Pietro L. Poliani

Inhibition of Th1 development and treatment of chronic-relapsing experimental allergic encephalomyelitis by a non-hypercalcemic analogue of 1,25-dihydroxyvitamin D3

1α,25‐dihydroxyvitamin D3 [1,25(OH)2D3] inhibits production of IL‐12, a cytokine involved in the development of Th1 cells and in the pathogenesis of Th1‐mediated autoimmune diseases. Here, we show that 1,25(OH)2D3 and a non‐hypercalcemic analogue are selective and potent inhibitors of Th1 development in vitro and in vivo without inducing a deviation to the Th2 phenotype. Administration of 1,25(OH)2D3 or its analogue prevents chronic‐relapsing experimental allergic encephalomyelitis (CR‐EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide 35u2009–u200955 (MOG35u2009–u200955) in Biozzi ABu2009/u2009H mice. The inhibition of EAE induction is associated with a profound reduction of MOG35u2009–u200955‐specific proliferation and Th1 cell development. Importantly, the non‐hypercalcemic analogue also provides long‐term protection from EAE relapses induced by immunization with spinal cord homogenate when administered for a short time at symptom onset or even after the first peak of disease. Neuropathological analysis shows a reduction of inflammatory infiltrates, demyelinated areas and axonal loss in brains and spinal cords of treated mice. These resuls indicate that inhibition of IL‐12‐dependent Th1 cell development is associated with effective treatment of CR‐EAE and suggest the feasibility of an approach based on low molecular weight inhibitors of IL‐12 production in the treatment of multiple sclerosis.

Intrathecal Delivery of IFN-γ Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-γ gene leads to persistent (up to 4 wk) CNS production of IFN-γ and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)35–55. Mice treated with the IFN-γ-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-γ-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-γ-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-γ was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-γ-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-γ in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

Fibroblast growth factor-II gene therapy reverts the clinical course and the pathological signs of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice.

The development of therapies aimed to promote remyelination is a major issue in chronic inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS), where the permanent neurological impairment is due to the axonal loss resulting from recurrent episodes of immune-mediated demyelination. Here, we show that the intrathecal injection of a herpes simplex virus (HSV) type-1 replication-defective multigene vector, engineered with the human fibroblast growth factor (FGF)-II gene (TH:bFGF vector), was able to significantly revert in C57BL/6 mice the clinicopathological signs of chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The treatment with the TH:bFGF vector was initiated within 1 week after the clinical onset of EAE and was effective throughout the whole follow-up period (ie 60 days). The disease-ameliorating effect in FGF-II-treated mice was associated with: (1) CNS production of FGF-II from vector-infected cells which were exclusively located around the CSF space (ependymal, choroidal and leptomeningeal cells); (2) significant decrease (Pu2009<u20090.01) of the number of myelinotoxic cells (T cells and macrophages) both in the CNS parenchyma and in the leptomeningeal space; and (3) significant increase (Pu2009<u20090.01) of the number of oligodendrocyte precursors and of myelin-forming oligodendrocytes in areas of demyelination and axonal loss. Our results indicate that CNS gene therapy using HSV-1-derived vector coding for neurotrophic factors (ie FGF-II) is a safe and non-toxic approach that might represent a potential useful ‘alternative’ tool for the future treatment of immune-mediated demyelinating diseases.

Central Nervous System Delivery of Interleukin 4 by a Nonreplicative Herpes Simplex Type 1 Viral Vector Ameliorates Autoimmune Demyelination

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.

