Slide 1

Good morning. Ladies and Gentlemen.

Slide 2

Well, this is an old nephrology forum from back in 1987 where I suggested that HUS and TTP are actually a variable expression of a single entity. The reason for me to suggest that and also the treatment was very similar in all plasma exchange and plasma infusion for both diseases essentially.

Slide 3

The pathology was very similar to detachment of endothelial from the basement membrane and creation of this space and a very reduced capillary lumen.

Slide 4

In ten years however, everything has changed and Doctor Moake in the New England Journal of Medicine said that a single laboratory test will enable physicians to distinguish TTP from HUS. So I was completely wrong, the field has changed rapidly.

Slide 5

You know HUS is a multisystemic disease of microangiopathic haemolytic anaemia, thrombocytopenia and predominant renal involvement with this kind of incidence all over the world with a peak in South America, Argentina in particular. This is largely due to an infection by a bacteria of E. coli of a particular strain that generates Shiga toxin/verotoxin that induces haemorrhagic colitis and this was discovered in 1983.

Slide 6

Everything about the pathogenesis of this disease was clarified between 1983-1984 when Karmali suggested that the E.coli was associated with sporadic cases of classical HUS of children. This is a beautiful picture showing how E-coli attach to the intestinal villi. This causes the disease in humans but not in cattle. E.coli colonises the bowel of cattle but does not cause the disease in cattle.

Slide 7

This Shiga toxin constituted by a subunit A and various B subunits that bind to specific receptors are internalised after receptor-mediated endocytosis and are capable of inactivating ribosomal RNA shutting down the protein synthesis and leading to cellular necrosis and cell death.

Slide 8

This is associated with microthrombi that required P-selectin, IL-8 and MCP1.

Slide 9

This is the reservoir, this is a picture from Argentina, this is the reservoir for the disease.

Slide 10

And during slaughter contamination by faecal material of even a small piece of meat can disseminate in a very impressive way millions of these patties can be contaminated.

Slide 11

And maybe this is the reason why Fidel Castro was looking at this American hamburger, maybe he wanted to know whether they were contaminated in such a way.

Slide 12

If you read books, the books say that blacks do not have HUS. That’s actually not true. This is an outbreak where thousands of patients were affected with bloody diarrhoea, they were all blacks, men who drunk untreated water while working in the fields.

Slide 13

What happened was that there was a profound drought.

Slide 14

And E.coli infected the carcasses of cattle that died after the prolonged drought and the heavy rain then washed these into the rivers and the cattle carcasses contaminated the surface water and the E.coli was isolated from the stools of patients, cattle dung and randomly collected water samples.

Slide 15

Now, a more recent outbreak has been reported in the United States. This is how many patients have been reported to have HUS with important sequela in different states and the source was fresh spinach.

Slide 16

What to do with the infectious disease? Essentially, the disease can go into remission and actually be cured by supportive measures, dialysis and – dialysis during the acute phase of the disease. An important modern thinking was that washing product before packaging is not sufficient, cooking food properly can eliminate the risk but undercooking is very common. The only reliable method is food irradiation. People are against food irradiation, they have the idea that this maybe toxic. Actually there are no data to support that and this is the only effective way in fact to protect people from these outbreaks in cases of food or water contamination.

Slide 17

There is another form of HUS, this is familial. It was reported for the first time in 1956 and it was reported as acute glomerulonephritis in infancy. There were two children and their male cousin who all died at 5 months of age. Clearly it was a familial disease.

Slide 18

This is called now atypical HUS, it’s much less common than the infectious form of the disease.

Slide 19

This gives you an idea of how many people can die in the same family.

Slide 20

Those are just a collection of families taken from the registry of atypical HUS that is based in Bergamo, Italy and was started as an Italian registry. Now we have patients referred from many countries from around the world.

Slide 21

Doctor Noris found that in those patients factor H, one of the complement modulatory factors are very low in affected patients and 50% of normal in healthy family members of these patients.

