Porphobilinogen deaminase deficiency in mice causes a neuropathy resembling that of human hepatic porphyria.

Acute intermittent porphyria (AIP) is a human disease resulting from a dominantly inherited partial deficiency of the heme biosynthetic enzyme, porphobilinogen deaminase (PBGD). The frequency of the trait for AIP is 1/10,000 in most populations, but may be markedly higher (1/500) in psychiatric patients. The clinical expression of the disease is characterized by acute, life-threatening attacks of 'porphyric neuropathy' that include abdominal pain, motor and sensory neurological deficits and psychiatric symptoms. Attacks are frequently precipitated by drugs, alcohol and low caloric intake. Identical symptoms occur in other hepatic porphyrias. To study the pathogenesis of the neurologic symptoms of AIP we have generated Pbgd-deficient mice by gene targeting. These mice exhibit the typical biochemical characteristics of human AIP, notably, decreased hepatic Pbgd activity, increased delta-aminolevulinic acid synthase activity and massively increased urinary excretion of the heme precursor, delta-aminolevulinic acid after treatment with drugs such as phenobarbital. Behavioural tests reveal decreased motor function and histopathological findings include axonal neuropathy and neurologic muscle atrophy.

Increased levels of inflammatory chemokines in amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is classically assumed to be a neurodegenerative disorder. Inflammation has been observed in CNS tissue in ALS patients. We investigated the expression and prognostic relevance of proinflammatory chemokines in ALS. METHODS: We analyzed nine chemokines, eotaxin, eotaxin-3, IL-8, IP-10, MCP-1, MCP-4, macrophage derived chemokine (MDC), macrophage inflammatory protein-1beta (MIP-1beta), and serum thymus and activation- regulated chemokine (TARC) in serum and cerebrospinal fluid (CSF) of 20 ALS- and 20 non-inflammatory neurological disease (NIND)-patients. RESULTS: MCP-1 and IL-8 levels in CSF in ALS were significantly higher than in NIND (1304 pg/ml vs. 1055 pg/ml, P = 0.013 and 22.7 pg/ml vs. 18.6 pg/ml, P = 0.035). The expression of MCP-1 and IL-8 were higher in CSF than in serum (P < 0.001). There was a trend towards higher MCP-1 CSF levels in ALS patients with shorter time between first symptoms and diagnosis (r = -0.407; P = 0.075). CONCLUSIONS: We confirmed previous findings of increased MCP-1 levels in CSF of ALS patients. Furthermore, increased levels of IL-8 in CSF suggest a stimulation of a proinflammatory cytokine cascade after microglia activation. We found a tendency for higher MCP-1 values in patients with a shorter diagnostic delay, who are known to have also a shorter survival. This may suggest an association of higher MCP-1 levels with rapidly progressing disease.

Motor neuropathy in porphobilinogen deaminase-deficient mice imitates the peripheral neuropathy of human acute porphyria.

Acute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias' enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD-/-) imitate acute porphyria through massive induction of hepatic delta-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD-/- mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD-/- mice exhibit a marked decrease in large-caliber (>8 microm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD-/- mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor delta-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy.

Genetic associations with brain cortical thickness in multiple sclerosis.

Multiple sclerosis (MS) is characterized by temporal and spatial dissemination of demyelinating lesions in the central nervous system. Associated neurodegenerative changes contributing to disability have been recognized even at early disease stages. Recent studies show the importance of gray matter damage for the accrual of clinical disability rather than white matter where demyelination is easily visualized by magnetic resonance imaging (MRI). The susceptibility to MS is influenced by genetic risk, but genetic factors associated with the disability are not known. We used MRI data to determine cortical thickness in 557 MS cases and 75 controls and in another cohort of 219 cases. We identified nine areas showing different thickness between cases and controls (regions of interest, ROI) (eight of them were negatively correlated with Kurtzke's expanded disability status scale, EDSS) and conducted genome-wide association studies (GWAS) in 464 and 211 cases available from the two data sets. No marker exceeded genome-wide significance in the discovery cohort. We next combined nominal statistical evidence of association with physical evidence of interaction from a curated human protein interaction network, and searched for subnetworks enriched with nominally associated genes and for commonalities between the two data sets. This network-based pathway analysis of GWAS detected gene sets involved in glutamate signaling, neural development and an adjustment of intracellular calcium concentration. We report here for the first time gene sets associated with cortical thinning of MS. These genes are potentially correlated with disability of MS.

