNA methylation at specific loci. The association between each of the quantitatively rated CAST subscale variables and DNA methylation at each CpG site was assessed using Pearson’s product–moment correlation.

Global DNA methylation analysis

Global levels of DNA methylation were quantified using the LUminometric Methylation Assay (LUMA).31 This method relies on DNA cleavage by methylation-sensitive and -insensitive restriction enzymes, followed by the quantification of the resulting restriction fragments using pyrosequencing.31 Positive controls, including both artificially methylated and artificially unmethylated samples, were included in all experimental steps to ensure unambiguous restriction enzyme digestions and to calibrate the experimental data, with each sample being processed in duplicate.

Fine mapping of DNA methylation using bisulphite pyrosequencing

Although the Illumina 27K array has been well validated for detecting differences in DNA methylation, we further tested specific regions nominated from the genome-wide microarray analysis using bisulphite pyrosequencing. Independent verification analyses were performed on two CpG sites (cg16474696, MGC3207; cg20507276, OR2L13) that demonstrated a large significant ASD-associated difference from the case versus control analysis. In each case, the assay spanned multiple CpG sites, including the specific CpG interrogated on the Illumina 27K array. Briefly, 500 ng DNA from each individual was independently treated with sodium bisulphite in duplicate using the EZ 96-DNA methylation kit as described above. Bisulphite-polymerase chain reaction amplification was performed in duplicate. Quantitative DNA methylation analysis was conducted using the PyroMark Q24 pyrosequencer (Qiagen, Valencia, CA, USA). The correlation between DNA methylation estimates obtained from Illumina 27K array and bisulphite pyrosequencing was assessed using Pearson’s moment–correlation coefficient. In addition, Sanger sequencing using BigDye v.3.1 terminator mix (Applied Biosystems, Foster City, CA, USA) was performed on the regions targeted by the MGC3207 pyrosequencing assay to ensure that the Illumina probe sequences and the primer binding sites for the pyrosequencing assay were free of any DNA sequence variation. The primers and assay conditions are given in Supplementary Table 2.

CNV analysis using genotyping arrays

Genomic DNA (200 ng) extracted from whole blood was genotyped using the Illumina HumanOmniExpress BeadChip (Illumina) targeting >730 000 single-nucleotide polymorphisms and Illumina GenomeStudio software was used to call genotypes based on predefined genotype cluster boundaries to denote cluster positions (HumanOmniExpress-12v1_C.egt). CNVs were identified from the genotyping data using two independent algorithms, PennCNV32 and QuantiSNP,33 with default parameters, and GC content signal preprocessing was applied. Stringent quality control steps were used to ensure that only high-confidence CNVs, that is, those >1 kb in size, covered by >5 probes and detected by both programs, were included for further analysis.

Results

ASD is not associated with systemic differences in global DNA methylation
As expected, within-twin patterns of DNA methylation were highly correlated across all MZ twin pairs (average within-twin r across all probes=0.99), indicating that ASD and related traits are not associated with systemic changes in epigenetic programming. Supplementary Figure 4a shows the correlation between genome-wide DNA methylation across all probes on the array and one example ASD-discordant MZ twin pair; data for the other ASD-discordant MZ pairs are available for download from http://epigenetics.iop.kcl.ac.uk/ASDTwins/. These data were corroborated by global DNA methylation analysis using LUMA, which identified no significant difference between affected ASD twins and their co-twins (affected ASD twins mean=65.1%, unaffected co-twins mean=65.9%; P=0.817) (Supplementary Figure 4b).

Site-specific DNA differences are widespread in MZ-discordant ASD twins

In contrast to global levels of DNA methylation, DNA methylation at individual CpG sites demonstrated considerable variability within ASD-discordant MZ twin pairs. Figure 1a shows the distribution of average absolute differences in DNA methylation (Δβ) within all MZ twins discordant for ASD and ‘control’ MZ twin pairs concordant for low autistic trait score (unaffected). The overall distribution of average within-pair DNA methylation differences showed a highly significant skew to the right in ASD-discordant twins (P<2.2e−16, Kolmogorov–Smirnov test), with a higher number of CpG sites demonstrating a larger average difference in DNA methylation. Using an analysis method designed to identify the largest and most significant differences in DNA methylation at individual CpG sites, we identified multiple CpG sites across the genome exhibiting significant ASD-associated differential DNA methylation (Table 1). Of note, variability at these sites appears to be specific to ASD-discordant twin pairs; for the 50 top-ranked ASD-associated DMRs, we observe significantly higher average within-pair differences for MZ twin pairs discordant for ASD (P<0.01; see Figure 1b). The top differentially methylated site (cg13735974) across all ASD-discordant MZ twin pairs located in the NFYC promoter was consistently hypermethylated in affected individuals compared with their unaffected co-twins (mean Δβ=0.08, range=0.04–0.10, P<0.0004). For the top 10 DMRs, Figure 2 indicates highly consistent differences across all six ASD-discordant twin pairs.

Large DNA methylation differences are observed at specific loci within individual ASD-discordant MZ twin pairs

Because ASD is a highly heterogeneous disorder,3 it is probable that many disease-associated DMRs are family-specific. We therefore screened for the largest family-specific DNA methylation differences within each discordant ASD twin pair, identifying numerous loci (average=37.4 per twin pair) showing large DNA methylation differences (Δβ0.15) within each discordant twin pair (Supplementary Figure 5 and Supplementary Table 3). Although the majority of DMRs of large magnitude are family-specific, several are common across two or more discordant twin pairs in the same direction: cg12164282, located in PXDN promoter, showed ASD hypomethylation in twin pair 2 (Δβ=−0.19) and twin pair 4 (Δβ=−0.28); cg04545708, located in exon 1 of C11orf1, showed ASD hypermethylation in both twin pair 3 (Δβ=0.23) and twin pair 6 (Δβ=0.35); cg20426860, located in exon 1 of TMEM161A, showed ASD hypermethylation in twin pair 4 (Δβ=0.21) and twin pair 6 (Δβ=0.27); and cg27009703, located in HOXA9 promoter, showed ASD hypermethylation in twin pair 1 (Δβ=0.19) and twin pair 4 (Δβ=0.21).

