Infectious myelitis.

Infections are an uncommon but very important etiology of myelitis as a correct diagnosis would allow for timely treatment and recovery. The term "myelitis" is generally used to describe an inflammatory pathologic process affecting the spinal cord and causing an interruption of the ascending and descending pathways, and, therefore, partial or complete loss of function. The onset may be acute or subacute, and the etiology may be cumbersome to determine. This article will review the most recently published literature regarding the infectious agents causing myelitis with an emphasis on diagnosis and treatment.

Rhombencephalitis / brainstem encephalitis.

Rhombencephalitis (RE) is a syndrome of multiple causes and multiple outcomes. Most authors now use the terms "rhombencephalitis" and "brainstem encephalitis" interchangeably even though anatomically they are slightly different. The etiologic categories of RE include infections, autoimmune diseases, and paraneoplastic syndromes (PNS). Listeria is the most common cause of infectious RE. Listeria RE primary occurs in healthy young adults. It usually occurs as a biphasic time course with a flu-like syndrome followed by brainstem dysfunction; 75% of patients have a cerebrospinal fluid (CSF) pleocytosis, and almost 100% have an abnormal brain MRI scan. Positive CSF and blood cultures are the most specific for diagnosis. Treatment primarily is with ampicillin. Enterovirus 71 is probably the second most common infectious cause of RE; however, 95% of cases have occurred in the Asian-Pacific region and there is no specific treatment. Herpes simplex virus (HSV) is the third most common infectious cause of RE, and about 80% of cases are caused by HSV1 and 20% by HSV2. About 50% only had involvement of the brainstem whereas the other 50% also had supratentorial involvement of the temporal and frontal lobes. Mortality with acyclovir treatment was 22% versus those not on acyclovir 75%. Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV6) have caused a few cases. The most common autoimmune etiology is Behçet disease. Over 90% of those with Behçet RE had abnormal MRI scans and 94% had a CSF pleocytosis. Treatment is with corticosteroids and immunosuppressive agents, but only 25% have complete recovery. Paraneoplastic causes are the third category of RE. Brain MRIs are usually normal; there is usually a CSF pleocytosis but the protein is usually normal. Often anti-neuronal antibodies can be found. Prognosis is poor and treatment is only partially beneficial. Because Listeria and HSV are the most common treatable acute causes of RE, we recommend empiric therapy with ampicillin and acyclovir for all cases after samples have been obtained from CSF and blood for cultures and the polymerase chain reaction (PCR). Antibiotics can be changed based upon MRI, culture results, PCR results, and antibody studies.

Quantitative differences in the immunomodulatory effects of Rebif and Avonex in IFN-ß 1a treated multiple sclerosis patients.

Interferon-ß (IFN-ß) is a current effective treatment for multiple sclerosis (MS) and exerts its therapeutic effects by down-modulating the systemic immune response and cytokine signaling. In clinical practice there are several formulations of interferon including a low dose of IFN-ß 1a formulation of 30 µg IM once weekly (Avonex) and a high dose formulation of 44 µg SC three times weekly (Rebif). Recent studies suggest that Rebif is more efficacious compared to Avonex in preventing relapses and decreasing MRI activity in relapsing remitting MS (RRMS) patients. This study examines whether there are quantitative gene expression changes in interferon-treated RRMS patients that can explain the difference in efficacy and side effects between Rebif and Avonex. Herein, RRMS patients were treated for three months with IFN-ß 1a and the levels of plasma cytokines and gene expression in peripheral blood mononuclear cells were examined. Thirty-two normal subjects were compared to thirty-two RRMS patients, of which ten were treated with Rebif and ten with Avonex. Rebif and Avonex both significantly and equally suppressed plasma TNF-α and IL-6 levels. Rebif suppressed IL-13 significantly more than Avonex. Rebif also significantly suppressed the levels of the chemokines CCL17 and RANTES, the protease ADAM8, and COX-2 at a higher degree compared to Avonex. The STAT1-inducible genes IP-10 and caspase 1 were significantly increased with Rebif compared to Avonex. In conclusion, the higher dosed, more frequently administered IFN-ß 1a Rebif when compared to IFN-ß 1a Avonex has more potent immunomodulatory effects. These quantitative results might relate to efficacy and side-effect profile of the two IFN-ß 1a formulations and provide prospective practical clinical tools to monitor treatment and adjust dosage.