Central nervous system gene therapy with interleukin-4 inhibits progression of ongoing relapsing-remitting autoimmune encephalomyelitis in Biozzi AB/H mice.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS) that might benefit from anti-inflammatory therapies. However, systemic delivery of anti-inflammatory drugs in MS patients has so far been disappointing, mostly due to the limited capacity of these molecules to enter the CNS. We injected into the cisterna magna (i.c.) of Biozzi AB/H mice affected by a relapsing–remitting form of experimental autoimmune encephalomyelitis (EAE), the animal model of MS, a non-replicative herpes simplex virus (HSV) type-1-derived vector containing the interleukin (IL)-4 gene (d120:LacZ:IL-4). CNS delivery of the d120:LacZ:IL-4 vector, after EAE onset, induced the in situ production of IL-4 by CNS-resident cells facing the cerebrospinal fluid (CSF) spaces and reduced by 47% (Pu2009<u20090.02) the disease-related deaths. compared with mice treated with the control d120:lacz vector, il-4-treated mice also showed a shorter duration of the first eae attack, a longer inter-relapse period, and a reduction in the severity and duration of the first relapse. protection from eae progression in il-4-treated mice was associated with activation of microglia in spinal cord areas where mrna content of the pro-inflammatory chemokines, macrophage chemoattractant protein-1 (mcp-1) and rantes, was reduced and that of the anti-inflammatory cytokine il-4 was increased. finally, cns-infiltrating mononuclear cells from il-4-treated mice produced lower levels of mcp-1 mrna compared with control mice. our results, showing that il-4 gene delivery using hsv-1 vectors induces protection from eae by in situ modulating the cytokine/chemokine-mediated circuits sustaining effector cell functions, indicate that the intrathecal ‘therapeutic’ use of nonreplicative hsv-1-derived vectors containing anti-inflammatory molecules might represent an alternative strategy in inflammatory diseases of the cns.

Delivery to the central nervous system of a nonreplicative herpes simplex type 1 vector engineered with the interleukin 4 gene protects rhesus monkeys from hyperacute autoimmune encephalomyelitis

Systemic administration of antiinflammatory molecules to patients affected by immune-mediated inflammatory demyelinating diseases of the central nervous system (CNS) has limited therapeutic efficacy due to the presence of the blood-brain barrier (BBB). We found that three of five rhesus monkeys injected intrathecally with a replication-defective herpes simplex virus (HSV) type 1-derived vector engineered with the human interleukin 4 (IL-4) gene were protected from an hyperacute and lethal form of experimental autoimmune encephalomyelitis induced by whole myelin. The intrathecally injected vector consistently diffused within the CNS via the cerebrospinal fluid and infected ependymal cells, which in turn sustained in situ production of IL-4 without overt immunological or toxic side effects. In EAE-protected monkeys, IL-4-gene therapy significantly decreased the number of brain as well as spinal cord inflammatory perivenular infiltrates and the extent of demyelination, necrosis, and axonal loss. The protective effect was associated with in situ downregulation of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1), upregulation of transforming growth factor beta (TGF-beta), and preservation of BBB integrity. Our results indicate that intrathecal delivery of HSV-1-derived vectors containing antiinflammatory cytokine genes may play a major role in the future therapeutic armamentarium of inflammatory CNS-confined demyelinating diseases and, in particular, in the most fulminant forms where conventional therapeutic approaches have, so far, failed to achieve a satisfactory control of the disease evolution.

Peripheral levels of caspase-1 mRNA correlate with disease activity in patients with multiple sclerosis; a preliminary study

The cysteine protease caspase-1 plays a crucial part in the inflammatory process due to its ability to proteolitically activate proinflammatory cytokine precursors, such as interleukin (IL)-1β and IL-18. Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS in which the pathogenic process is mainly orchestrated by proinflammatory cytokines. u2003The role of caspase-1 in multiple sclerosis was evaluated by measuring its mRNA levels in peripheral blood mononuclear cells (PBMCs) from seven patients with relapsing-remitting multiple sclerosis every 15 days over a 1 year period. The recorded levels were compared with clinical and MRI evidence of disease activity. Brain MRI was performed monthly in each patient. u2003Caspase-1 mRNA levels were significantly increased in PBMCs from patients with multiple sclerosis compared with healthy controls (p<0.001). In patients with multiple sclerosis, a twofold to threefold increase of caspase-1 mRNA mean level was found in the week preceding an acute attack (p<0.05). The magnitude of caspase-1 mRNA increase correlated with the number of new (p=0.01) but not persisting gadolinium enhancing brain MRI lesions. u2003In conclusion, caspase-1 might be involved in the immune mediated process underlying CNS inflammation and might represent a suitable peripheral immunological marker of disease activity in multiple sclerosis.

