Absence of merkel cell polyoma virus in cutaneous melanoma.

Recently, merkel cell polyoma virus (MCPyV) has been described in 80% of merkel cell carcinomas (MCC). Similar to MCC, melanoma incidence is increased in immuno-suppressed patients. We hypothesized that MCPyV may play a role in melanoma development as well. We selected 95 archival, paraffin-embedded primary melanomas. DNA was obtained from micro-dissected tissue and amplified with PCR primer sets specific for the MCPyV T-antigen locus (LT1 and LT3) and for the VP1 gene. None of the 95 melanoma samples did show LT1, LT3, or VP1 fragment amplification. In conclusion, there is no evidence that MCPyV infection plays a role in cutaneous melanoma development.

Improved outcome of EAN, an animal model of GBS, through amelioration of peripheral and central inflammation by minocycline.

Experimental autoimmune neuritis (EAN) is a widely used animal model of the human acute inflammatory demyelinating polyradiculoneuropathy, which is the most common subtype of Guillain-Barré Syndrome. EAN is pathologically characterized by breakdown of the blood-nerve barrier, infiltration of reactive immune cells, local inflammation, demyelination in the peripheral nervous system and mechanical allodynia. Minocycline is known to have neuroprotective and anti-inflammatory effects. Furthermore, relieve of neuropathic pain following minocycline administration was observed in a variety of animal models. Here, we investigated the effects of minocycline on rat EAN. Suppressive treatment with minocycline (50 mg/kg body weight daily immediately after immunization) significantly attenuated the severity and duration of EAN. Macrophage and T-cell infiltration and demyelination in sciatic nerves of EAN rats treated with minocycline were significantly reduced compared to phosphate-buffered saline (PBS)-treated EAN rats. mRNA expressions of matrix metallopeptidase-9, inducible nitric oxide synthase and pro-inflammatory cytokines interleukin-1 beta and tumour necrosis factor-alpha in EAN sciatic nerves were greatly decreased by administration of minocycline as well. Furthermore, minocycline attenuated mechanical allodynia in EAN rats and greatly suppressed spinal microglial activation. All together, our data showed that minocycline could effectively suppress the peripheral and spinal inflammation (immune activation) to improve outcome in EAN rats, which suggests that minocycline may be considered as a potential candidate of pharmacological treatment for autoimmune-mediated neuropathies.

Global hypomethylation defines a sub-population of reactive microglia/macrophages in experimental traumatic brain injury.

Global alterations in gene expression have been observed in different traumatic brain injury (TBI) models and are considered of crucial importance to the development of subsequent tissue injury and repair. Cytosine methylation is a well-known process of endogenous DNA modification in mammals and the primary mechanism responsible for changes in epigenetic gene expression. Here we have investigated the early global spatio-temporal changes of the status of cellular DNA methylation in a rat TBI model by immunohistochemistry and analyzed the effects of dexamethasone on these changes. Global cellular hypomethylation was seen as early as day 1 in pannecrosis and day 2 in peripannecrosis following TBI. A sub-population of reactive microglia/macrophages was identified as the major source of hypomethylated cells by double-staining experiments. Further, peripheral administration of dexamethasone suppressed this lesional hypomethylation at day 2 post-injury. In sum, our data suggest that lesional hypomethylation defines a sub-population of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Polyguanosine motif increases cellular oligonucleotide uptake in the brain cortex.

Cellular uptake and localization of fluorescein isothiocyanate-labelled or biotin-labelled CpG-oligonucleotides (oligonucleotides containing unmethylated CpG dinucleotides within specific flanking bases) with or without 3'-end continuous guanosines (polyG motif) were studied in the rat cortex. Twenty minutes after intracerebral microinjection, labelled CpG-oligonucleotide accumulated in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cells localized near the injection site. In the brain, polyG motif significantly increased the cellular uptake of phosphodiester CpG-oligonucleotide (P < 0.05) but not phosphothioate CpG-oligonucleotide (P > 0.05). These data not only provide in-vivo evidence for the local uptake and distribution of oligonucleotides in the brain cortex but also indicate that phosphodiester CpG-oligonucleotides containing a 3'-end polyG motif show an increased cellular uptake and thus might be promising alternate analogues for application to the brain.

Distribution of Foxp3(+) T-regulatory cells in experimental autoimmune neuritis rats.

T-regulatory cells expressing the forkhead box transcription factor 3 (Foxp3) play essential roles in immune homeostasis. Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. We investigated the distribution of Foxp3(+) cells in sciatic nerves, spleen and lymph nodes of EAN rats, and the influence of FTY720 on the localization of Foxp3(+) cells in EAN rats. In sciatic nerves of EAN rats, accumulation of Foxp3(+) cells was not seen during the pre-symptomatic phase (until Day 9) or during early or peak disease activity. In contrast, Foxp3(+) cell accumulation was regularly seen in the recovery from neurologic disease, suggesting a contribution of Foxp3(+) cells to the resolution of EAN. FTY720 was given at onset of EAN (Day 10) until Day 18. Following FTY720 administration, percentages of Foxp3(+) cells in EAN rats were increased in circulating blood, but reduced in lymph nodes compared to the PBS control. FTY720 treatment suppressed total numbers but increased percentages of Foxp3(+) cells in sciatic nerves of EAN rats. These data not only suggests a protective role of Foxp3(+) cells in EAN but also provides a potential way to alter localization of Foxp3(+) cells in vivo.