DNA methylation differences are observed in MZ twins discordant for ASD-related traits

We detected significant DNA methylation differences between MZ twin pairs discordant for the three ASD-associated traits: that is, social autistic traits (N=9 MZ pairs), autistic RRBIs (N=9 MZ pairs) and communication autistic traits (N=8 MZ pairs). The top-ranked DMRs for each trait are shown in Supplementary Figure 6 and Supplementary Table 4. Interestingly, these included several genes previously implicated in the aetiology of ASD, including GABRB3, AFF2, NLGN2, JMJD1C, SNRPN, SNURF, UBE3A and KCNJ10.
As ASD is composed of a triad of all three impairments, we also examined if any CpG sites are differentially methylated across all discordant twin pairs (N=32 pairs, 64 individuals), regardless of their focal impairment. The top DMRs across all discordant twin pairs are shown in Supplementary Figure 7 and Supplementary Table 5. The top-ranked DMR located in the promoter region of PIK3C3 (cg19837131) was significantly hypomethylated in affected individuals compared with their unaffected co-twins (mean Δβ=−0.04, P<0.00004). Interestingly, while the overall average difference at this locus is small, the range of within-twin methylation difference is much greater (Δβ ranges from −0.12 to 0.6) and that the direction of effect is strikingly consistent across the majority of individual twin pairs, with 25 out of 32 discordant pairs (78%) demonstrating trait-related hypomethylation (Supplementary Figure 7).

Between-group analyses identified additional ASD-associated DMRs

Our study design also permitted us to examine group-level DNA methylation differences between ASD cases and controls. Unlike the within-pair discordant MZ twin design, between-group DNA methylation differences can be attributable to both genetic and environmental factors. Given the known gender difference in DNA methylation across the X chromosome, these analyses were restricted to probes on the autosomes (N=22 678) to minimize gender-induced biases.

Numerous DNA methylation differences were observed between ASD cases and controls. Supplementary Table 6a and Supplementary Figure 8 highlight the CpG sites showing the largest absolute DNA methylation differences (mean Δβ0.15) between ASD cases and unrelated control samples. The top case–control ASD-associated DMR was located upstream of MGC3207 (cg16474696), which was significantly hypomethylated in ASD cases compared with control samples (mean Δβ=−0.24, P<0.0002). In addition to MGC3207, large significant ASD-associated differences were observed in several other loci, including CpG sites near OR2L13 (cg20507276; mean Δβ=0.18) and C14orf152 (cg20022541; mean Δβ=−0.16, data not shown). Verification experiments were conducted on MGC3207 and OR2L13 using bisulphite-pyrosequencing confirming a high correlation (r=0.91 and 0.86, for MGC3207 (total N=33) and OR2L13 (total N=35), respectively) in DNA methylation levels, detected using the Infinium microarray and pyrosequencing platforms. Although our list of DMRs was stringently filtered to exclude probes containing known polymorphic SNPs,29 several of the top-ranked case–control DMRs, including cg16474696 and cg20507276, demonstrated patterns of DNA methylation consistent with DNA sequence effects, suggesting that they may be mediated by cis effects on DNA methylation34 or potentially reflect technical artefacts caused by uncatalogued sequence variation in probe binding sequences. To exclude the latter for MGC3207, we sequenced genomic DNA across the DMR in a range of samples showing differential methylation and identified no obvious polymorphic DNA sequence variation in the immediate vicinity of the probe.

Epigenetic differences identified between sporadic and familial ASD cases

ASD is an aetiologically heterogeneous syndrome and can occur both as a sporadic and a familial disorder. Recent CNV analyses report considerably higher frequencies of de novo variation in simplex compared with multiplex ASD families,35 suggesting that they represent genetically distinct classes. To test whether these are epigenetically distinct, we compared DNA methylation between individuals with sporadic ASD (where ASD is reported in only one member of the MZ twin pair; N=6) and individuals with familial ASD (as observed in concordant ASD MZ twin pairs; N=10). The genes most proximal to the 50 top-ranked differentially methylated CpG sites from this analysis are listed in Supplementary Table 6b. The top differentially methylated CpG site (cg07665060) is located upstream of C19orf33, which was significantly hypomethylated in individuals affected by sporadic ASD compared with those affected by familial ASD (mean Δβ=−0.12, P<0.0008) (Supplementary Figure 9). Interestingly, significant DNA methylation differences were also observed near several genes that have been previously implicated in ASD, including MBD4, AUTS2 and MAP2.

There is some overlap in DMRs across analytical groups

Table 2 provides a full list of top-ranked DMRs demonstrating overlap between analytical groups and highlighting their potential relevance to different autism-associated phenotypes. Interestingly, the top-ranked locus from the ASD-discordant twin analysis, located near NFYC, was also differentially methylated in the case–control analysis (mean Δβ=0.04, P<0.003). Furthermore, we identified significant DNA methylation differences in the MBD4 promoter in both ASD-discordant twin analysis and sporadic versus familial ASD analysis, suggesting that MBD4 methylation may have functional relevance to sporadic ASD. For each of the 50 top-ranked probes in each analysis category, Supplementary Table 7 lists their corresponding rank across the other analysis groups; although there is some overlap across groups (and each ranked list is positively, although modestly, correlated; Supplementary Table 8), few CpG sites are consistently altered across multiple analytical groups.

Quantitative autistic trait scores are correlated with DNA methylation at multiple CpG sites

Supplementary Figure 1 shows the distribution of total CAST and its three trait subscale scores across our samples. Initial analyses highlighted a strong correlation between DNA methylation and CAST score at multiple CpG sites (Supplementary Table 9). Further analysis showed that many of these correlations are influenced by extreme DNA methylation levels and phenotypic scores exhibited by one male ASD-concordant MZ twin pair (Figure 3a and Supplementary Figure 10). These twins are extreme outliers for CAST score (both scored 29 out of a maximum score of 31) and DNA methylation at multiple CpG sites (Supplementary Figure 11), and both have a history of pervasive developmental problems, with severe behavioural phenotypes and early-appearing IQ deficits, with special deficits in language. Given the existing link between highly penetrant CNVs and severe ASD, we tested whether the extreme patterns of DNA methylation in these two twins were associated with the presence of genomic alterations. Interestingly, high-density SNP microarray analysis revealed significant structural genomic alterations at multiple loci, with CNVs detected in regions previously implicated in ASD (Supplementary Table 10).