Molecular characterization of the murine Coh locus: an amino acid difference at position 117 confers high and low coumarin 7-hydroxylase activity in P450coh.

Coumarin 7-hydroxylase (P450coh) and steroid 15 alpha-hydroxylase (P450(15 alpha) are encoded by members within the mouse 2A subfamily. Since P450coh activity is regulated by the Coh locus, we characterized P450coh cDNAs in strains having high coumarin 7-hydroxylase activity (CohH homozygote) including 129/J and DBA/2J, and compared them with P450coh cDNAs in low activity strains (CohL homozygote) C57BL/6J, C3H/HeJ and AKR/J. The nucleotide sequences of these two cDNAs differ by a single base, which results in an amino acid difference at position 117 (Val in P450cohH and Ala in P450cohL). The CohH phenotype exhibits approximately 10-fold greater Vmax and four-fold lower Km values than those in the CohL. Male 129 AKF1/J expresses approximately equal amounts of P450cohH and P450cohL mRNAs, associated with two Coh alleles. The levels of P450coh and P450(15 alpha) mRNAs in the F1 offspring suggested that a trans-acting factor(s) appeared to regulate the expressions of the P450 genes. A recent duplication in the ancestral mouse established the line of descent to P450(15 alpha) from the ancestral P450coh gene. During evolution, amino acid substitutions have selectively occurred at positions which alter the enzyme's substrate specificity and increase in the specific activity. Consistent with an important role of natural selection in the evolution of these genes is the relatively high nonsynonomous substitution rates on the P450(15 alpha) and the P450coh branches. As a result of these evolution events, the gene family consists of members which exhibit an extremely high degree of structural similarity, but very divergent hydroxylase activities and modes of regulation.

Metabolism of nomifensine after oral and intravenous administration.

The metabolism of nomifensine was studied after single oral and intravenous administration and after 2 weeks of oral dosing. The three principal metabolites reached maximum plasma concentrations rapidly (in 1 to 1.5 hours) after nomifensine administration. Less than 10% was detected as a free, unconjugated form. All three metabolites were eliminated rapidly (elimination t1/2 values between 6.8 and 9.0 hours). Only very low concentrations of free metabolites were found in plasma after 24 hours of nomifensine administration. AUC values for free metabolites were between 0.27 to 0.46 hr X mumol/L after all nomifensine schedules. Two weeks of dosing had no significant influence on the elimination t1/2 or AUC values of the metabolites, indicating no change in the hydroxylation and methylation reactions. In addition, there were no changes in the conjugation reactions during prolonged nomifensine dosing. Nomifensine has a very short t1/2 and no tendency for accumulation after repeated doses. We conclude that nomifensine's clinical pharmacokinetic profile is not significantly changed by the kinetic behavior of its three main metabolites after the usual maintenance doses.

Disposition of nomifensine after acute and prolonged dosing.

The pharmacokinetics of nomifensine were studied after single oral and intravenous doses. The effect of prolonged oral dosing on the pharmacokinetics of nomifensine was also evaluated. Nomifensine was rapidly absorbed from the gastrointestinal tract. The peak concentration of free nomifensine (0.18 mumol/L) was reached at 1.13 hours after dosing. The highest concentration after the intravenous dose was 1.21 mumol/L. The elimination t1/2 after a single dose was about 4 hours regardless of the route of administration. Nomifensine was extensively distributed in body fluids and tissues, with an apparent volume of distribution of 8.69 L/kg. The AUC of free nomifensine after oral dosing was only 26.5% of that after intravenous infusion. Absorption from the gastrointestinal tract was complete, and the AUCs of total nomifensine were equal after all treatments. The main reason for limited bioavailability seems to be extensive first-pass metabolism during the absorption process. The AUC of free nomifensine decreased substantially (from 0.78 to 0.32 hr X mumol/L) and the elimination t1/2 was shortened (from 4.39 to 2.11 hours) after a 2-week dosing period. These effects suggest marked induction of the metabolizing enzymes. An increase in nomifensine dosage may be needed in some patients to maintain a full therapeutic effect.

Neurofilament heavy chain in CSF correlates with relapses and disability in multiple sclerosis.

