Autoreactivity against matrilin-1 in a patient with relapsing polychondritis.

Relapsing polychondritis (RP) is a rare inflammatory disease of cartilage. Chondritis of the auricular, nasal, and tracheal cartilages predominates in this disease, suggesting a response to a tissue-specific antigen. One potential antigen is matrilin-1, a cartilage matrix protein found uniquely in the tracheal, auricular, and nasal cartilage of adults. We describe herein a patient with RP who had both a humoral and a cellular immune response directed toward the cartilage matrix protein matrilin-1.

A study of topiramate pharmacokinetics and tolerability in children with epilepsy.

The pharmacokinetic and safety profile of topiramate as adjunctive therapy was assessed in pediatric patients with epilepsy in an open-label, 4-week, single-center study. Six children from each of the following age groups were enrolled: 4-7 years, 8-11 years, and 12-17 years. Patients received topiramate 1 mg/kg/day for 1 week, with subsequent progressive weekly increases in dosage to 3, 6, and then 9 mg/kg/day or 800 mg/day, whichever was less. Topiramate oral plasma clearance (CI/F) was independent of dose, and steady-state plasma concentrations increased in proportion to dose. Weight-normalized topiramate CL/F was higher (P = 0.003) in pediatric patients receiving enzyme-inducing concomitant antiepileptic drugs (AEDs) (mean = 70.1 ml/minute/70 kg) than in those not receiving enzyme-inducing AEDs (mean = 33.1 mL/ minute/kg). Topiramate CL/F in children was approximately 50% greater than that observed in adults regardless of the type of concomitant AED therapy. Thus steady-state plasma topiramate concentrations for the same mg/kg dose will be approximately 33% lower in pediatric patients than in adult patients. The most frequently reported treatment-emergent adverse events considered related to topiramate therapy included anorexia, fatigue, and nervousness, and no patient discontinued therapy. This study indicates that, in children 4-17 years of age, topiramate has linear pharmacokinetics, 50% higher clearance than in adults, and is generally well tolerated.

Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis.

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.

Oncostatin M stimulates excessive extracellular matrix accumulation in a transgenic mouse model of connective tissue disease.

Oncostatin M (OM), a member of the IL-6 gene family, stimulates a variety of functions implicated in wound repair. Transgenic mice that express this cytokine in islet beta-cells develop a connective tissue disorder that typifies excessive healing with severe fibrosis and lymphocytic infiltration. To compare this phenotype with the normal progression of connective tissue disease, we measured the expression patterns of genes encoding proinflammatory cytokines, fibrogenic cytokines, and ECM components by in situ hybridization. To test whether the OM effect was caused by its ability to regulate IL-6, we crossed the OM transgene into IL-6-deficient mice. Our data suggest that the fibrosis in these animals is not a secondary consequence of inflammation, or IL-6 expression, but is a direct effect by OM on extracellular matrix production. In a separate experiment, we observed that OM could regulate vasoactive intestinal peptide gene expression in the neurons that innervate the transgenic pancreas. This nerve healing response, in combination with its fibrogenic activity, suggests that OM functions downstream of inflammation in the wound repair cascade. These transgenic mice represent a useful model in which the fibroproliferative phase of connective tissue disease is uncoupled from inflammation.

Assessing pharmacokinetic and pharmacodynamic interactions in clinical trials of antiepileptic drugs.

An understanding of the pharmacokinetic and pharmacodynamic properties of a drug is a basic requirement for its clinical use. The investigations of these properties and their timing are fairly clearly defined in the drug development process. Without fundamental knowledge of the pharmacokinetics and pharmacodynamics of a drug, a physician could not use it appropriately, nor would a regulatory agency be likely to approve its use. Information about the interactions of a new antiepileptic drug with other antiepileptic drugs also aids a physician and is required, in varying degrees, by regulatory agencies. The amount of this information that is needed depends, in part, on the class of drug and the population for which the agent is intended. Because antiepileptic drugs are often used as polytherapy and generally are developed first for this use, their interaction potential must be part of the thought process in their development. The correct time to obtain this information, however, is not clearly defined. The analytic methodology to investigate the pharmacokinetic profile of an NCE exists and is fairly sophisticated. This methodology has enabled the development of study designs to investigate pharmacokinetic interactions. Because the plasma concentration of an antiepileptic drug may be increased or decreased as a result of pharmacokinetic interactions with concomitant antiepileptic drugs, it is of great importance to know about the specific interaction potential of an antiepileptic drug early in its development. Recent studies have confirmed the importance of investigating pharmacokinetic interactions in phase I before proceeding into phases II and III. Without this information, study results are often difficult to interpret; with this knowledge, study designs can be modified to minimize the confounding effect. A methodology exists to investigate the pharmacodynamic effects of an antiepileptic drug at receptors in cell cultures and in animal models of seizures; however, no procedure has been established to evaluate the short-term or immediate clinical pharmacodynamic effect of an antiepileptic drug, as has been done for other classes of drugs and other diseases. The clinical effect that is sought in trials with antiepileptic drugs is a reduction in seizures with little toxicity. The methodology to investigate the effect of seizure reduction over time has been used repeatedly with minor variations in the development of all the new antiepileptic drugs. However, no study has evaluated the effect of pharmacodynamic interactions among antiepileptic drugs on seizure reduction. Some studies have purported to show an interaction effect on adverse events, and assumptions are made about pharmacodynamic interactions. Although the information regarding pharmacodynamic interactions is important and existing trial designs could evaluate this, there has been no perceived need to carry out such trials. This information is less accessible than pharmacokinetic interaction information. Moreover, pharmacodynamic interactions, as opposed to pharmacokinetic interactions, are probably unidirectional and lead only to increased effects. Although it would be preferable to have this knowledge, an antiepileptic drug can be used effectively without it; over time, the information about pharmacodynamics will be inferred. Thus, conducting pharmacokinetic interaction studies with antiepileptic drugs early in their development as part of phase I is essential, whereas obtaining pharmacodynamic interaction information can be deferred.

Gene expression of collagen types IIA and IX correlates with ultrastructural events in early osteoarthrosis: new applications of the rabbit meniscectomy model.

OBJECTIVE: To examine the phenotypic expression and geographic distribution of collagens in early stages of osteoarthrosis and their relationship to ultrastructural events in cartilage. METHODS: In situ hybridization was used to localize articular expression of total type II (A+B) and type IX collagen at 2 and 4 weeks in the rabbit meniscectomy model of osteoarthrosis. The expression of the developmental marker collagen IIA was analyzed at the same time points. Articular cartilage structure was examined by scanning electron microscopy. RESULTS: Little difference was found in total type II or type IX collagen gene expression for operated versus control limbs at 2 weeks. Gene expression for collagen types IIA and IX was found to be site-specific by the 4 week period and was largely limited to the meniscectomy site. At 4 weeks, this activity was correlated with site-specific alterations in chondrocyte morphology, qualitative changes in the collagen matrix, and articular surface delamination on microscopy. CONCLUSION: Gene expression for collagen types IIA and IX is site-specific and correlates with ultrastructural changes in cartilage in this model of early osteoarthrosis. We present the first known report of the distribution of type IX collagen gene expression in any model of osteoarthrosis. These findings support the central importance of matrix interactions in osteoarthrosis and suggest that early phases of repair involve re-expression of a developmental sequence by chondrocytes.

Topiramate monotherapy for partial onset seizures.

PURPOSE: Evaluation of topiramate (TPM) as monotherapy in patients with uncontrolled partial onset seizures. METHODS: A total of 48 patients were evaluated in a double-blind, parallel-group trial. During a 56-day baseline period, patients had at least eight partial onset seizures while being treated with one or two standard antiepileptic drugs (AEDs). After 1-2 weeks of open-label treatment with TPM 100 mg/day, patients were randomly assigned, in equal proportions, to receive double-blind therapy with TPM 100 or 1,000 mg/day in a 5-week conversion and an 11-week monotherapy period. The study endpoint was completion of 112 study days (success) or fulfillment of one or more exit criteria: doubling of average 28-day or highest 2-day baseline seizure rate, a generalized tonic-clonic seizure (GTCS) if none had occurred at baseline, or significant prolongation of generalized seizure duration. RESULTS: Time until exit was longer (p = 0.002) and success frequency was higher (p = 0.005) with TPM 1,000 as compared with 100 mg/day. Seizure-rate reductions of > or = 50, > or = 75, or 100% were achieved by 46, 25, and 13% of the 1,000-mg/day group, respectively, as compared with 13, 8, and 0% of the 100-mg/day group, respectively. Most adverse events (AE) were mild or moderate in severity. CONCLUSIONS: Monotherapy with TPM 1,000 mg/day for partial onset seizures with or without secondarily generalized seizures was effective, with a favorable safety profile.