DNA methylation at multiple CpG sites remained significantly correlated with CAST scores even after this extreme twin pair was excluded from analyses (Supplementary Table 11 and Figure 3b), suggesting that they do not necessarily represent epigenetic/phenotypic ‘outliers’ but have DNA methylation levels (and phenotypic scores) at the extreme end of a true quantitative spectrum. For example, there is a strong correlation between DNA methylation at cg07753644 in P2RY11 and total CAST score in both analyses (with extreme twin pair: r=0.44; P=0.000009; without extreme twin pair: r=0.35; P=0.0006). Furthermore, DNA methylation at cg16279786 in the known ASD susceptibility locus, NRXN1, is significantly correlated with social autistic trait score in both analyses with (r=−0.41; P=0.00003) and without (r=−0.28; P=0.007) the extreme twin pair.

Discussion

This study represents the first comprehensive analysis of DNA methylation differences in MZ twins discordant for ASD and autism-related traits using a genome-wide approach. We report ASD-associated DNA methylation differences at numerous CpG sites, with some DMRs consistent across all discordant twin pairs for each diagnostic category and others specific to one or two twin pairs, or one or two autism-related traits. Although sporadic cases of ASD appear to be epigenetically distinct to familial cases of ASD, some DMRs are common across both discordant MZ twin and case–control analyses. We also observed that DNA methylation at multiple CpG sites was significantly correlated with quantitatively rated autistic trait scores, with our analyses identifying one MZ twin pair, concordant for a very severe autistic phenotype, that appear to represent epigenetic outliers at multiple CpG sites across the genome. Interestingly, both individuals harbour numerous CNVs in genomic regions previously implicated in autism. Given the important role of epigenetic mechanisms in regulating gene expression, it is plausible that, like CNVs, methylomic variation could mediate disease susceptibility via altered gene dosage. Our hypothesis-free experimental design allowed us to identify disease-associated DNA methylation differences at loci not previously implicated in ASD, although we also found evidence for epigenetic changes at several genes previously implicated in autism.

Our findings have several implications for our understanding about the aetiology of ASD. First, they document the presence of numerous DNA methylation differences in MZ twins discordant for ASD and ASD-related traits, as well as between autistic individuals and control samples. This concurs with findings from a previous ASD-discordant twin study23 and further supports the association of variable DNA methylation with phenotypic differences between genetically identical individuals.21, 22 Second, the observed DNA methylation differences in MZ twins discordant for ASD and ASD-related traits, who are otherwise matched for genotype, shared environment, age, sex and other potential confounders, highlight the role of non-shared environmental and stochastic factors in the aetiology of autism. These findings concur with mounting data suggesting that environmentally mediated effects on the epigenome may be relatively common and important for disease.36 Third, our data suggest that although DNA methylation at some CpG sites is consistently altered across the entire set of discordant twins, differences at other CpG sites are specific to certain symptom groups, with considerable overall epigenetic heterogeneity between the three domains of autistic traits. These findings are in line with recent genetic research demonstrating significant genetic heterogeneity between the three core symptoms of ASD.4, 6 Fourth, the analysis of individual ASD-discordant twin pairs suggests that there is also considerable familial heterogeneity, with rare epigenetic alterations of large magnitude being potentially associated with ASD. These findings are not entirely surprising given the known heterogeneous nature of ASD revealed by molecular genetic studies,3 with an important role for highly penetrant rare genomic alterations, especially de novo mutations. Fifth, the identification of significant correlations between DNA methylation and autism symptom scores across our sample cohort suggests that there is a quantitative relationship between the severity of the autistic phenotype and epigenetic variation at certain loci. This reinforces the view of autism as the quantitative extreme of a phenotypic spectrum and highlights the potential use of epigenetic biomarkers as a predictor for severity of symptoms, although the accuracy, sensitivity and specificity of such predictors would require extended investigation. Finally, in addition to implicating a number of novel genes in the aetiology of ASD, we identified ASD-associated differential DNA methylation in the vicinity of multiple loci previously implicated in the pathogenesis of autism in genetic studies, including AFF2, AUTS2, GABRB3, NLGN3, NRXN1, SLC6A4 and UBE3A (see Supplementary Table 12 for a comprehensive list).

This study has several strengths. First, our unique sample consisted of MZ twin pairs discordant for autism and ASD-related-traits, in addition to age-matched concordant MZ twin pairs (for both ASD and low CAST score). It allowed us to perform a comprehensive analysis of the role of DNA methylation in ASD and ASD-related traits controlling for genotype, age, sex and other potential confounders. Second, by undertaking a genome-wide approach using a robust and reliable array platform, we were able to uncover phenotype-relevant differentially methylated loci in genomic regions that are both novel and have been previously associated with ASD. Third, our analysis of 32 discordant MZ twin pairs is relatively large compared with other discordant twin studies performed for other complex disease phenotypes; in this regard, for example, the only other ASD-discordant twin study assessed only three MZ twin pairs.23 Finally, we were able to complement our discordant-twin analyses by assessing group-level differences between ASD cases and controls, and also examining the relationship between DNA methylation and quantitatively rated trait scores across our entire sample cohort.

