Slide 1

Dear Colleagues, Mrs Chairmans, first of all I would like to thank the organizers of the Meeting for this kind invitation.

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I would like to share with you this afternoon a particular topic. Could NF-kB be a target for treatment of glomerulonephritis?
To be or no to be? NF-kB is the Answer. That’s the title of a wonderful review published two years ago. But what is NF-kB? It’s a ubiquitous transcription factor involved in the regulation of the expression of various genes encoding for flogistic, sclerotic and survival factors.
For this reason it could be a good target for therapeutic approaches in GN.

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NF-kB’s activation has been detected in non-proliferative, as well as in proliferative disease and during the sclerotic progression of glomerulonephritis.

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We showed that there is an activation in PBMC isolated from IgA nephropathy patients, suggesting that it could play a pathogenetic role.

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What does NF-kB do?  In quiescent cells NF-kB  is present in the cytoplasm bound to its inhibitor, IkBα. Under a stimulus, there is an activation of the IKK complex, that induces a phosphorylation of  IkBα, that leaves NF-kB and allow it to enter the nucleus and binding specific sequences, starting the transcription of specific genes.

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IkBα undergoes polyubiquitination and then is degraded by proteasomes.

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Are there strategies to inhibit the NF-kB pathway? There are many.
First of all we can block the IKK complex in different ways: we will see how we can block the activity of this complex.
We can block the translocation of NF-kB into the nucleus.

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We saw the role of IKK complex. A part of this complex, called NEMO, is a regulator protein that blocks the activity of IKKα and β. A small peptide called “NEMO binding domain”, NBD, has been produced and has been used to facilitate the cellular uptake of the NBD. The final effect is the blocking of IKKα and β activity.   

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In a model of chronic inflammatory rheumatoid arthritis induced in mice by injection of collagen type II, the injection of NEMO NBD was able to reduce the chronic inflammation.

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IKK β is the most important component of the IKK complex. Hence it is a potential target suitable for the treatment of inflammatory disease, while IKKα is critical for antibody production. Hence, IKKα inhibition could be useful for preventing autoimmune diseases mediated by B cells.

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There are many inhibitors of IKK β, and of particular note thalidomide, that has been used in lupus nephritis and multiple myeloma. But several other molecules have been produced.

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Egido’s group showed that by inhibiting IKK β activity with gliotoxin and parthenolide, in both in vitro and in vivo studies, there is a block of NF-kB but not of AP-1

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These studies have also shown an inhibition of the genes regulated by NF-kB, and in a model of anti Thy 1.1, as we heard before, gliotoxin and parthenolide, inhibitors of IKK, induced a downregulation of NF-kB, MCP-1, iNOS, with an improvement in the disease clinical pattern and in histology.

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Another way to block the activity of NF-kB is the use of up-regulators of IkBα, such as IL-10, IL-11 or IL-13

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We demonstrated that in unconditioned mesangial cells, NF-kB is present in the cytoplasm. Treating mesangial cells with aberrantly glycosylated IgA we showed a nuclear translocation of NF-kB, meaning NF-kB activation, that was blocked or, at least, was partially blunted by the coincubation with IL-10

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Another possibility is to use proteasome inhibitors.

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As I told you, the proteasome, through the polyubiquitination system, degrades IkBα, inducing a stabilization of this inhibitor and then an NF-kB inhibition.

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One of the proteasome inhibitor mostly used today is bortezomib in multiple myeloma. NF-kB plays a key role in the pathogenesis and progression of multiple myeloma. In preclinical evidence was showed that bortezomib was able to induce inhibition of myeloma cells proliferation in vitro and induction of apoptosis in these cells.

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It was able to block the transcription factor in these tumour cells.

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Bortezomib by inhibiting NF-kB plays an anti-proliferative and pro-apoptotic effect in stromal myelomatosus plasmocytes.

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In this disease reduction in IL6 exerts anti-angiogenetic effect and a reduction in both T lymphocytes proliferation and release in Th1 cytokines.

