Lorenzo’s Oil for Nervous Breakdowns

 

A Happy New Year to all our readers – and indeed to anyone who isn’t a member of that merry band!

What better way to start than with a salute to the miracles of modern science by talking about how the lives of a group of young boys have been saved by one such miracle.

However, as is almost always the way in science, this miraculous moment is merely the latest step in a long journey. In retracing those steps we first meet a wonderful Belgian – so, when ‘name a famous Belgian’ comes up in your next pub quiz, you can triumphantly produce him as a variant on dear old Eddy Merckx (of bicycle fame) and César Franck (albeit born before Belgium was invented). As it happened, our star was born in Thames Ditton (in 1917: his parents were among the one quarter of a million Belgians who fled to Britain at the beginning of the First World War) but he grew up in Antwerp and the start of World War II found him on the point of becoming qualified as a doctor at the Catholic University of Leuven. Nonetheless, he joined the Belgian Army, was captured by the Germans, escaped, helped by his language skills, and completed his medical degree.

Not entirely down to luck

This set him off on a long scientific career in which he worked in major institutes in both Europe and America. He began by studying insulin (he was the first to suggest that insulin lowered blood sugar levels by prompting the liver to take up glucose), which led him to the wider problems of how cells are organized to carry out the myriad tasks of molecular breaking and making that keep us alive.

The notion of the cell as a kind of sac with an outer membrane that protects the inside from the world dates from Robert Hooke’s efforts with a microscope in the 1660s. By the end of the nineteenth century it had become clear that there were cells-within-cells: sub-compartments, also enclosed by membranes, where special events took place. Notably these included the nucleus (containing DNA of course) and mitochondria (sites of cellular respiration where the final stages of nutrient breakdown occurs and the energy released is transformed into adenosine triphosphate (ATP) with the consumption of oxygen).

In the light of that history it might seem a bit surprising that two more sub-compartments (‘organelles’) remained hidden until the 1950s. However, if you’re thinking that such a delay could only be down to boffins taking massive coffee breaks and long vacations, you’ve never tried purifying cell components and getting them to work in test-tubes. It’s a process called ‘cell fractionation’ and, even with today’s methods, it’s a nightmare (sub-text: if you have to do it, give it to a Ph.D. student!).

By this point our famous Belgian had gathered a research group around him and they were trying to dissect how insulin worked in liver cells. To this end they (the Ph.D. students?!) were using cell fractionation and measuring the activity of an enzyme called acid phosphatase. Finding a very low level of activity one Friday afternoon, they stuck the samples in the fridge and went home. A few days later some dedicated soul pulled them out and re-measured the activity discovering, doubtless to their amazement, that it was now much higher!

In science you get odd results all the time – the thing is: can you repeat them? In this case they found the effect to be absolutely reproducible. Leave the samples a few days and you get more activity. Explanation: most of the enzyme they were measuring was contained within a membrane-like barrier that prevented the substrate (the chemical that the enzyme reacts with) getting to the enzyme. Over a few days the enzyme leaked through the barrier and, lo and behold, now when you measured activity there was more of it!

Thus was discovered the ‘lysosome’ – a cell-within-a cell that we now know is home to an array of some 40-odd enzymes that break down a range of biomolecules (proteinsnucleic acidssugars and lipids). Our self-effacing hero said it was down to ‘chance’ but in science, as in other fields of life, you make your own luck – often, as in this case, by spotting something abnormal, nailing it down and then coming up with an explanation.

In the last few years lysosomes have emerged as a major player in cancer because they help cells to escape death pathways. Furthermore, they can take up anti-cancer drugs, thereby reducing potency. For these reasons they are the focus of great interest as a therapeutic target.

Lysosomes in cells revealed by immunofluorescence.

Antibody molecules that stick to specific proteins are tagged with fluorescent labels. In these two cells protein filaments of F-actin that outline cell shape are labelled red. The green dots are lysosomes (picked out by an antibody that sticks to a lysosome protein, RAB9). Nuclei are blue (image: ThermoFisher Scientific).

Play it again Prof!

In something of a re-run of the lysosome story, the research team then found itself struggling with several other enzymes that also seemed to be shielded from the bulk of the cell – but the organelle these lived in wasn’t a lysosome – nor were they in mitochondria or anything else then known. Some 10 years after the lysosome the answer emerged as the ‘peroxisome’ – so called because some of their enzymes produce hydrogen peroxide. They’re also known as ‘microbodies’ – little sacs, present in virtually all cells, containing enzymatic goodies that break down molecules into smaller units. In short, they’re a variation on the lysosome theme and among their targets for catabolism are very long-chain fatty acids (for mitochondriacs the reaction is β-oxidation but by a different pathway to that in mitochondria).

Peroxisomes revealed by immunofluorescence.