This study also has a number of limitations that should be considered when interpreting the results. First, although this is the largest and most comprehensive study of epigenetic variation in ASD performed to date, the sample size for each subgroup is small, in part because truly discordant MZ twin pairs are relatively rare. Although none of the reported differentially methylated loci reached a Bonferroni-corrected P-value cutoff (P=2.13E–05 for discordant-twin analysis and P=2.20E–05 for between-group analysis), this statistical approach is likely to be too conservative, especially given the non-independence of CpG sites37 and the small numbers of samples tested in each group. In this study, a combined analytic approach, taking into account the significance and the extent of methylation change, was used to identify differentially methylated loci that have potentially real, biological relevance to ASD. This analytic approach is widely used in genome-wide gene expression studies and is reported to produce gene lists of higher reproducibility and biological relevance compared with the convention method that relies solely on statistical significance.30 This notion is supported by the identification of differentially methylated loci near numerous genes previously implicated in ASD. Nonetheless, given the relatively small subgroup sample size, replication in larger samples is needed. Second, genome-wide DNA methylation profiling was performed on DNA extracted from whole blood, controlled for cell count, rather than the brain. Unfortunately, there is no archived collection of post-mortem brain samples from ASD-discordant MZ twins. Although there are known tissue-specific differences in DNA methylation profiles, recent studies suggest that disease-associated epimutations may be detectable across tissues,38 and our recent work suggests that some between-individual epigenetic variation is conserved across brain and blood.39 Furthermore, ASD-associated epimutations have been demonstrated to be detectable both in the brain and in peripheral tissues (that is, blood).13, 23 Moreover, our identification of DMRs in the vicinity of genes previously implicated in autism supports the notion that disease-relevant gene network and pathways can be identified from peripheral samples. Nonetheless, it would be informative for future studies to assess whether disease-associated epimutations reported from this study are also present in brain samples from ASD patients. Third, informations pertaining to the amniotic and chorionic status of our twin samples are unavailable, preventing us from further dissecting the epigenetic similarity/dissimilarity between twins sharing their placenta and/or amniotic sac. Fourth, the genome-wide platform used for this study (the Illumina 27K array), although robust and highly reliable,28 has a somewhat limited density of probe coverage, assaying only one or two CpG sites per gene. Future studies should take advantage of recent advances in genomic profiling technology and perform a more in-depth examination of methylomic differences associated with ASD. Finally, it is difficult to draw conclusions about causality for any of the ASD-associated DMRs identified in this study, in part, because we do not have corresponding RNA expression data, or DNA samples from the twins taken before they became discordant for ASD. It is thus plausible that many of the identified changes have occurred downstream of ASD, for example, resulting from exposure to medications commonly used to treat autistic symptoms. In fact, there is mounting evidence that many drugs used to treat neuropsychiatric disorders induce epigenetic changes.40 Such medication-induced changes could still be interesting; an understanding of the pathways via which these drugs work may provide information about the neurobiological processes involved in disease. The ideal study design, however, would assess DNA methylation changes in the brain longitudinally during individuals’ transition into ASD, although such a study does not appear feasible at present.

In summary, this is the first large-scale study to examine the role of genome-wide DNA methylation in ASD and ASD-related traits. Our findings show that: (1) there are numerous DNA methylation differences between MZ twins discordant for ASD and ASD-related traits, as well as between autistic individuals and control samples; (2) many of these DMRs are located in the vicinity of both novel genes and loci that have been previously implicated in ASD; (3) the nature of ASD-associated epimutations is complex with high heterogeneity between individuals; (4) there is high epigenetic heterogeneity between the triad of impairments that define ASD; and (5) there is a quantitative relationship between the severity of the autistic phenotype and DNA methylation at specific CpG sites across the genome. Overall, our findings from this study provide further support for the potential role of DNA methylation in ASD and ASD-related traits.

To review the figures you can access the full study here http://www.nature.com/mp/journal/vaop/ncurrent/full/mp201341a.html
Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits

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Some women actually have men on the brain By Melissa Healy, Los Angeles Times
http://www.latimes.com/health/boostershots/la-heb-women-brain-microchimerism-20120926,0,6446716.story
For the Booster Shots Blog
September 27, 2012

For decades after a woman has carried a male child in her womb or shared her mother’s womb with a brother, she carries a faint but unmistakable echo of that intimate bond: male fetal DNA that lodges itself in the far recesses of her brain.

That astonishing finding, published Wednesday in the journal Public Library of Science One (PLoS One), suggests that the act of having a child is no mere one-way transmission of genetic material and all that goes with it: There is an exchange of DNA that passes into the part of us that makes us who we are. That, in turn, may alter a woman’s health prospects in ways her own DNA never intended.

In the study, researchers from the Fred Hutchinson Cancer Research Center and the University of Washington examined, post-mortem, the brains of 59 women. In 63% of the brains, they found fetal DNA that could only have come from a male. While scattered throughout the brain, the genetic traces of this other individual were clustered heavily in the brain’s hippocampus — a region crucial to the consolidation of memories — and in the parietal and temporal lobes of the brain’s prefrontal cortex, areas that play roles in sensation, perception, sensory integration and language comprehension.

When a person takes on the DNA of another, as happens, for instance, in bone marrow transfusions, she is called a “chimera” — in mythology, a beast that is the fusion of two or more creatures. The discovery that a person can carry the fetal DNA of another person has given rise to a variant: This is dubbed microchimerism.

This line of research, says rheumatologist J. Lee Nelson, coauthor of the study, “suggests we need a new paradigm of the biological self” and how it is formed. We think of ourselves as the product of two biological parents and a one-time roll of the genetic dice. That, says Nelson, appears to be wrong: In the womb, we may also pick up the DNA of older siblings left over from their stay, or of a fetal twin who never made it to daylight. In the course of our lives, we may take on the DNA of the sons we bear, or even of the sons we conceived and miscarried. And that DNA can stay with us long after our big brothers have moved on and our sons have grown up and moved away.

The sources of our DNA “are much more diverse than we know,” said Nelson in an interview. And these exchanges of DNA may play an evolutionary role far greater than we have ever imagined, she added. Walt Whitman once wrote, “I contain multitudes,” and Nelson says she and her colleagues now glean new meanings from the observation.

The new study shows that this evolutionary X-factor is also at work in the brain.

It hasn’t been many years since scientists first learned that a baby’s DNA could cross the placental barrier from baby to mother and lodge itself in her blood and organs. The current study finds that it can also penetrate the vaunted “blood-brain barrier,” which is thought to protect the brain from toxins and foreign invaders.

Once there, Nelson said, the DNA of another person may alter a woman’s propensity to certain brain diseases — conferring protection in some cases and vulnerability in others. It may carry the switches that turn brain cancers on — or off. It may harden the brain against trauma or psychiatric disease — or make it less resilient. Future research will need to determine how, say, carrying a male fetus may influence a mother’s likelihood of developing Alzheimer’s disease or auto-immune diseases such as multiple sclerosis.

http://www.latimes.com/health/boostershots/la-heb-women-brain-microchimerism-20120926,0,6446716.story

Male Microchimerism in the Human Female Brain
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045592

William F. N. Chan1¤*, Cécile Gurnot1, Thomas J. Montine2, Joshua A. Sonnen2, Katherine A. Guthrie1, J. Lee Nelson1,3
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America, 2 Department of Pathology, University of Washington, Seattle, Washington, United States of America, 3 Division of Rheumatology, University of Washington, Seattle, Washington, United States of America

Abstract

In humans, naturally acquired microchimerism has been observed in many tissues and organs. Fetal microchimerism, however, has not been investigated in the human brain. Microchimerism of fetal as well as maternal origin has recently been reported in the mouse brain. In this study, we quantified male DNA in the human female brain as a marker for microchimerism of fetal origin (i.e. acquisition of male DNA by a woman while bearing a male fetus). Targeting the Y-chromosome-specific DYS14 gene, we performed real-time quantitative PCR in autopsied brain from women without clinical or pathologic evidence of neurologic disease (n = 26), or women who had Alzheimer’s disease (n = 33). We report that 63% of the females (37 of 59) tested harbored male microchimerism in the brain. Male microchimerism was present in multiple brain regions. Results also suggested lower prevalence (p = 0.03) and concentration (p = 0.06) of male microchimerism in the brains of women with Alzheimer’s disease than the brains of women without neurologic disease. In conclusion, male microchimerism is frequent and widely distributed in the human female brain.

Introduction

During pregnancy, genetic material and cells are bi-directionally exchanged between the fetus and mother [1], following which there can be persistence of the foreign cells and/or DNA in the recipient [2], [3]. This naturally acquired microchimerism (Mc) may impart beneficial or adverse effects on human health. Fetal Mc, which describes the persistence of cells and/or DNA of fetal origin in the mother acquired during pregnancy, has been associated with several different autoimmune diseases as well as implicated in tissue repair and immunosurveillance [4]–[6].

Although there is a broad anatomical distribution of Mc in humans that varies in prevalence and quantity [7]–[13], whether the human brain harbors fetal Mc and with what frequency is not known. Fetal Mc has recently been described in the mouse brain [14], [15]. In limited studies, maternal Mc was described in the human fetal brain [9].

In this study, we performed real-time quantitative PCR (qPCR) to detect and quantify male DNA in multiple brain regions of women, targeting the Y-chromosome-specific DYS14 gene sequence as a marker for Mc of fetal origin. Deceased female subjects had no clinical or pathologic evidence of neurologic disease. We also tested brain specimens from women with Alzheimer’s disease (AD) for Mc. This is because AD has been reported as more prevalent in parous vs. nulliparous women [16], [17], increasing with higher number of pregnancies that also correlated with a younger age of AD onset [17], [18].

Methods

Subjects and Specimens

This study was approved by the institutional review board of the Fred Hutchinson Cancer Research Center (Number 5369; Protocol 1707). Subjects of the study were women without neurologic disease or with AD, totaling 59 deceased individuals. Twenty-six women had no neurologic disease. Thirty-three women had AD (Table 1). Brain autopsy specimens from these women came from one of two institutions: the Department of Pathology at the University of Washington in Seattle, Washington, or the Harvard Brain Tissue Resource Center established at McLean Hospital in Belmont, Massachusetts. Specimens from the University of Washington were obtained from adult women who had no clinical history of neurologic disease within two years of death and whose brain histology showed no evidence of disease, and from women who were diagnosed with probable AD during life [19] and met the National Institute on Aging-Reagan Institute consensus criteria for a neuropathologic diagnosis of AD [20]. Similarly, specimens from the Harvard Brain Tissue Resource Center were obtained from adult women without neurologic disease or who met clinical and pathologic criteria for AD. Age at death ranged from 32 to 101 (Table 1). Age at disease onset was known for subjects with AD from the University of Washington (median: 77 years; range: 64–93 years). Following autopsy, brain specimens were either formalin fixed or frozen in liquid nitrogen. Depending on availability, samples from two to twelve brain regions were obtained from each subject. Brain regions investigated included frontal lobe, parietal lobe, temporal lobe, occipital lobe, cingulate gyrus, hippocampus, amygdala, caudate, putamen, globus pallidus, thalamus, medulla, pons, cerebellum, and spinal cord. Subjects with AD contributed more specimens per person than subjects without neurologic disease, but this was not statistically significant (means: 3.6 vs. 2.5, respectively; p = 0.05). Combining subjects from both institutions, subjects with AD were significantly older at death (p<0.001); the post-mortem intervals (PMIs) were not significantly different (p = 0.06; Table 1). The most likely source of male Mc in female brain is a woman’s acquisition of male DNA from pregnancy with a male fetus. Limited pregnancy history was available on the subjects; pregnancy history on most subjects was unknown. Nine women were known to have at least one son, eight with AD and one without neurologic disease. Two women were known to have no history of having sons, one with AD and one without neurologic disease.

DNA Extraction

Genomic DNA was extracted from brain tissues using the QIAamp® DNA Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s tissue protocol.

Real-time qPCR

Male DNA was quantified in female brain tissues by amplifying the Y chromosome-specific sequence DYS14 (GenBank Accession X06325) [21] using the TaqMan® assay and the ABI Prism® 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Primer and probe sequences for quantifying DYS14 [22], as well as preparation of standard curves, composition of the qPCR mixture and thermal profile [23] have all been described previously. Square of the correlation coefficient for all standard curves was always greater than 0.99. Every experiment included no template controls to test for male DNA contamination during plate setup and all controls were negative. A minimum of six wells was tested for each specimen. Mean Ct was 36, with a range between 30 and 39 for all specimens except those of B6388, which was between 26 and 29. A representative amplification plot is provided in (Figure S1). Only wells in which amplification occurred at Ct0.5 gEq/105. Thus, estimates of male Mc might be lower than the true values. On the other hand, since detection of male DNA did not account for Mc potentially contributed by female fetuses, this could result in underestimation of the overall Mc in the brain. HLA-specific qPCR, as previously reported [25], [26], is another approach to Mc detection that is not sex-dependent. It requires participation of family members which was not possible for the current studies. As a supplementary study, we tested autopsied brain from a female systemic sclerosis patient by HLA-specific qPCR for whom we had familial HLA genotyping, targeting the child’s paternally transmitted HLA as previously described [26], [27]. These data are provided in (Tables S1 and S2). All qPCR data were analyzed using the 7000 System Sequence Detection Software.

Statistics

Subject and Mc measurement characteristics were compared across groups by Chi-squared test for categorical data and t-test for continuous data. Mc prevalence and concentrations were analyzed according to disease status. A logistic regression model was used to estimate the association between Mc prevalence and disease status, with adjustment for total gEq tested, age at death, and PMI. The estimates were also adjusted for possible correlation between repeated measures from the same subject via generalized estimating equations. Association was reported as an odds ratio (OR) along with p value to indicate significance. As an example, OR of 0.30 could be interpreted to say that the odds of having AD for a subject who tested positive for Mc was 70% lower than the odds for a subject who tested negative. We also analyzed Mc concentrations as the outcome in Poisson log-linear regression models, assuming that the number of gEq detected as Mc was directly proportional to the number of total gEq tested. By definition, Mc occurs rarely, thus the data distribution is skewed to the right. We utilized negative-binomial models to account for the high degree of over-dispersion in the data; interpretation of the resulting estimates is identical to those of a Poisson model. Adjustments for potential confounders and for possible correlation between repeated measures were made as described above. The rate ratio (RR), along with p value to indicate significance, was used to compare the observed rates of Mc detection in the two groups. As an example, RR of 0.30 could be interpreted to say that the rate of Mc detection in subjects with AD was 70% lower than the rate of Mc detection in subjects without neurological disease. Secondary analysis was conducted to determine whether disease status was associated with Mc prevalence or concentration in a subset of samples from brain regions thought to be most affected by AD. Furthermore, we investigated whether Mc prevalence or concentration correlated with the Braak stage, which describes the extent of neurofibrillary tangles in subjects with AD [28], or with HLA-DRB1*1501, a human leukocyte antigen allele that has been reported in association with AD [29]. Two-sided p-values from regression models were derived from the Wald test. Analyses were performed on SAS software version 9 (SAS Institute, Inc., Cary, NC).

Results

Mc Prevalence and Concentration According to Brain Regions

he median number of specimens tested per subject was three, with a range of one to 12. Table 2 summarizes the specimen-level prevalence of male Mc according to brain region for all subjects. Per brain region, between two and 35 specimens were tested for male DNA. Although there were few specimens available, we did not detect male DNA in the frontal lobe and the putamen, and found the highest prevalence in the medulla. Considering all subjects together, Mc concentrations ranged from 0–512.5 gEq/105, with a median value of 0.2 and a 90th percentile of 3.7 gEq/105 (Figure 1). One subject from the Harvard Brain Tissue Resource Center who was without neurologic disease (coded as B6388; age of death 69 years) had three specimens with the highest concentration values in our dataset (296.1, 481.8, and 512.5 gEq/105 in the temporal lobe, cingulate gyrus, and pons, respectively). Using fluorescence in situ hybridization, we indeed found rare male cells in the brain of B6388 (Figure S2). The remaining concentration values in the dataset were 29.4 gEq/105 or less. Regarding the relationship between pregnancy history and Mc prevalence, five of nine subjects who were known to have at least one son harbored male Mc in at least one of their brain regions (Table S3). All positive individuals had AD; among the negatives were three with AD and one without neurologic disease. One of two women without history of having sons was also positive for male Mc in her brain and without neurologic disease; the negative individual had AD.

Prevalence and Concentration of Male Mc in Human Brain: Women without Neurologic Disease or with AD

Of 183 specimens, 64 (35%) tested positive for Mc (Table 2). Eighteen of 26 subjects without neurologic disease (69%) had at least one positive value, with 30 positive results in 65 specimens (46%). Nineteen of 33 subjects with AD (58%) had at least one positive value, with 34 positive results in 118 specimens (29%). The estimated OR from a univariate model was 0.47 (95% confidence interval (CI) 0.21–1.08, p = 0.08). After adjustment for total gEq tested, age at death, and PMI, AD was significantly associated with lower Mc prevalence: OR 0.40 (95% CI 0.17–0.93, p = 0.03). Thus, the odds of having AD for a subject who tested positive for Mc was 60% lower than the odds for a subject who tested negative. When Mc concentrations were analyzed according to whether subjects had no neurologic disease or had AD, the estimated RR from an adjusted model was 0.05 (95% CI 0.01–0.39, p = 0.004). However, exclusion of brain specimens from subject B6388, who was without neurologic disease and whose level of male Mc was 10-fold greater than the next highest concentration from a different subject, changed the estimate dramatically: RR 0.41 (95% CI 0.16–1.05, p = 0.06). Thus, the rate of Mc detection in subjects with AD was 59% lower than the rate of Mc detection in subjects without neurological disease, but was not statistically significant. Age at death was also not statistically significantly associated with Mc prevalence, either in univariate or adjusted models (adjustments for disease status and total gEq tested; p = 0.79). However, any relationship between age at death and male Mc from prior pregnancies with a male fetus could not be evaluated because pregnancy history and the time interval from pregnancies to death were generally unknown from our subjects.

Prevalence and Concentration of Male Mc in Brain Regions Affected by AD

We conducted a secondary analysis considering specimens only from the five brain regions thought to be most affected by AD: amygdala, hippocampus, frontal lobe, parietal lobe, and temporal lobe [30], [31]. Considering only these regions, 12 of 24 subjects without neurologic disease (50%) had at least one positive value, with 12 positive results in 24 specimens (50%). Thirteen of 33 subjects with AD (39%) had at least one positive value, with 15 positive results in 44 specimens (34%). The adjusted OR describing the association of Mc prevalence and disease status was 0.48 (95% CI 0.14–1.62, p = 0.23). Therefore, the odds of having AD for a subject who tested positive for Mc in brain regions most affected by this disease was 52% lower than the odds for a subject who tested negative, but was not statistically significant. However, none of the subjects without neurologic disease contributed specimens of the amygdala or the frontal lobe. Comparing Mc concentrations across groups, excluding one specimen from subject B6388, the adjusted RR was 0.27 (95% CI 0.13–0.56, p<0.001). Thus, the proportion of positive specimens was not significantly different between groups, but Mc concentrations in this subset of brain specimens from subjects with AD tended to have lower values than those found in subjects without neurologic disease. In other analyses, there was no significant association between Mc prevalence or concentration and the Braak stage (Table 1; p = 0.99 and 0.93, respectively), and no significant association between Mc prevalence and HLA-DRB1*1501 (8 of 11 DR15-bearing subjects positive for Mc (73%) vs. 16 of 31 subjects without DR15 who also had Mc (52%); p = 0.13).