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A new proteasome inhibitor has been recently proposed, and in Torino we are trying to use these proteasome inhibitor, already used in HIV patients, starting a trial in FSGS steroid resistant patients.

Slide 23

Another possibility to block NF-kB is the use of decoy ODN. The receptor mediated activation induces, as I told you, an activation of the transcription factor that are phosphorylated, able then to translocate into the nucleus and start the transcription of specific mRNA. If specific sequences, complementary to the genome to which the transcription factors bind, are injected into the cells, there is a block in the specific gene transcription.

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In vivo administration of these decoy suppresses experimental crescentic glomerulonephritis,

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and expression of CD45 positive cells in mice treated with TNFα.

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Another possibility is to use antioxidants that act as scavengers for ROS, classic activator of NF-kB. But oxidants act also by altering the cysteine of the kinase activation loop of the IKK complex, then blocking the kinase activity. They reacts also with cysteine residues on NF-kB, already bound to DNA, inducing a detachment and blocking its activity.

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We studied mesothelial cells in vitro treated with products of non enzymatic glycosylation.

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N-Acetylcysteine induced a specific inhibition of NF-kB activation, as you can see in this confocal microscopy.

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Cells in basal condition are in panel A, after treatment with AGE in panel B,

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and reduction of NF-kB activation after treatment with antioxidants.

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So, I told you this afternoon, that there are many strategies to inhibit NF-kB activation by working upstream on IKK, by blocking IkBα, by downregulating NF-kB nuclear function, by using antioxidants and so on.

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But my take home message is, OK, NF-kB could be a targeted therapy for diseases, but be careful because many of the strategies used to inhibit NF-kB are not specific. NF-kB plays a physiologic role in the regulation of the immune system and carcinogenesis control.
The use of decoy ODN seem to be the most promising tool to inhibit specifically NF-kB only in the inflamed sick tissue.

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Thank you for your attention and thanks to all my group.

Slide 34

Chairman: Thank you very much Alessandro for this excellent review. Any comments, question?

Question: I have the idea that blockade of the rennin-angiotensin system has a strong inhibitory effect on the nuclear factor-kB. Am I right or not?

Dr Amore: You are completely right. Ang II is one of the main activator of NF-kB system.
However there is the possibility that angiotensin IV, that is an escape way production when you use ACE inhibitors, is an activator of NF-kB as well: then ACE-inhibitors could not completely block NF-kB system.

Question: Spironolactone and anti-aldosteronic agent?

Dr Amore: They do the same.

Question: You suggested at the end that maybe the ODN would be the most efficient thing clinically, but remember the treatment may need to be given for a very long time over in chronic inflammatory diseases and I wondered the small molecules inhibitors of IKKα or IKKβ might be something that would be effective and would relatively selective for inflamed tissue. I don’t think they would ……I don’t
 Known, I’m asking.

Dr Amore: Yes you’re perfectly right, and I completely agree with you, but my feeling is that a combination of actions with Martin, that is not here now, and somebody who created in vitro ODN to block NF-kB when it’s already into the nucleus is the definite answer.
When ODN are injected and NF-kB is attached to the DNA, ODN find the place occupied and cannot exert their effect. They should be injected before the interaction of NF-kB and genome, they should compete with the specific genome sequence and then continuously chronically injected.
The problem is that in vivo the situation is completely different than in vitro.
In vitro you create the disease and you know in which moment you can induce the remission, you can manipulate what you created.
In vivo you have in front of  you a patients and you don’t know when the disease started, which is the activation state of NF-kB. My message was OK, NF-kB could be the future target,  but let’s block NF-kB only when this block is useful and further studies need to clarify the timing  of injection in chronic disease.

Question: Thank you.

Chairman: Ok if there are no more questions I sense people are getting tired and restless. I’d like to thank all of the speakers for some tremendous talks this afternoon. I Thank my Co-Chair and thank the audience for sticking with us, thanks very much indeed.