Topiramate as adjunctive therapy in refractory partial epilepsy: pooled analysis of data from five double-blind, placebo-controlled trials.

A pooled analysis of data from five similarly designed double-blind, placebo-controlled trials of topiramate (TPM) as add-on therapy in patients with partial epilepsy was performed. The pooled analysis allowed evaluation of efficacy end points and response to treatment for a number of study subgroups not statistically evaluable in the individual study analyses due to limited sample sizes. The five trials included 534 patients, 360 who received TPM at target dosages of 200-1,000 mg daily and 174 who received placebo. In the intent-to-treat pooled analysis, TPM was significantly (p < or = 0.01) superior to placebo in reducing total seizures by > or = 75% or by 100%. When seizure types were evaluated independently, TPM significantly (p < or = 0.001) reduced the frequency of simple partial, complex partial, and secondarily generalized seizures. TPM was significantly (p < 0.001) better than placebo regardless of gender, patient age, baseline seizure rate, and concomitant AEDs. The efficacy of TPM in partial epilepsy is consistent across efficacy end points and across strata defined by study population characteristics.

Topiramate placebo-controlled dose-ranging trial in refractory partial epilepsy using 200-, 400-, and 600-mg daily dosages. Topiramate YD Study Group.

We conducted a randomized double-blind comparison of three doses of the novel antiepileptic drug (AED) topiramate (200, 400, and 600 mg/day) and placebo as adjunctive therapy in patients with refractory partial onset epilepsy receiving one or two other AEDs at therapeutic concentrations. A total of 181 patients completed the 12-week baseline phase and were randomized to double-blind therapy. Median percent reductions from baseline in average monthly seizure rate, the principal efficacy evaluation, were 13% for placebo, 30% for topiramate 200 mg/day, 48% for topiramate 400 mg/day, and 45% for topiramate 600 mg/day. For the seizure rate comparison of active drug to placebo p values were: topiramate 200 mg/day, p = 0.051; topiramate 400 mg/day, p = 0.007; topiramate 600 mg/day, p < 0.001. Percent responders ( > or = 50% reduction in seizure rates) were 18% for placebo, 27% for topiramate 200 mg/day, 47% for topiramate 400 mg/day (p = 0.013), and 46% for topiramate 600 mg/day (p = 0.027). A significant (p = 0.003) reduction in secondarily generalized seizures compared with placebo treatment was also documented with topiramate. Topiramate plasma concentrations were closely related to dosage, and there were no significant interactions between topiramate and other AEDs. The minimal effective dose of topiramate in this study population was approximately 200 mg/day. Mild or moderate CNS symptoms were the primary treatment-emergent adverse events, but treatment-limiting adverse events occurred in only 9% of patients given topiramate compared with 7% given placebo. Results of this initial well-controlled study in patients indicate that topiramate is a very promising new AED.

Porphyromonas gingivalis lipopolysaccharide is poorly recognized by molecular components of innate host defense in a mouse model of early inflammation.

Porphyromonas gingivalis is a gram-negative bacterium that is associated with periodontitis. It has been hypothesized that destruction of bone and periodontal connective tissue is associated with colonization of the subgingival crevicular space by P. gingivalis, although how these bacteria overcome innate host defenses is largely unknown. To examine the early cellular and molecular events of P. gingivalis interaction with host tissues, we compared lipopolysaccharide (LPS) isolated from this bacterium with Escherichia coli LPS, a potent inflammatory mediator, in a mouse model of acute inflammation. In these studies, mice were given intramuscular injections of either P. gingivalis LPS or E. coli LPS and then sacrificed after 4 h. Reverse transcriptase-PCR analysis showed that expression of mRNAs for E- and P-selectins was higher in E. coli LPS-injected muscles than in P. gingivalis LPS-injected or control phosphate-buffered-saline-injected muscles. Similarly, monocyte chemotactic protein 1 and fibroblast-induced cytokine mRNAs were expressed in E. coli LPS-injected muscles whereas their expression was reduced or absent in P. gingivalis LPS-injected samples. These results were confirmed by in situ hybridization whereby stronger hybridization for selectin mRNAs was observed in the endothelium of capillaries from E. coli LPS-injected samples than in that from P. gingivalis LPS-injected muscles. In addition, many monocytes expressing monocyte chemotactic protein 1 mRNA and polymorphonuclear leukocytes expressing fibroblast-induced cytokine mRNA were observed in E. coli LPS-injected muscles whereas only a few cells were identified in P. gingivalis LPS-injected muscles. These results demonstrate that compared with E. coli, P. gingivalis has a low biologically reactive LPS as measured by its weak activation of inflammation. This may allow P. gingivalis to evade innate host defense mechanisms, resulting in colonization and chronic disease.

Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non-cartilaginous tissues during early mouse development.

Type II collagen, generally considered to be characteristic of cartilage, has been localized in specific non-cartilaginous structures during embryogenesis and development of the skeleton. Type II procollagen is synthesized in two different forms generated by alternative splicing of exon 2 in the precursor mRNA transcript. One form (type IIA procollagen) contains a large cysteine-rich domain in the NH2-terminal propeptide, while the second form (type IIB procollagen) does not. These two forms are spatially expressed during development and chondrogenesis with the type IIB procollagen mRNA primarily expressed by chondrocytes while the IIA form is expressed in chondroprogenitor cells (Sandell et al. [1991] J. Cell Biol. 114:1307-1319). The present study demonstrates that the early non-cartilage expression, by somites, mesenchymal and epithelial cells, is predominantly the alternate splice form, type IIA procollagen mRNA. Later in development, the type IIB mRNA splice form is expressed by chondrocytes. During the development of intramembranous bones, such as the mandible, type IIA procollagen mRNA is also expressed. In this tissue, the splice form does not switch to type IIB mRNA and no cartilage is formed. These results show that expression of type IIA mRNA, whether by epithelial or mesenchymal cells, precedes formation of overt skeletal structures.

Induction of arthritis with monoclonal antibodies to collagen.

mAb were developed from DBA/1 mice immunized with chick type II collagen. A total of 69 IgG antibodies was isolated and characterized. The majority (36%) reacted with a CNBr-derived peptide CB11 previously identified as containing a major immunogenic and arthritogenic epitope(s). Seven of the antibodies reactive with CB11 crossreacted strongly with mouse type II collagen. These were administered to DBA/1 mice in an attempt to induce arthritis. Individual antibodies were able to induce mild lesions consisting of minimal synovial proliferation but not overt arthritis. However, a combination of antibodies induced severe arthritis with marked destruction of articular cartilage. The minimal effective combination consisted of three antibodies. Arthritis developed within 48 to 72 h after injection of the antibodies and persisted for the duration of the observation period of 3 wk. Antibody levels were measured at intervals and persisted for the 3 wk observation period although at diminishing levels. Competitive binding assays demonstrated that each of the effective antibodies bound independently suggesting that some spatial or quantitative relationship was important possibly related to their ability to activate complement.

Characterization of type V collagenase (gelatinase) in synovial fluid of patients with inflammatory arthritis.

Gelatin degrading matrix metalloproteinases in synovial fluid from 21 patients with inflammatory arthritis were shown to consist of two distinct gene products, 92 and 70 kDa gelatinases. The gelatinolytic activity of 92 kDa enzyme, which is released from stimulated neutrophils, was positively correlated to neutrophil count in the fluid. By contrast, 70 kDa molecule did not correlate with neutrophil cell count. Purification of these enzymes revealed they could degrade type XI collagen, a cartilage component resistant to interstitial collagenase. The elevated levels of 92 kDa gelatinase in rheumatoid arthritis samples compared to osteoarthritis suggest a role of this enzyme in cartilage destruction.

SWR mice are resistant to collagen-induced arthritis but produce potentially arthritogenic antibodies.

SWR mice are resistant to collagen-induced arthritis but produce antibodies to type II collagen. To determine if these antibodies have arthritogenic potential, serum from collagen-immunized mice was concentrated and passively transferred to DBA/1 mice. The recipients developed severe arthritis within 72 hours. To evaluate the role of complement, SWR mice were bred with congenic inbred B10.D2/oSn (complement deficient) and B10.D2/nSn (complement normal) mice. Collagen-immunized (SWR x B10.D2/nSn)F1 mice had high levels of C5 and were susceptible to arthritis, while (SWR x B10.D2/oSn)F1 mice were deficient in C5 and were resistant to arthritis.

The role of C5 and T-cell receptor Vb genes in susceptibility to collagen-induced arthritis.

Collagen-induced arthritis (CIA) is a rodent arthritis model in which immunization with heterologous type II collagen induces an inflammatory polyarthritis. Susceptibility to the disease is mediated by major histocompatibility complex (MHC) genes as well as genes at other loci. Previous studies of the SWR/J mouse strain, which is resistant to CIA despite bearing the susceptible H-2q haplotype, have suggested that this resistance is the result of a deletion of T-cell receptor (Tcr) Vb gene segments which is carried by this strain. Other studies have implicated a deficiency in complement component C5 as the cause for the resistance. In order to assess the relative importance of these two genes in susceptibility to CIA, and to provide an estimate of the number of independent genes involved in the disease, we analyzed 196 F2 progeny of a (DBA/1 x SWR/J) cross for arthritis susceptibility, and expression of both C5 and Tcr genes. Thirty of the F2 progeny developed arthritis. All of the arthritic mice had at least one copy of the wild-type C5 allele, while the Tcr-Vb haplotypes were distributed in Mendelian fashion. These results demonstrate that C5 sufficiency is an absolute requirement for CIA, but that Tcr-Vb genes located within the SWR deletion have little influence. Genetic analysis of the incidence rate suggests that there is polygenic control of susceptibility to CIA and that in addition to H-2, 5-6 other independent loci (including C5) may be involved.

The role of stromelysin in the cartilage destruction that accompanies inflammatory arthritis.

Articular cartilage from arthritic joints of rats immunized with type II collagen is severely depleted of proteoglycans. Depletion begins within 48 hours after the onset of inflammation, prior to extensive pannus formation, and may represent a critical first step in cartilage destruction. We have immunolocalized stromelysin, an enzyme that is believed to play a major role in the pathologic degradation of proteoglycans, in the joints of rats with collagen-induced arthritis. Immunoperoxidase staining of frozen tissue sections demonstrated the presence of stromelysin in both the synovium and chondrocytes. In contrast, collagenase was localized primarily to the pannus-cartilage junction. Neither enzyme was detectable in joints from normal animals. To test the hypothesis that chondrocytes respond directly to inflammatory mediators by increasing the production of stromelysin, isolated chrondrocytes were incubated with various concentrations of interleukin-1. The culture media were also assayed for the presence of stromelysin by immunoreactivity on Western blots and by analysis of enzymatic activity on casein substrate gels. A 3-fold increase in a doublet of proteins synthesized in response to 10 units/ml of interleukin-1 was observed. These proteins also immunoreacted with the stromelysin antibody and degraded casein. Northern blotting results established that the increased levels of stromelysin were accompanied by increases in stromelysin-specific messenger RNA levels. These results suggest that stromelysin is responsible for proteoglycan degradation in early inflammatory arthritis, and that chondrocytes may play a direct role in the earliest stages of the degradation of their own matrices.

Histochemical and biochemical studies of the hepatopancreas peroxidase of the freshwater crayfish, Cambarus robustus.

Crayfish are among the few invertebrate species reported to possess endogenous peroxidase activity. The enzyme is found within the hepatopancreas, the principal digestive and absorptive organ of the crustacean body. Cambarus robustus, a species found in abundance in the streams of western New York, was used in this study. Homogenates of 18 hepatopancreases were assayed for peroxidase activity using guaiacol as the substrate. Although present in all organs, peroxidase activity displayed a greater than 50-fold difference between the two extremes (0.05-;2.72 units/mg protein). Histochemical examination using diaminobenzidine revealed peroxidase activity within a line of cells extending along the distal two-thirds of the lengths of all hepatopancreatic tubules. The cells function to synthesize the enzyme, sequester it within vacuoles of increasing size, and eventually secrete it into the tubule lumen. Since the tubule is constantly renewed by distal mitotic activity and concomitant proximal exfoliation, this histochemical technique permits not only the examination of the ontogeny of this peroxidase-positive cell line, but also offers additional insight into the mechanism of hepatopancreatic tubule renewal.