Interferon-beta treatment in multiple sclerosis attenuates inflammatory gene expression through inducible activity of the phosphatase SHP-1.

Interferon-beta is a current treatment for multiple sclerosis (MS). Interferon-beta is thought to exert its therapeutic effects on MS by down-modulating the immune response by multiple potential pathways. Here, we document that treatment of MS patients with interferon beta-1a (Rebif) results in a significant increase in the levels and function of the protein tyrosine phosphatase SHP-1 in PBMCs. SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and CNS demyelination as evidenced in mice deficient in SHP-1. In order to examine the functional significance of SHP-1 induction in MS PBMCs, we analyzed the activity of proinflammatory signaling molecules STAT1, STAT6, and NF-kappaB, which are known SHP-1 targets. Interferon-beta treatment in vivo resulted in decreased NF-kappaB and STAT6 activation and increased STAT1 activation. Further analysis in vitro showed that cultured PBMCs of MS patients and normal subjects had a significant SHP-1 induction following interferon-beta treatment that correlated with decreased NF-kappaB and STAT6 activation. Most importantly, experimental depletion of SHP-1 in cultured PBMCs abolished the anti-inflammatory effects of interferon-beta treatment, indicating that SHP-1 is a predominant mediator of interferon-beta activity. In conclusion, interferon-beta treatment upregulates SHP-1 expression resulting in decreased transcription factor activation and inflammatory gene expression important in MS pathogenesis.

SHP-1 deficiency and increased inflammatory gene expression in PBMCs of multiple sclerosis patients.

Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination.

Phosphorothiolate analogues of phosphatidylinositols as assay substrates for phospholipase C.

Accurate measurement of phosphatidylinositol-specific phospholipase C (PI-PLC) activity is important in view of the key role of this enzyme in signal-transduction pathways. In this work we synthesized enantiomerically pure phosphorothiolate analogues of all natural PI-PLC substrates, including those of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2), 4-phosphate (PI-4-P), 5-phosphate (PI-5-P) and unphosphorylated PI, in both long- and short-chain versions. The enzymatic cleavage of these substrates produces thiol analogues of diacyl glycerol, which can be quantified by UV absorbance after treatment with dipyridyl disulfide. The monodisperse dihexanoyl derivatives are suitable substrates for PI-PLC assay: they give rise to high enzyme activity, and provide excellent linear kinetic responses. For all substrates, we found a good linear correlation between the reaction rate and the amount of enzyme; this indicated the suitability of this assay for enzyme quantification. The short-chain substrates enable the enzyme specificity with variously phosphorylated inositol head groups to be established--unobstructed by substrate aggregation, "scooting" kinetics on micelles, or surface dilution effects. The kinetic results indicated allosteric behavior of PLC for all substrates tested. We found that substrates phosphorylated at the inositol 4-position (phosphorothiolate analogues of PI-4,5-P2 and PI-4-P) displayed very similar kinetic properties, and were cleaved with approximately 20- to 30-fold higher activity than the 4-nonphosphorylated substrates (analogues of PI-5-P and PI). Hence it appears that interactions between the enzyme and the 4-phosphate group of the substrate, but not its 5-phosphate group, is important for PI-PLC catalysis. In addition, the binding affinities of all four substrate types were found to be quite similar; this indicates that the energy of enzyme interaction with the 4-phosphate group is directed almost entirely to catalysis.

Post-infectious encephalomyelitis.

The term post-infectious encephalomyelitis (PIEM) is frequently used interchangeably with acute disseminated encephalomyelitis (ADEM), although technically PIEM occurs after a known infection whereas with ADEM there is no antecedent infection. PIEM represents one of the primary demyelinating disorders of the central nervous system, along with multiple sclerosis and Devic's disease. There is no specific diagnostic test for any of these conditions and at onset it may be difficult to differentiate between ADEM and the first attack of multiple sclerosis. However, there are clinical and magnetic resonance imaging features that allow differentiation between PIEM/ADEM and a relapsing disease such as multiple sclerosis. Some patients improve spontaneously; most improve with methylprednisolone. If that fails, plasma exchange or intravenous immunoglobulin may be effective.

Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase.

A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.

Vibrationally enhanced hydrogen tunneling in the Escherichia coli thymidylate synthase catalyzed reaction.

The enzyme thymidylate synthase (TS) catalyzes a complex reaction that involves forming and breaking at least six covalent bonds. The physical nature of the hydride transfer step in this complex reaction cascade has been studied by means of isotope effects and their temperature dependence. Competitive kinetic isotope effects (KIEs) on the second-order rate constant (V/K) were measured over a temperature range of 5-45 degrees C. The observed H/T ((T)V/K(H)) and D/T ((T)V/K(D)) KIEs were used to calculate the intrinsic KIEs throughout the temperature range. The Swain-Schaad relationships between the H/T and D/T V/K KIEs revealed that the hydride transfer step is the rate-determining step at the physiological temperature of Escherichia coli (20-30 degrees C) but is only partly rate-determining at elevated and reduced temperatures. H/D KIE on the first-order rate constant k(cat) ((D)k = 3.72) has been previously reported [Spencer et al. (1997) Biochemistry 36, 4212-4222]. Additionally, the Swain-Schaad relationships between that (D)k and the V/K KIEs reported here suggested that at 20 degrees C the hydride transfer step is the rate-determining step for both rate constants. Intrinsic KIEs were calculated here and were found to be virtually temperature independent (DeltaE(a) = 0 within experimental error). The isotope effects on the preexponential Arrhenius factors for the intrinsic KIEs were A(H)/A(T) = 6.8 +/- 2.8 and A(D)/A(T) = 1.9 +/- 0.25. Both effects are significantly above the semiclassical (no-tunneling) predicted values and indicate a contribution of quantum mechanical tunneling to this hydride transfer reaction. Tunneling correction to transition state theory would predict that these isotope effects on activation parameters result from no energy of activation for all isotopes. Yet, initial velocity measurements over the same temperature range indicate cofactor inhibition and result in significant activation energy on k(cat) (4.0 +/- 0.1 kcal/mol). Taken together, the temperature-independent KIEs, the large isotope effects on the preexponential Arrhenius factors, and a significant energy of activation all suggest vibrationally enhanced hydride tunneling in the TS-catalyzed reaction.

Application of Brønsted-type LFER in the study of the phospholipase C mechanism.

Phosphatidylinositol-specific phospholipase C cleaves the phosphodiester bond of phosphatidylinositol to form inositol 1,2-cyclic phosphate and diacylglycerol. This enzyme also accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable model enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship (LFER). In this work, we conducted a study of Brønsted-type relationship (log k = beta(lg) pK(a) + C) to compare mechanisms of enzymatic and nonenzymatic reactions, confirm the earlier proposed mechanism, and assess further the role of hydrophobicity in the leaving group as a general acid-enabling factor. The observation of the high negative Brønsted coefficients for both nonenzymatic (beta(lg) = -0.65 to -0.73) and enzymatic cleavage of aryl and nonhydrophobic alkyl inositol phosphates (beta(lg) = -0.58) indicates that these reactions involve only weak general acid catalysis. In contrast, the enzymatic cleavage of hydrophobic alkyl inositol phosphates showed low negative Brønsted coefficient (beta(lg) = -0.12), indicating a small amount of the negative charge on the leaving group and efficient general acid catalysis. Overall, our results firmly support the previously postulated mechanism where hydrophobic interactions between the enzyme and remote parts of the leaving group induce an unprecedented negative-charge stabilization on the leaving group in the transition state.