As in the lysosome image, F-actin is red. The green spots here are from an antibody that binds to a peroxisome protein (PMP70). Nuclei are blue (image: Novus Biologicals)

Cell biology fans will by now have worked out that our first hero in this saga of heroes is Christian de Duve who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and George Palade.

A wonderful Belgian. Christian de Duve: physician and Nobel laureate.

Hooray!

Fascinating and important stuff – but nonetheless background to our main story which, as they used to say in The Goon Show, really starts here. It’s so exciting that, in 1992, they made a film about it! Who’d have believed it?! A movie about a fatty acid!! Cinema buffs may recall that in Lorenzo’s Oil Susan Sarandon and Nick Nolte played the parents of a little boy who’d been born with a desperate disease called adrenoleukodystrophy (ALD). There are several forms of ALD but in the childhood disease there is progression to a vegetative state and death occurs within 10 years. The severity of ALD arises from the destruction of myelin, the protective sheath that surrounds nerve fibres and is essential for transmission of messages between brain cells and the rest of the body. It occurs in about 1 in 20,000 people.

Electrical impulses (called action potentials) are transmitted along nerve and muscle fibres. Action potentials travel much faster (about 200 times) in myelinated nerve cells (right) than in (left) unmyelinated neurons (because of Saltatory conduction). Neurons (or nerve cells) transmit information using electrical and chemical signals.

The film traces the extraordinary effort and devotion of Lorenzo’s parents in seeking some form of treatment for their little boy and how, eventually, they lighted on a fatty acid found in lots of green plants – particularly in the oils from rapeseed and olives. It’s one of the dreaded omega mono-unsaturated fatty acids (if you’re interested, it can be denoted as 22:1ω9, meaning a chain of 22 carbon atoms with one double bond 9 carbons from the end – so it’s ‘unsaturated’). In a dietary combination with oleic acid  (another unsaturated fatty acid: 18:1ω9) it normalizes the accumulation of very long chain fatty acids in the brain and slows the progression of ALD. It did not reverse the neurological damage that had already been done to Lorenzo’s brain but, even so, he lived to the age of 30, some 22 years longer than predicted when he was diagnosed.

What’s going on?

It’s pretty obvious from the story of Lorenzo’s Oil that ALD is a genetic disease and you will have guessed that we wouldn’t have summarized the wonderful career of Christian de Duve had it not turned out that the fault lies in peroxisomes.

The culprit is a gene (called ABCD1) on the X chromosome (so ALD is an X-linked genetic disease). ABCD1 encodes part of the protein channel that carries very long chain fatty acids into peroxisomes. Mutations in ABCD1 (over 500 have been found) cause defective import of fatty acids, resulting in the accumulation of very long chain fatty acids in various tissues. This can lead to irreversible brain damage. In children the myelin sheath of neurons is damaged, causing neurological defects including impaired vision and speech disorders.

And the miracle?

It’s gene therapy of course and, helpfully, we’ve already seen it in action. Self Help – Part 2 described how novel genes can be inserted into the DNA of cells taken from a blood sample. The genetically modified cells (T lymphocytes) are grown in the laboratory and then infused into the patient – in that example the engineered cells carried an artificial T cell receptor that enabled them to target a leukemia.

In Gosh! Wonderful GOSH we saw how the folk at Great Ormond Street Hospital adapted that approach to treat a leukemia in a little girl.

Now David Williams, Florian Eichler, and colleagues from Harvard and many other centres around the world, including GOSH, have adapted these methods to tackle ALD. Again, from a blood sample they selected one type of cell (stem cells that give rise to all blood cell types) and then used genetic engineering to insert a complete, normal copy of the DNA that encodes ABCD1. These cells were then infused into patients. As in the earlier studies, they used a virus (or rather part of a viral genome) to get the new genetic material into cells. They choose a lentivirus for the job – these are a family of retroviruses (i.e. they have RNA genomes) that includes HIV. Specifically they used a commercial vector called Lenti-D. During the life cycle of RNA viruses their genomes are converted to DNA that becomes a permanent part of the host DNA. What’s more, lentiviruses can infect both non-dividing and actively dividing cells, so they’re ideal for the job.

In the first phase of this ongoing, multi-centre trial a total of 17 boys with ALD received Lenti-D gene therapy. After about 30 months, in results reported in October 2017, 15 of the 17 patients were alive and free of major functional disability, with minimal clinical symptoms. Two of the boys with advanced symptoms had died. The achievement of such high remission rates is a real triumph, albeit in a study that will continue for many years.

In tracing this extraordinary galaxy, one further hero merits special mention for he played a critical role in the story. In 1999 Jesse Gelsinger, a teenager, became the first person to receive viral gene therapy. This was for a metabolic defect and modified adenovirus was used as the gene carrier. Despite this method having been extensively tested in a range of animals (and the fact that most humans, without knowing it, are infected with some form of adenovirus), Gelsinger died after his body mounted a massive immune response to the viral vector that caused multiple organ failure and brain death.