OBJECTIVE: Neurodegeneration is now accepted as a pathologic hallmark of multiple sclerosis (MS). We sought to discover whether CSF levels of neurofilament heavy chain protein (NfH(SMI35)) correlate with disability, disease activity, or specific stages of MS. METHODS: An electrochemiluminescence immunoassay was used to retrospectively measure NfH(SMI35) in CSF of patients with clinically isolated syndrome (CIS) (n = 63), relapsing-remitting multiple sclerosis (RRMS) (n = 39), secondary progressive multiple sclerosis (SPMS) (n = 25), primary progressive multiple sclerosis (PPMS) (n = 23), or controls (n = 73). Cell count and CSF levels of immunoglobulin and albumin were also measured. RESULTS: CSF levels of NfH(SMI35) increased with age in controls (r(s) = 0.50, p < 0.0001) and CIS (r(s) = 0.50, p < 0.0001); this effect was less pronounced in RRMS (r(s) = 0.35, p = 0.027) and absent in SPMS/PPMS. After age correction, NfH(SMI35) levels were found to be higher in all disease stages compared to control. Relapses were associated with higher CSF NfH(SMI35) values compared with stable disease. NfH(SMI35) levels correlated with EDSS scores in patients with CIS and RRMS (r(s) = 0.33, p = 0.001), and during relapse (r(s) = 0.35, p = 0.01); the correlation was most prominent in RRMS during relapse (r(s) = 0.54, p = 0.01). This was not the case for any of the other CSF markers examined. CONCLUSIONS: Neuronal loss is a feature of aging, and the age-dependent increase of CSF NfH(SMI35) suggests that this loss accelerates over time. For MS, increased NfH(SMI35) levels reflect the superimposed presence of further neurodegenerative processes. Evaluation of NfH(SMI35) levels is likely to provide a useful surrogate for measuring the rate of neurodegeneration in MS. Furthermore, the dissociation of NfH(SMI35) levels with biomarkers of inflammation suggests that the mechanisms responsible for their production are at least partly independent.

FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis.

BACKGROUND: The oral immunomodulator FTY720 has shown efficacy in patients with relapsing multiple sclerosis (MS). FTY720 functionally antagonizes sphingosine 1-phosphate receptor-1 (S1P1) on T cells and consequently inhibits S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Little is known about the phenotype and function of T cells remaining in peripheral blood during long-term FTY720 treatment. METHODS: T cells from FTY720-treated, interferon-beta (IFNbeta)-treated and untreated patients with MS, and healthy donors (HD) were analyzed with respect to T cell subpopulation composition, proliferation, and cytokine production. RESULTS: In FTY720-treated patients (n = 16), peripheral blood CD4+ and CD8+ T cell counts were reduced by approximately 80% and 60% when compared to the other groups (IFN beta: n = 7; untreated: n = 5; HD: n = 10). This related to selective reduction of naive (CCR7+CD45RA+) and central memory (CCR7+CD45RA-) T cells (TCM), and resulted in a relative increase of peripheral effector memory (CCR7-CD45RA- [TEM] and CCR7-CD45RA+ [TEMRA]) T cells. The remaining blood T cell populations displayed a reduced potential to secrete IL-2 and to proliferate in vitro, but rapidly produced interferon-gamma upon reactivation, confirming a functional TEM/TEMRA phenotype. Neither FTY720 nor FTY720-P directly suppressed proliferation or cytokine production by T cells. CONCLUSION: Therapeutic dosing of FTY720 reduces naïve T cells and TCM, but not TEM, in blood, without affecting T cell function. This is presumably because naive T cells and TCM express the homing receptor CCR7, allowing recirculation to secondary lymphoid tissues on a regular basis and, thus, trapping of the cells by FTY720 in lymph nodes.

Qualitative and quantitative analysis of antibody response against IFNbeta in patients with multiple sclerosis.

To date, inter- and intra-laboratory consistency of binding assays for measuring anti-interferon (IFN)beta antibodies has not been assessed. In this investigation, two independent laboratories tested a library of 80 serum specimens obtained from multiple sclerosis (MS) patients treated with IFNbeta. For binding antibodies (BAbs) evaluations, each laboratory used both a capture-ELISA (cELISA) and an enzyme-immuno-assay (EIA), which is commercially available. Samples were also tested for neutralizing antibodies (NAbs). Data demonstrated good intra-laboratory reliability (r(pearson) > or = 0.86), and a good overall agreement between the results obtained from the two centers, using both the cELISA (69/80 of observed agreements) and the EIA (67/80). Accordingly, kappa coefficients (K) showed good concurrence (K > or = 0.651). There was also substantial agreement between cELISA and EIA measurements, as performed in both centers (Orbassano, 66/80, K = 0.631; Basel, 70/80, K = 0.717). However, by comparing NAbs and BAbs titers obtained with both assays, we found that a high degree of BAb-negative samples were positive in NAb-assay. Thus, our study does not support the usefulness of ELISA-based BAb assays as a screening tool for NAbs. Otherwise, BAb-assays can be used as a confirmation test, indicating that the decrease of the biological effects is due to antibodies. In this context, both ELISA-based assays are equally reliable techniques.

Gelatinase B [matrix metalloproteinase (MMP)-9] and collagenases (MMP-8/-13) are upregulated in cerebrospinal fluid during aseptic and bacterial meningitis in children.

We investigated the protein expression of gelatinases [matrix metalloproteinase (MMP)-2 and -9] and collagenases (MMP-8 and -13) in cerebrospinal fluid (CSF) from patients with bacterial (BM, n = 17) and aseptic (AM, n = 14) meningitis. In both, MMP-8 and -9 were increased in 100% of patients, whereas MMP-13 was detectable in 53% and 82% respectively. Three patients with clinical signs of meningitis, without CSF pleocytosis, scored positive for all three MMPs. MMP-8 appeared in two isoforms, granulocyte-type [polymorphonuclear cell (PMN)] and fibroblast/macrophage (F/M) MMP-8. Analysis of kinetic changes from serial lumbar punctures showed that these MMPs are independently regulated, and correlate only partly with CSF cytosis or levels of the endogenous inhibitor, tissue inhibitor of matrix metalloproteinase-1. In vitro, T cells, peripheral blood mononuclear cells (PBMCs) and granulocytes (PMN) release MMP-8 and -9, whereas MMP-13 could be found only in the former two cell types. Using models of exogenous (n-formyl-Met-Leu-Phe, T cell receptor cross-linking) and host-derived stimuli (interleukin-2), the kinetics and the release of the MMP-8, -9 and -13 showed strong variation between these immune cells and suggest release from preformed stocks. In addition, MMP-9 is also synthesized de novo in PBMCs and T cells. In conclusion, invading immune cells contribute only partially to MMPs in CSF during meningitis, and parenchymal cells are an equally relevant source. In this context, in patients with clinical signs of meningitis, but without CSF pleocytosis, MMPs seem to be a highly sensitive marker for intrathecal inflammation. The present data support the concept that broad-spectrum enzyme inhibition targeting gelatinases and collagenases is a potential strategy for adjunctive therapy in infectious meningitis.

Differential regulation of metzincins in experimental chronic renal allograft rejection: potential markers and novel therapeutic targets.

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

Selective and sensitive high-performance liquid chromatographic assay for the metabolites of nomifensine in human plasma.

A selective high-performance liquid chromatographic method for the determination of the three metabolites of nomifensine in human plasma is described. All metabolites and the internal standard, mexiletine, are extracted with diethyl ether and then back-extracted into an acidic aqueous phase. After subsequent extraction into diethyl ether the metabolites are analysed by high-performance liquid chromatography. A reversed-phase C18 column is used with a mobile phase of dioxane-methanol-potassium phosphate buffer (pH 2.25). The sensitivity of the method is 0.007 micromol/l for all metabolites. Extraction efficiencies are 84.6%, 75.8%, and 78.2% for 4'-hydroxynomifensine, 4'-hydroxy-3'-methoxynomifensine and 3'-hydroxy-4'-methoxynomifensine, respectively. The reproducibility of the method is good, the coefficients of variation (%) varying between 2.1% and 9.9% in the concentration range 0.05-1.00 micromol/l. The procedure was applied to human plasma samples from a volunteer who had received a single oral dose of nomifensine. The method is accurate and sensitive for pharmacokinetic studies on the metabolites of nomifensine.

High-performance liquid chromatographic determination of bupivacaine in human serum.

