Fingolimod attenuates experimental autoimmune neuritis and contributes to Schwann cell-mediated axonal protection.

BACKGROUND Fingolimod, a sphingosine-1-phosphate receptor modulator with well-described immunomodulatory properties involving peripheral immune cell trafficking, was the first oral agent approved for the treatment of relapsing remitting multiple sclerosis. Analogous immunomodulatory treatment options for chronic peripheral autoimmune neuropathies are lacking. METHODS We tested fingolimod in the animal model of experimental autoimmune neuritis in Lewis rat. Six to eight-week-old female rats were immunized with P2 peptide and from this day on treated with fingolimod. Histology of the sciatic nerve was done to analyze T cell and macrophage cell count, intercellular adhesion molecule (ICAM) and amyloid precursor protein (APP) expression, as well as apoptotic Schwann cell counts. RESULTS Preventive oral treatment with 0.1 mg/kg up to 3 mg/kg fingolimod once daily dissolved in rapeseed oil completely ameliorated clinical neuritis signs. It reduced circulating peripheral blood T cells and infiltrating T cells and macrophages in the sciatic nerve, whereas at the same time, it preserved blood-nerve barrier impermeability. Most importantly, fingolimod showed beneficial properties on the pathogenic process as indicated by fewer apoptotic Schwann cells and a lower amount of amyloid precursor protein indicative of axonal damage at the peak of disease course. CONCLUSIONS Taken together, orally administered low-dose fingolimod showed an impressive immunomodulatory effect in the rat model of experimental autoimmune neuritis. Our current observations introduce fingolimod as an attractive treatment option for neuritis patients

1,25-OH vitamin D and AKT-inhibition increase glucocorticoid induced apoptosis in a model of T-cell acute lymphoblastic leukemia (ALL).

In acute lymphoblastic leukemia (ALL), steroid resistance and hypovitaminosis D are both associated with a poor prognosis. We show that methylprednisolone, calcitriol and the AKT-inhibitor MK-2206 have a synergistic effect on the apoptosis of steroid resistant T-ALL cells. Compared to methylprednisolone monotherapy, calcitriol increases methylprednisolone induced apoptosis dose-dependently (1.37-1.92-fold; p < 0.05). Pre-incubation with calcitriol increases the apoptotic effect of MK-2206 even further (3.6-fold; p < 0.05). It also potentiates synergism between MK-2206 and methylprednisolone (vehicle control 38% vs. calcitriol 58%, p < 0.01). The combination of calcitriol and AKT inhibition should be investigated further as treatment options for steroid resistance in T-ALL

Dimethyl Fumarate Ameliorates Lewis Rat Experimental Autoimmune Neuritis and Mediates Axonal Protection

<div><p>Background</p><p>Dimethyl fumarate is an immunomodulatory and neuroprotective drug, approved recently for the treatment of relapsing-remitting multiple sclerosis. In view of the limited therapeutic options for human acute and chronic polyneuritis, we used the animal model of experimental autoimmune neuritis in the Lewis rat to study the effects of dimethyl fumarate on autoimmune inflammation and neuroprotection in the peripheral nervous system.</p><p>Methods and Findings</p><p>Experimental autoimmune neuritis was induced by immunization with the neuritogenic peptide (amino acids 53–78) of P2 myelin protein. Preventive treatment with dimethyl fumarate given at 45 mg/kg twice daily by oral gavage significantly ameliorated clinical neuritis by reducing demyelination and axonal degeneration in the nerve conduction studies. Histology revealed a significantly lower degree of inflammatory infiltrates in the sciatic nerves. In addition, we detected a reduction of early signs of axonal degeneration through a reduction of amyloid precursor protein expressed in axons of the peripheral nerves. This reduction correlated with an increase of nuclear factor (erythroid derived 2)-related factor 2 positive axons, supporting the neuroprotective potential of dimethyl fumarate. Furthermore, nuclear factor (erythroid derived 2)-related factor 2 expression in Schwann cells was only rarely detected and there was no increase of Schwann cells death during EAN.</p><p>Conclusions</p><p>We conclude that immunmodulatory and neuroprotective dimethyl fumarate may represent an innovative therapeutic option in human autoimmune neuropathies.</p></div

Dimethyl fumarate did not induce Nrf2 in Schwann cells at the peak of EAN course.

<p>Representative photos of double (merge) Nrf2 positive Schwann cells (S100 positive) staining for sciatic nerve transverse sections of rats (n = 6/group) treated with DMF 15mg/kg, 45mg/kg and methylcellulose. No statistical significant increase between Nrf2 positive Schwann cells for DMF-treated vs. methylcellulose-treated rats on day 16 p.i. was detected (insets depict details of staining). Scale bars indicate 50μm.</p

Clinical EAN course under dimethyl fumarate treatment.