Slide 22

In 1988 Tim Goodship documented that in 3 large families with HUS that there is an area on chromosome 1q32 where factor H gene is mapped and he found that this area segregated with the disease. Affected members and obligate carriers within one family were found by mutation analysis to have a point mutation in factor H causing arginine to Glycine changes.

Slide 23

Factor H plays a pivotal role in the regulation of the alternative pathway of complement activation. It binds to the surface of cells, of bacterial cells and human endothelium if you wish, to heparan sulphate and you can bind to C3b. It is a circulating molecule that interferes with factor B binding to C3b and in this way it modulates the activation of complement. It is produced mainly in the liver I’d like you to realise that, as a single polypeptide glycoprotein and circulates in plasma at this particular concentration. Among other complement modulatory proteins MCP acts as a cofactor for factor I-mediated cleavage of C3b to inactive C3b. Unlike factor H which is circulating, MCP is membrane bound and has an intracytoplasmic tail and an extracellular domain that is responsible for this activity.

Slide 24

Published data on factor H mutation in a typical HUS includes 100 different mutations identified in almost 120 patients. The mutation frequency is 40% in the familial form and 13-17% in sporadic forms. Those mutations have been described essentially by 3-4 groups in the world including our own group and the majority of the mutations affect the recognition domain of the molecule that is important for factor H to bind to proteoglycans on the surface of the endothelium and to C3b.

Slide 25

When we expressed factor H mutated protein found in HUS patients, we were able to document together with Doctor Ziegfeld in Germany a reduced affinity to heparin as tested by heparin-chromatography column and to surface bound C3b. Mutation in this particular part of the molecule by reducing the capacity of factor H to bind proteoglycans and C3b would favour, we believe the occurrence of microvascular endothelial damage in the presence of complement activating trigger.

Slide 26

This is just to show you how a single mutation change, exactly the one that we have identified in some patients in the SCR 20 of the molecule, affects endothelial cell binding. You see the capacity of factor H to bind endothelial cells in the case of wild type factor H, mutated factor H which has been expressed exactly with the same mutation as patients has almost no binding you see the control that has no binding at all for comparison.

Slide 27

Marina Botto has created a murine model of spontaneous HUS in London. They have generated Factor H deficient mice which is transgenic for a mutant mouse factor H protein which lacks the five C terminal domains, exactly the recognition domain where the majority of mutations in atypical HUS have been found. These mice whose factor H lacks the 16-20 part the terminal part of the molecule, molecules develop spontaneous HUS with red cell fragmentation, reduced platelet count, elevated blood urea and glomerular histology with features of thrombotic microangiopathy. This is one of  the unbelievable ways you know the gene, you know the mutation, you identify a knockout animal for exactly that particular mutation after you have expressed the mutation in patients and show the physiology of that and you reproduce the model which will be extremely important because now at least 3 groups in the world, 3 different companies are trying to develop a complement modulating material in order to treat patients. Before that we are now testing whether this material, for instance, an anti-factor 5 antibody or an anti-factor B antibody all will be available soon.

Slide 28

We now are testing these particular new drugs in this model in order to see whether they will be able to cure the disease in mice which is exactly mimicking what happens in patients. So far what you can do with these patients is to infuse plasma and you see that it is enough to go with plasma infusion up to 15% of normal and this is the concentration you reach from being undetectable when the disease is active in order to normalise the haptaglobin level but we are expecting much more from this complement modulator material. The majority of these patients, 80% of them anyhow progress to terminal renal failure and they need dialysis or transplantation.

Slide 29

Transplantation of course, is the option for these patients and back in 1986 during one of the ASN meetings, it was in Philadelphia I was approached by Michael Mauer who told me ‘Every patient in whom I do a transplant with HUS has recurrence of the disease’. Ten years later Doctor Gagnadoux and colleagues from Paris published that not a single patient on which they do transplant has recurrence of the disease.

