Adenosine A2A receptor agonist ameliorates EAE and correlates with Th1 cytokine-induced blood brain barrier dysfunction via suppression of MLCK signaling pathway.

INTRODUCTION: Multiple sclerosis (MS) disease activity is associated with blood-brain barrier (BBB) disruption, which is mediated by inflammatory cytokines released by CD4+ lymphocytes. To assess the effects of adenosine A2A receptors on BBB permeability in vitro and in vivo. METHODS: A2A receptor expression was detected by immunostaining in experimental autoimmune encephalomyelitis (EAE) C57BL/6 mice immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 , and human MS brain. F-actin and the tight junction protein Claudin-5 were assessed in endothelial cells treated with an A2A receptor specific agonist (CGS-21680) after Th1 cytokine stimulation. EAE mice were divided into control and CGS-21680 (50 µg/kg, i.p., daily) groups. Disease scores were recorded daily to evaluate neurological impairment. The effects of A2A receptor on inflammation and demyelination were assessed after euthanasia by immunostaining or histology; BBB permeability was measured by sodium fluoride (Na-F) and FITC-dextran amounts. RESULTS: Endothelial A2A receptor was detected in demyelination areas of MS brain samples. In EAE lesions, A2A receptor was expressed in the endothelium in association with immune cell infiltration. Treatment with CGS-21680 counteracted the effects of Th1 cytokines on endothelial cells in vitro, preventing the reduction of tight junction protein expression and stress fiber formation. The effects of A2A receptor activation were correlated with MLCK phosphorylation signaling repression. In EAE, A2A receptor agonist decreased BBB permeability and inhibited EAE neurologic deficiency in mice. CONCLUSIONS: A2A receptor activation at EAE onset helps reduce the effects of Th1 stimulation on BBB permeability, indicating that A2A receptor mediates BBB function in CNS demyelinated disease.

Progressive multiple sclerosis cerebrospinal fluid induces inflammatory demyelination, axonal loss, and astrogliosis in mice.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination and neurodegeneration throughout the CNS, which lead over time to a condition of irreversible functional decline known as progressive MS. Currently, there are no satisfactory treatments for this condition because the mechanisms that underlie disease progression are not well understood. This is partly due to the lack of a specific animal model that represents progressive MS. We investigated the effects of intracerebroventricular injections of cerebrospinal fluid (CSF) derived from untreated primary progressive (PPMS), secondary progressive (SPMS), and relapsing/remitting (RRMS) MS patients into mice. We found discrete inflammatory demyelinating lesions containing macrophages, B cell and T cell infiltrates in the brains of animals injected with CSF from patients with progressive MS. These lesions were rarely found in animals injected with RRMS-CSF and never in those treated with control-CSF. Animals that developed brain lesions also presented extensive inflammation in their spinal cord. However, discrete spinal cord lesions were rare and only seen in animals injected with PPMS-CSF. Axonal loss and astrogliosis were seen within the lesions following the initial demyelination. In addition, Th17 cell activity was enhanced in the CNS and in lymph nodes of progressive MS-CSF injected animals compared to controls. Furthermore, CSF derived from MS patients who were clinically stable following therapy had greatly diminished capacity to induce CNS lesions in mice. Finally, we provided evidence suggesting that differential expression of pro-inflammatory cytokines present in the progressive MS CSF might be involved in the observed mouse pathology. Our data suggests that the agent(s) responsible for the demyelination and neurodegeneration characteristic of progressive MS is present in patient CSF and is amenable to further characterization in experimental models of the disease.

Detection and toxin typing of Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples by PCR.

Since current microbiology methods are not suitable to detect Clostridium perfringens in formalin-fixed, paraffin-embedded tissue samples, we developed a PCR assay to detect toxin-encoding genes and the 16S rRNA gene of C. perfringens. We successfully detected and genotyped C. perfringens in tissue sections from two autopsy cases.

Fulminant and fatal gas gangrene of the stomach in a healthy live liver donor.

A 57-year-old male with a history of hypercholesterolemia and anxiety but otherwise in good health volunteered to donate the right lobe of his liver to his brother. The operation was performed uneventfully, without transfusion. Postoperatively he did well, until he developed tachycardia, profound hypotension, and coffee ground emesis on postoperative day 3. Despite resuscitative measures, he arrested and expired. Autopsy demonstrated gas gangrene of the stomach as the underlying cause of the hemorrhage and numerous colonies of Gram-positive bacilli were identified. Subsequent polymerase chain reaction (PCR) analysis identified these bacteria to be Clostridium perfringens (C. perfringens) type D. This patient's death was devastating, both to his family and his medical team. The impact of his death has transcended that of an individual occurrence. In conclusion, herein we present the facts and discuss this extraordinary example of florid clostridial infection and toxin-mediated shock. It was completely unexpected and probably unpreventable, and its cause was almost inconceivable.