Discussion

n this study, we provide the first description of male Mc in female human brain and specific brain regions. Collectively with data showing the presence of male DNA in the cerebrospinal fluid [32], our results indicate that fetal DNA and likely cells can cross the human blood-brain barrier (BBB) and reside in the brain. Changes in BBB permeability occur during pregnancy [33] and may therefore provide a unique opportunity for the establishment of Mc in the brain. Also unique to our study are the findings that male Mc in the human female brain is relatively frequent (positive in 63% of subjects) and distributed in multiple brain regions, and is potentially persistent across the human lifespan (the oldest female in whom male DNA was detected in the brain was 94 years).

That Mc can penetrate the human BBB and reside in the brain was first indicated by murine studies that showed the presence of both foreign cells and DNA in mouse brains [14], [15]. However, prevalence of brain Mc in mice has not been well defined, as the frequency reported varies depending on the study [14], [15], [34]–[36], and in one investigation, Mc was not observed [37]. Similar to mouse data, our study of humans found that brain Mc was not present in all individuals tested. Even in those who showed positivity overall, not all of their brain regions had Mc. Mc concentration also showed considerable variability. Overall, our data complement and extend on other reports describing Mc in the general human population, in peripheral blood and at the level of the tissue/organ studied within and between subjects [9]–[13]. It is currently not possible to meaningfully compare Mc prevalence or concentration in human brain to other tissues because other tissues were not available from our subjects. Moreover, prior studies that evaluated Mc in other organs used diverse methods, some of which were not quantitative.

The most likely source of male Mc in female brain is acquisition of fetal Mc from pregnancy with a male fetus. In women without sons, male DNA can also be acquired from an abortion or a miscarriage [22], [23], [38]–[40]. The pregnancy history was unknown for all but a few subjects in the current studies, thus male Mc in female brain could not be evaluated according to specific prior pregnancy history. In addition to prior pregnancies, male Mc could be acquired by a female from a recognized or vanished male twin [41]–[43], an older male sibling, or through non-irradiated blood transfusion [44].

Because AD is more prevalent in women than men and an increased risk has been reported in parous vs. nulliparous women and correlated with younger age of onset [16]–[18], we also investigated male Mc in women with AD. AD is a neurodegenerative disease characterized by elevated levels of amyloid plaques, cerebrovascular amyloidosis, and neurofibrillary tangle [30]. Our results suggesting women with AD have a lower prevalence of male Mc in the brain and lower concentrations in regions most affected by AD were unexpected. However, the number of subjects tested was modest and, as discussed previously, pregnancy history was largely unknown. The explanation for decreased Mc in AD, should this observation be replicated in a larger study, is not obvious. In other diseases, both beneficial and detrimental effects of Mc of fetal origin have been described depending on several factors including the specific type and source of Mc [6]. A significant limitation of the current study was the inability to distinguish the type and source of male Mc, and further studies that distinguish genetically normal from abnormal Mc would be of potential interest.

At present, the biological significance of harboring Mc in the human brain requires further investigation. Mc appears to persist in the blood, bone, and bone marrow for decades [2], [45] and is present among different hematopoietic lineages [46]. Moreover, Mc appears to integrate and generate specific cell types in tissues [10], [11], [47]–[49]. In murine studies, fetal Mc in the maternal brain has been observed to resemble perivascular macrophages, neurons, astrocytes, and oligodendrocytes both morphologically and phenotypically and occupy the respective niches [15], [36]. Thus, it is possible that Mc in the brain is able to differentiate into various mature phenotypes or undergoes fusion with pre-existing cells and acquires a new phenotype, as suggested by murine and human studies in which bone marrow-derived cells circulated to the brain and generated neuronal cells by differentiation, or fused with pre-existing neurons [50]–[53]. Lastly, a few studies have reported an association between parity and decreased risk of brain cancer, raising the possibility that Mc could contribute to immunosurveillance against tumorigenic cells as has been suggested for some other types of malignancy [6], [54]–[56].

In conclusion, male Mc is frequent and widely distributed in the human female brain. Although the relationship between brain Mc and health versus disease requires further study, our findings suggest that Mc of fetal origin could impact maternal health and potentially be of evolutionary significance.

For more on this important study http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0045592

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The Man Working To Reverse-Engineer Your Brain – February 29, 2012 NPR
http://www.npr.org/2012/02/29/147190092/the-man-working-to-reverse-engineer-your-brain

Our brains are filled with billions of neurons, entangled like a dense canopy of tropical forest branches. When we think of a concept or a memory — or have a perception or feeling — our brain’s neurons quickly fire and talk to each other across connections called synapses.

How these neurons interact with each other — and what the wiring is like between them — is key to understanding our identity, says Sebastian Seung, a professor of computational neuroscience at MIT.

Seung’s new book, Connectome: How the Brain’s Wiring Makes Us Who We Are, explains how mapping out our neural connections in our brains might be the key to understanding the basis of things like personality, memory, perception and ideas, as well as illnesses that happen in the brain, like autism and schizophrenia.

“These kinds of disorders have been a puzzle for a long time,” says Seung. “We can look at other brain diseases, like Alzheimer’s disease and Parkinson’s disease, and see clear evidence that there is something wrong in the brain.”

But with schizophrenia and autism, there’s no clear abnormality during autopsy dissections, says Seung.

“We believe these are brain disorders because of lots of indirect evidence, but we can’t look at the brain directly and see something is wrong,” he says. “So the hypothesis is that the neurons are healthy, but they are simply connected together or organized in an abnormal way.”

One current theory, says Seung, is that there’s a connection between the wiring that develops between neurons during early infancy and developmental disorders like schizophrenia and autism.