This was, of course, a huge set-back for gene therapy. Despite this, the field has advanced significantly in the new century, both in methods of gene delivery (including over 400 adenovirus-based gene therapy trials) and in understanding how to deal with unexpected immune reactions. Even so, to this day the Jesse Gelsinger disaster weighs heavily with those involved in gene therapy for it reminds us all that the field is still in its infancy and that each new step is a venture into the unknown requiring skill, perseverance and bravery from all involved – scientists, doctors and patients. But what better encouragement could there be than the ALD story of young lives restored.

It’s taken us a while to piece together the main threads of this wonderful tale but it’s emerged as a brilliant example of how science proceeds: in tiny steps, usually with no sense of direction. And yet, despite setbacks, over much time, fragments of knowledge come together to find a place in the grand jigsaw of life.

In setting out to probe the recesses of metabolism, Christian de Duve cannot have had any inkling that he would build a foundation on which twenty-first century technology could devise a means of saving youngsters from a truly terrible fate but, my goodness, what a legacy!!!

References

Eichler, F. et al. (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. The New England Journal of Medicine 377, 1630-1638.

 

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Obesity and Cancer

Science, you could say, comes in two sorts. There’s the stuff we more or less understand – and there’s the rest. We’re pretty secure with the earth being round and orbiting the sun, the heart being a pump connected to a network of tubes that keeps us alive, DNA carrying the genetic code – and a few other things. But human beings are curious souls and we tend to be fascinated by what we don’t know and can’t see – why the Dance of the Seven Veils caught on, I guess.

Scientists are, of course, the extreme example – they spend their lives pursuing the unknown (and, as Fred Hoyle gloomily remarked, they’re always wrong and yet they always go on). But in this media era they pay a public price for their doggedness because they get asked the pressing questions of the moment. Is global warning going to finish us off soon, why is British sport generally so poor and – today’s teaser – does being fat make you more likely to get cancer?

A few facts go a long way

The major cancers have become familiar because the numbers afflicted are so staggering – but the one good thing is that the epidemiology can tell us something about the disease. Thus for cancers of the bowel, endometrium, kidney, oesophagus and pancreas and also for postmenopausal breast cancer there is clear evidence that being overweight or obese makes you more susceptible. In other words, if you compare large groups with those cancers to equally large numbers without, the disease groups contain significantly more people who are fat. We should add that the above list is conservative. A number of other cancers are almost certainly more common in those who are overweight (brain, thyroid, liver, ovary, prostate and stomach tumours as well as multiple myeloma, leukaemia, non-Hodgkin lymphoma and malignant melanoma in men).

Sizing up the problem

The usual measure is Body Mass Index (BMI) – your weight (in kilograms) divided by the square of your height (in metres). A BMI of 25 to 29.9 and you’re overweight; over 30 is obese. In England in 2009 just over 61% of adults and 28% of children (aged 2-10) were overweight or obese and of these, 23% of adults and 14% of children were obese. And every year these figures get bigger.

How big is the risk?

Impossible to say exactly – for one thing we don’t know how long you need to be exposed to the risk (i.e. being overweight) for cancer to develop but in 2010 just over 5% of the total of new cancer cases in the UK was due to excess weight. That’s another conservative estimate, but it means at least 17,000 out of 309,000 cases, with bowel and breast cancers being the major sites.

What’s going on?

Showing an association is a good start but the important thing is to find out which molecules make that link. For obesity and cancer detail remains obscure but broad outlines are emerging, summarised in the sketch. In obesity fat (adipose) cells increase in both number and size (so it’s a double problem: more cells – and the fat cells themselves are fatter). As this happens other cells are recruited to adipose tissue and, from this cellular cooperative, signalling proteins are released that have the potential to drive tumours. This picture is similar to that of the microenvironment of tumours themselves, where many types of cell infiltrate the new growth. Initially this inflammatory and immune response aims to kill the tumour but if it fails the balance of signalling shifts so that it actually helps the tumour grow. In addition to signals from fat cells themselves, obesity is usually associated with increased levels of circulating growth hormones (e.g., insulin) and of lipids, both of which may also promote tumour development.

Thus many signals with cancerous potential arise in obese individuals. In principle these could initiate tumour growth or they could accelerate it in cancers that have started to develop independently of obesity. So it is complicated – but at least as new signalling strands emerge they offer new targets for drug therapy.

In obesity abnormal signals from fatty tissue can combine with others arising from perturbed metabolism to help cancers develop

Reference

World Cancer Research Fund (WCRF) Panel on Food, Nutrition, Physical Activity, and the Prevention of Cancer (WCRF, 2007).