Cytokines and immunity in multiple sclerosis: the dual signal hypothesis

Multiple sclerosis (MS) is considered an immune-mediated disease of the central nervous system (CNS) sustained by a chronic inflammatory process leading to patchy demyelination and axonal loss. However, the inflammatory triggering event as well as the target of the pathogenic process in MS are still partially unknown. We report evidence that a local inflammatory process occurring in the CNS (considered as a reaction of blood vessels in vascularized living tissue to a local injury leading to the accumulation of fluid and blood cells) along with a concomitant, but possibly unrelated, peripheral inflammatory event may trigger a CNS-specific autoimmune reaction cascade sustaining the MS pathogenesis. In the CNS, inflammatory mediators (mainly cytokines) act either as regulatory (i.e. activation of glial cells, shaping the autoimmune response) or effector molecules (i.e. myelinotoxicity, oligodendrotoxicity). In the periphery, inflammatory cytokines induce, in a bystander fashion, activation of monocytes and T cells. Among this latter cell population there are myelin-specific T cells belonging to the normal autoimmune repertoire that home to the CNS where they may trigger the continuous recruitment of effector cells (macrophages) from the periphery. The concept that two concomitant, but possibly unrelated, inflammatory events, occurring in the CNS and in the periphery, represent the crucial elements sustaining MS, might reveal a more comprehensive view (dual signal hypothesis) of the entire etiopathogenic process underlying this disease.

Cytokine therapy in immune-mediated demyelinating diseases of the central nervous system: a novel gene therapy approach.

Pro-inflammatory cytokines play a crucial role in the regulatory and effector phase of the immune-mediated mechanism sustaining multiple sclerosis pathogenesis (MS) thus supporting the use of anti-inflammatory cytokines as a therapeutic option. Systemic administration of cytokines shows, however, limited therapeutic efficacy and undesirable/unpredictable side-effects. We have developed a non-toxic system to deliver cytokines within the central nervous system (CNS) based on the intrathecal (i.c.) administration of non-replicative herpes simplex (HSV) type-1-derived viral vectors engineered with heterologous cytokine genes. Compared to controls, mice affected by experimental autoimmune encephalomyelitis (EAE) and i.c. injected with an HSV-1-derived vector containing the gene of the anti-inflammatory cytokine IL-4 showed a significant amelioration of clinical and pathological EAE signs. A decreased mRNA expression of the monocyte chemoattractant protein-1 (MCP-1) by mononuclear CNS-infiltrating cells was also observed. Peripheral T cells from IL-4-treated mice were not affected both in their antigen-specific proliferative response and in the cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1-derived vectors is a feasible therapeutic strategy and might represent an alternative approach for the treatment of immune-mediated demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached within the CNS and the absence of side-effects on the peripheral immune system. The short-lasting cytokine production in the CNS after a single vector administration (4 weeks) is the limiting factor of this novel technology which, although promising, has to be improved.

Cytokine gene therapy of autoimmune demyelination revisited using herpes simplex virus type-1-derived vectors

The peripheral delivery of drugs in patients affected by central nervous system (CNS)-confined diseases is therapeutically ineffective due to the presence of the blood–brain barrier which forms an inaccessible wall to the majority of CNS targeting molecules. When molecules with an anti-inflammatory profile have been systemically administered to patients affected by a chronic inflammatory demyelinating disease of the CNS, such as multiple sclerosis (MS), results have been disappointing. A successful therapeutic approach in MS should therefore consider the delivery of anti-inflammatory molecules directly into the CNS in order to inhibit blood-borne CNS-confined mononuclear cells which act as ultimate effector cells directly destroying oligodendrocytes and/or releasing myelinotoxic substances. Biological and physical vectors engineered with heterologous genes coding for immunomodulatory cytokines with an anti-inflammatory profile might represent the appropriate tool to deliver therapeutic genes into the CNS of patients with MS. So far, cytokine gene therapy has never been attempted in MS, but encouraging results have been obtained in the animal model of MS, experimental autoimmune encephalomyelitis (EAE), using viral vectors or plasmids engineered with cytokine genes and then injected systemically, either in the blood stream or circulating encephalitogenic T cells, or into the CNS. Here, we critically discuss the various attempts made in EAE using gene therapy protocols based on the delivery of immunomodulatory cytokine genes. Special emphasis is put on the use of non-replicative herpes simplex type-1 (HSV)-derived vectors engineered with the gene of the immunomodulatory cytokine interleukin (IL)-4.