“In autism, the development of the brain is hypothesized to go awry sometime before age 2, maybe in the womb,” he says. “In schizophrenia, no one knows for sure when the development is going off course. We know that schizophrenia tends to emerge in early adulthood, so many people believe that something abnormal is happening during adolescence. Or it could be that something is happening much earlier and it’s not revealed until you become an adult.”

What scientists do know, he says, is that the wiring of the brain in the first three years is critical for development. Infants born with cataracts in poor countries that don’t have the resources to restore their eyesight remain blind even after surgery is performed on them later in life.

“No matter how much they practice seeing, they can never really see,” says Seung. “They recover some visual function, but they are still blind by comparison to you and me. And one hypothesis is that the brain didn’t wire up properly when they were babies, so by the time they become adults, there’s no way for the brain to learn how to see properly.”

At birth, he says, you are born with all of the neurons you will ever have in life, except for neurons that exist in two specific areas of the brain: the dentate gyrus of the hippocampus, which is thought to help new memories form, and the olfactory bulb, which is involved in your sense of smell.

“The obvious hypothesis [is] that these two areas need to be highly plastic and need to learn more than other regions, and that’s why new neurons have to be created — to give these regions more potential for learning,” says Seung. “But we don’t really have any proof of that hypothesis.”

But not everything is set in stone from birth. The complex synaptic connections that allow neurons to communicate with one another develop after babies have left the womb.

“As far as we know, this is happening throughout your life,” he says. “Part of the reason that we are lifelong learners — that no matter how old you get, you can still learn something new — may be due to the fact that synapse creation and elimination are both continuing into adulthood.”

Connectomes: Reverse-Engineering The Brain

Only one organism has had its full connectome — or neural map — mapped out by neuroscientists. It’s a tiny worm no bigger than a millimeter, but it took scientists more than a dozen years to map out its 7,000 neural connections. They started out by using the world’s most powerful knife and slicing the worm into slices a thousand times thinner than a human hair. They then put each slice in an electron microscope and created a 3-D image of the worm’s nervous system. That’s when the true labor started, says Seung.

“That’s when [neuroscientists had to] go through all these images and trace out the paths taken by all of the branches of the neurons and find the synapses, and compile all that information to create the connectome,” he says.

Each of the worm’s 300 neurons had between 20 and 30 connections. In comparison, humans have 10,000 connections of neurons — and billions of neurons. And scientists still aren’t sure what the various pathways in a worm’s nervous system mean.

“We’re still far away from understanding the worm,” says Seung. He says that scientists would like to eventually map a 1-millimeter cube of a human brain or a mouse brain, which contains 100,000 neurons and a billion connections.

“The imaging of all of those slices of brain can be automated and made much more reliable,” he says. “And now we have computers that are getting better at seeing.”

So far, though, neuroscientists have only mapped the neural connections of a piece of a mouse retina, which is very thin.

“What we know in the retina is a catalog of the types of neurons,” he says. “The next challenge is to figure out what are the rules of connection between these types of neurons. And that’s where we still don’t know a whole lot.”

Mapping more of these connections, he says, will tell us a lot about brain function and possible pathways that can be treated.

“I don’t want to promise too much, and my goal right now is simply to see what is wrong,” he says. “That’s not in itself a cure. But obviously it’s a step toward finding better treatments. The analogy I make is the study of infectious diseases before the microscope. You could see the symptoms, but you couldn’t see the microbes — the bacteria that caused disease. We’re in an analogous stage with mental disorders. We see the symptoms, but we don’t have a clear thing we can look at in the brain and say, ‘This is what’s wrong.’ ”

http://www.npr.org/2012/02/29/147190092/the-man-working-to-reverse-engineer-your-brain

Bernard Weiss
http://www2.envmed.rochester.edu/envmed/tox/faculty/weiss.html

Neurobiology and Behavior

Our brains, the ultimate product of millions of years of evolution, are what make us human. But over the past few decades, scientists have discovered that many chemicals in our environment threaten the integrity of our brains. Thousands more have never been studied for their effects. We know some of the outcomes: reduced intelligence and cognitive function, increased antisocial tendencies, impaired senory and motor function, and elevated risks of neurodegenerative disorders such as Parkinson’s disease.

Most of these chemicals are ubiquitous and persistent. We are exposed throughout our lifetimes. But some periods of life are more vulnerable than others. Early development is an especially perilous time for exposure to toxic chemicals. The brain is exquisitely sensitive during this period because of the many paths by which it expands and differentiates on the path to maturity. Cells divide and proliferate; they migrate to specific target areas; they grow connections to other cells to form massive neural networks; neurotransmitter systems take root. All these processes are candidates for interference by toxic chemicals. All are reflected in neurobehavioral outcomes that can be measured when organisms mature to a stage at which they can be tested by procedures that are sensitive to such interference. Late in life, we enter another period of enhanced vulnerability. We are not as able as during earlier periods to compensate for toxic processes and many of our organ systems operate at diminished capacity. It is also a period when these reduced capacities may begin to reflect the damage inflicted earlier in life.

My own research aims to relate behavioral measures to neurotoxicant exposure. Behavioral research occupies a special role in safety assessment because it offers the ability to trace changes in function as organisms mature and age. Among the endpoints of salient interest to regulatory agencies such as EPA and to chemical and pharmaceutical manufacturers are learning capacity, other aspects of cognitive capacity, motor and sensory performance, and differences between males and females.

My efforts have spanned a variety of agents: metals such as mercury and manganese; solvents such as toluene and methanol; air pollutants such as ozone; adventitious contaminants such as dioxin; and endocrine disruptors, which include common ingredients in consumer products such as phthalates.

For more on Bernard Weiss and his research see below.

School of Medicine & Dentistry
Molecular Toxicology & Environmental Medicine Cluster
Ph.D. Program in Toxicology

Bernard Weiss
Professor of Environmental Medicine
and Pediatrics;
Environmental Health Sciences Center,
and Center for Reproductive Epidemiology.
B.A. 1949 (New York University)
Ph.D. 1953 (University of Rochester)

http://www2.envmed.rochester.edu/envmed/tox/faculty/weiss.html

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