A selective high-performance liquid chromatographic method for the determination of bupivacaine in human serum is described. The technique is based on a single extraction of the drug from alkalinized serum with a mixture of hexane-isopropanol-chloroform. Desmethyldoxepin is used as internal standard. The chromatographic system consists of a home-packed Nucleosil C8 (10 microns) column; the mobile phase is acetonitrile--0.05 M potassium phosphate buffer (pH 3.3) (28:72, v/v). The method can accurately measure serum bupivacaine concentrations down to 20 micrograms/l using 500 microliters of sample. The coefficient of variation for intra-assay variability of bupivacaine is 2.1% (n = 13) and for inter-assay variability of bupivacaine 5.7% (n = 11) at 1.00 mg/l. The calibration graph is linear over the range 0.02-5.00 mg/l and the extraction efficiency is 91.8 +/- 3.8% (+/- S.D., n = 7). The method is accurate and sensitive for both clinical and pharmacokinetic studies on bupivacaine in man. The method is applied to the analysis of serum samples obtained from orthopaedic patients during both spinal and epidural analgesia.

Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino-acid residue.

As a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015 alpha and P450coh, for example, which are expressed in female liver and male kidney cells, catalyse 15 alpha-hydroxylation of delta 4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues. To determine the structural basis of the different substrate specificities of P45015 alpha and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.

Bupivacaine with and without adrenaline in interscalene brachial plexus blockade. Studies in patients with rheumatoid arthritis.

The action of adrenaline on the pharmacokinetics of bupivacaine has been tested during two successive interscalene brachial plexus blocks in 10 patients with rheumatoid arthritis. The mean venous serum Cmax of bupivacaine after using it with or without adrenaline 1:200000 were 1.49 +/- 0.41 micrograms ml-1 and 2.46 +/- 0.85 micrograms ml-1, respectively. In spite of relatively high total serum concentrations, we could not detect any evidence of toxicity from bupivacaine. Significant tachycardia was seen after bupivacaine with adrenaline, but systolic and diastolic arterial pressures did not change significantly in any session. Marked subjective side effects were noticed only after bupivacaine with adrenaline (shivering twice and palpitations once). The serum protein bound fraction of bupivacaine was higher in rheumatic patients than in our healthy controls: 97.1 +/- 2.4% and 91.3 +/- 3.6%, respectively. Thus bupivacaine as a local anaesthetic agent seems to be even safer in patients with rheumatoid arthritis than in normal healthy volunteers, because of lower free fraction in the former.

Rapid high-pressure liquid chromatographic method for analysis of phenoxymethylpenicillin in human serum.

A selective high-pressure liquid chromatographic method for the determination of phenoxymethylpenicillin in human serum is described. The technique is based on the single extraction of the drug from acidified serum with diethyl ether. Chloramphenicol is used as internal standard. The chromatographic system consists of a reversed-phase C-18 column; the mobile phase is acetonitrile-0.01 M potassium acetate buffer (20:80 [vol/vol]; pH 6.5). The method can accurately measure serum penicillin concentrations down to 30 micrograms/liter with 500 microliter of sample. The coefficient of variation for intraassay variability of penicillin is between 1.5 and 4.9% in the range of 0.125 to 16.00 mg/liter. The extraction efficiency is 78.5 +/- 6.8% (+/- standard deviation; n = 9), and the calibration graph is linear in the concentration range studied. Pharmacokinetic data, obtained with the present method from seven healthy volunteers, are presented.

Lack of effect of cimetidine on the pharmacokinetics of bupivacaine in healthy subjects.

1. The possibility of a pharmacokinetic interaction between the H2-receptor antagonist cimetidine and the long-acting local anaesthetic agent bupivacaine was studied in seven healthy, non-smoking volunteers. 2. The study consisted of two sessions at a minimum interval of 4 days. In a randomized, crossover fashion, the volunteers received bupivacaine HCl 1.4 mg kg-1 by i.m. injection at two occasions, once after no premedication, and once after two oral doses of 400 mg cimetidine. The concentrations of bupivacaine and its metabolites, 4'-hydroxybupivacaine and desbutylbupivacaine, were assayed by h.p.l.c., in serum up to 8 h and in urine fractions up to 24 h. 3. No influence of cimetidine on the pharmacokinetics of bupivacaine or on the serum cumulation of urinary recovery of its measured metabolites was detected. 4. These data suggest that cimetidine may be used safely as a premedication before local anaesthetic procedures with bupivacaine.