Slide 30

So people were confused about publications and what happened in fact was that the Paris cases were essentially D+ HUS the infectious form of the disease while patients of Michael Mauer were from we know all of typical HUS the majority of them do recur after transplantation and this is because factor H is circulating. You transplant the kidney, the abnormality remains in the circulation, the disease does recur.

Slide 31

Because of that we thought that since the gene is expressed mainly in the liver and proteins are synthesised by the liver, we could transplant kidney and liver in order for the liver to generate normal factor H and protect the kidney from recurrences. The first case was successful then we had a case that was unfortunately fatal.

Slide 32

Now in the literature there are at least 4 cases that I know of in which combined kidney and liver transplantation associated with extensive exchange before surgery have been associated with good renal and liver function after 27-13 and 6 months. In another case you have good renal and liver function at 15 months, so this now is a very complicated way I hope we will do much better when complement modulatory proteins will become available.

Slide 33

Other mutations have been found in a typical HUS. Factor B and Factor C3 mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to activation by complement regulators.

Slide 34

This is another mutation that we and Doctor Goodship reported for the first time. It is a mutation in MCP for the first time in a 25 non Shiga toxin HUS patients and in a 20 year old woman with a history of recurrent HUS and in her brother.

Slide 35

Those are the mutations reported so far by the various groups in the molecule, 25 mutations so far in 32 patients. The frequency is almost 13%. They can cause reduced protein expression of cell membrane or defective membrane modulatory activity.

Slide 36

I know of 5 patients with MCP mutation that have been transplanted, none of them recurred. This is a case where genetics is so important in orienting what you can do with patients with this particular disease. The reason for these patients not to recur I believe is that MCP is a membrane bound protein as opposite to Factor H which is circulating. You transplant the kidney and when you transplant a normal kidney, you have normal MCP attached on the endothelium of this particular kidney.

Slide 37

These are the first patients we transplanted with HUS many years ago.

Slide 38

Now we know that it was MCP. At that time we did not know. These patients had no recurrence of the disease and she had recently a baby. The very similar disease thrombotic thrombocytopenic purpura is a disease of microangiopathic haemolytic anaemia, thrombocytopenia and microvascular thrombosis. One of the problems is that in TTP thrombi have abundance of Von Willebrand factor with very modest fibrinogen/fibrin present in biopsies, for instance.

Slide 39

In 2001 the gene for a specific protease which is capable of cleaving vWF, ADAMTS13 was mapped on chromosome 9q34 and 12 mutations were identified.

Slide 40

ADAMTS13 is capable of cleaving the large multimers that normally are generated by endothelial cells secreted form Weibel-Palade body. If you have no ADAMTS13 theoretically you are incapable of fragmenting VWF that circulates as a large molecule and causes platelet aggregation and thrombosis.

Slide 41

This was the dogma in the absence of ADAMTS13 unusually large multimers are not cleaved and cause intravascular platelet aggregation.

Slide 42

Not true we found fragmentation of VWF even in patients who have no ADAMTS13.

Slide 43

We have a patient who had factor H mutation and ADAMTS13 deficiency, two different mutations. These patients had neurological and renal problems. The sister who only has an ADAMTS13 mutation only had neurological abnormalities.

Slide 44

Another case shows difficulties in diagnosing TTP from HUS in this particular case.

Slide 45

There are reports of ADAMTS13 deficiency in HUS and complement activation in TTP.

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Slide 48

Strong deposition of C3b can occur on endothelial cells exposed to sera of patients with acute TTP.

Slide 49

Both give rise to the same lesion.

Slide 50

I don’t think that a single laboratory test may enable physicians to distinguish TTP from HUS any longer. I think that in fact, they are two different clinical expressions of this very same disease.

Slide 51

In TTP ADAMTS13 deficiency generates unusually large VWF molecules and platelet activation which now we know very recent data is associated with complement activation. In HUS factor H deficiency is associated with loss of complement regulation, C3b deposition on endothelial cells, endothelial damage and microthrombi.

Slide 52

Thank you very much.