<p>EAN was induced in Lewis rats by immunisation on day 0 with P2 peptide 53–78 plus CFA. Rats received DMF diluted in 0,08% methylcellulose in tap water at doses of 15 mg/kg, 30mg/kg and 45mg/kg twice daily from day 0 to day 23-post immunisation by oral gavage. Control rats received 0,08% methylcellulose in tap water only. Mean values and SEM are depicted, ROC Area under curve (AUC) 45mg/kg vs. methylcellulose, n = 8 * p<0,05. The experiment was repeated 2 times with similar results.</p

Dimethyl fumarate induced Nrf2 at the peak of EAN course.

<p>(A) Representative photos of Nrf2 staining for sciatic nerve transverse sections of rats (n = 6/group) treated with DMF 15mg/kg (d-f), 45mg/kg (g-i) and methylcellulose-treated animals (a-c), showing an increase of Nrf2 positive cells for 45mg/kg DMF-treated rats. Pictures a, d and g show nuclear stain (DAPI), pictures b, e and h Nrf2 stain and pictures c, f and i indicate double staining. Scale bars indicate 100μm. (B) Percentage of Nrf2 positive staining per sciatic nerve section measured by immunofluorescent staining on day 16 p.i. from EAN rats (n = 6/group) receiving DMF at different doses (15mg/kg, 45mg/kg/day) and methylcellulose-treated rats. Mean values and SEM are depicted (*p<0,05).</p

Dimethyl fumarate improved proximal and distal nerve conduction.

<p>(A) Representative CMAP (compound motor action potentials) traces during EAN course at days −1 and 16 p.i. showing a conduction block for methylcellulose-treated rats at day 16 p.i. whereas for 45 mg/kg DMF-treated rats no conduction block was recorded. (B) Representative F-wave traces after distal stimulation showing prolonged F-waves latencies only for the methylcellulose-treated group at day 16 p.i. in comparison to day -1. Rats treated with 45mg/kg did not show any significant differences in the F-wave latencies between day -1 and 16 p.i. The black vertical line defines the motor (M) response and the F (F-wave) response latency. On the left of the red vertical line applies the M response regarding distance (horizontally, ms) and vertically (mV) and on the right of the red vertical line applies the F response data (ms, mV), (M: M response, F: F response, D: distance of one side of the dotted lined squares). (C) After proximal and distal stimulation of the sciatic nerve the conduction velocity was calculated. A statistical significant reduction of the MNCV (motor nerve conduction velocity) appeared for the control group and the 15mg/kg group (p<0,0001 ***, n = 10), but no difference in the MNCV was seen for the 45mg/kg DMF treated group indicating a protective role of DMF against demyelination. Mean values and SEM are depicted.</p

Dimethyl fumarate reduced inflammatory infiltrates of T cells and macrophages in sciatic nerves of EAN rats.

<p>(A) Rats were daily force fed with DMF or tap water and 16 days p.i. (at expected disease maximum), sciatic nerves were isolated and stained for CD3⁺ cells (a, b, c) and CD68⁺ cells (macrophages) (d, e, f). Representative photos of sciatic nerves in transverse sections of methylcellulose-treated animals (a and d), 15 mg/kg DMF-treated animals (b and e) and 45mg/kg DMF treated animals (c and f). Scale bars indicate 100μm. (B) Mean numbers of T cells per mm² sciatic nerve sections and B. Mean numbers of macrophages (CD68⁺) per mm² sciatic nerve sections as calculated by immunohistochemistry on day 16 p.i. from EAN rats (n = 6/group) receiving orally DMF at different doses (15mg/kg, 45mg/kg/day) and methylcellulose-treated rats. Mean values and SEM are depicted (** p<0,005, ***p<0,0001). The experiment was repeated 2 times with similar results.</p

Dimethyl fumarate reduced early axonal damage at the peak of EAN course.

<p>(A) Representative photos of APP (amyloid precursor protein) staining for sciatic nerve transverse sections of rats (n = 6/group) treated with DMF 15mg/kg (b, e), 45mg/kg (c, f) and methylcellulose-treated animals (a, d), showing an reduction of APP positive cells for DMF-treated rats. Scale bars indicate 100μm for a-c and 50μm for d-f. (B) Mean numbers of APP positive cells per mm² sciatic nerve sections as calculated by immunohistochemistry on day 16 p.i. from EAN rats (n = 6/group) receiving orally DMF at different doses (15mg/kg, 45mg/kg/day) and methylcellulose-treated rats. Mean values and SEM are depicted (*p<0,05). The experiment was repeated 2 times with similar results.</p