Expression of interleukin-16 in sciatic nerves, spinal roots and spinal cords of experimental autoimmune neuritis rats.

Experimental autoimmune neuritis (EAN) is a well-known animal model of Guillain-Barré Syndrome. In this study, we studied the spatiotemporal expression of interleukin-16 (IL-16) in the nervous system of EAN rats and pharmacological effects of minocycline on IL-16 expressions in EAN rats. In sciatic nerves and dorsal/ventral roots of EAN rats, IL-16+ cells, identified as macrophages and T cells, were mainly found to concentrate around blood vessels. However, in spinal cords, IL-16+ microglial cells were mainly found in lumbar dorsal horns. Massive IL-16+ cell accumulation in sciatic nerves and spinal roots was temporally correlated with severity of neurological signs of EAN. Furthermore, a strong correlation of IL-16+ cell accumulation with local demyelination in perivascular areas of sciatic nerves, and significant reduction of IL-16+ cell numbers in sciatic nerves and spinal cords by minocycline suggested a pathological contribution of IL-16+ cells in EAN. Taken together, robust IL-16+ cell accumulation in the nervous system and its temporal correlation with severity of neurological signs in EAN might suggest a pathological role of IL-16 in EAN, which makes IL-16 a potential pharmacological target.

Dexamethasone suppresses infiltration of RhoA+ cells into early lesions of rat traumatic brain injury.

Inflammatory cell infiltration is a major part of secondary tissue damage in traumatic brain injury (TBI). RhoA is an important member of Rho GTPases and is involved in leukocyte migration. Inhibition of RhoA and its downstream target, Rho-associated coiled kinase (ROCK), has been proven to promote axon regeneration and function recovery following injury in the central nervous system (CNS). Previously, we showed that dexamethasone, an immunosuppressive corticosteroid, attenuated early expression of three molecules associated with microglia/macrophages activation following TBI in rats. Here, the effects of dexamethasone on the early expression of RhoA have been investigated in brains of TBI rats by immunohistochemistry. In brains of rats treated with TBI alone, significant RhoA+ cell accumulation was observed at 18 h post-injury and continuously increased during our observed time period. The accumulated RhoA+ cells were distributed to the areas of pannecrosis and selective neuronal loss. Most accumulated RhoA+ cells were identified as active microglia/macrophages by double-labelling. Dexamethasone (1 mg/kg body weight) was intraperitoneally injected on day 0 and 2 immediately following brain injury. Numbers of RhoA+ cells were significantly reduced on day 1 and 2 following administration of dexamethasone but returned to vehicle control level on day 4. However, dexamethasone treatment did not change the proportion of RhoA+ cells. These observations suggest that dexamethasone has only a transient effect on early leukocyte recruitment.

FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves.

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune demyelinating inflammatory disease of the peripheral nervous system (PNS). T cells and macrophages are essential for the initiation and development of EAN. FTY720 acts as an agonist of sphingosine-1-phosphate receptors, resulting in inhibition of lymphocyte egress from secondary lymphoid tissues and thymocytes from thymus. This investigation describes the immunosuppressive effects of FTY720 in EAN, the animal model of autoimmune neuropathies. FTY720 (1 mg/kg body weight) completely suppressed paraparesis if administrated from the day of immunization. Furthermore, FTY720 greatly reduced the severity and duration of EAN even when administrated after the appearance of the first neurological sign. T cell, B cell, and macrophage infiltration and demyelination of sciatic nerves were significantly decreased in FTY720-treated EAN. Therefore, FTY720 might be a potential candidate for treatment of inflammatory neuropathies.

Expression of RhoA by inflammatory macrophages and T cells in rat experimental autoimmune neuritis.

RhoA is one of the best-studied members of Rho GTPases. Experimental autoimmune neuritis (EAN), which is characterized by infiltration of T cells and macrophages into the peripheral nervous system, is an autoantigen-specific T-cell-mediated animal model of human Guillain-Barré Syndrome. In this study, RhoA expression has been investigated in the dorsal/ventral roots of EAN rats by immunohistochemistry. A significant accumulation of RhoA+ cells was observed on Day 12, with a maximum around Day 15, correlating to the clinical severity of EAN. In dorsal/ventral roots of EAN, RhoA+ cells were seen in perivascular areas but also in the parenchyma. Furthermore, double-labelling experiments showed that the major cellular sources of RhoA were reactive macrophages and T cells. In conclusion, this is the first demonstration of the presence of RhoA in the dorsal/ventral roots of EAN. The time courses and cellular sources of RhoA together with the functions of RhoA indicate that RhoA may function to facilitate macrophage and T-cell infiltration in EAN and therefore could be a potential therapeutic target.

FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury.

FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo-compatibility complex class II (MHC-II) and endothelial-monocyte activating polypeptide II (EMAP-II). Accumulation of MHC-II(+) or EMAP-II(+) cells became significant 1 day after injury and continuously increased during the early time periods. Further, double-staining experiments confirmed that the major cellular sources of MHC-II were reactive macrophages, however MHC-II(+) cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC-II(+) macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP-II(+) macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP-II(+) and MHC-II(+) reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI.