Consensus definitions and application guidelines for control groups in cerebrospinal fluid biomarker studies in multiple sclerosis.

The choice of appropriate control group(s) is critical in cerebrospinal fluid (CSF) biomarker research in multiple sclerosis (MS). There is a lack of definitions and nomenclature of different control groups and a rationalized application of different control groups. We here propose consensus definitions and nomenclature for the following groups: healthy controls (HCs), spinal anesthesia subjects (SASs), inflammatory neurological disease controls (INDCs), peripheral inflammatory neurological disease controls (PINDCs), non-inflammatory neurological controls (NINDCs), symptomatic controls (SCs). Furthermore, we discuss the application of these control groups in specific study designs, such as for diagnostic biomarker studies, prognostic biomarker studies and therapeutic response studies. Application of these uniform definitions will lead to better comparability of biomarker studies and optimal use of available resources. This will lead to improved quality of CSF biomarker research in MS and related disorders.

Improvement of field matching in segmented-field electron conformal therapy using a variable-SCD applicator.

The purpose of the present study is to demonstrate that the use of an electron applicator with energy-dependent source-to-collimator distances (SCDs) will significantly improve the dose homogeneity for abutted electron fields in segmented-field electron conformal therapy (ECT). Multiple Coulomb scattering theory was used to calculate and study the P(80-20) penumbra width of off-axis dose profiles as a function of air gap and depth. Collimating insert locations with air gaps (collimator-to-isocenter distance) of 5.0, 7.5, 11.5, 17.5 and 19.5 cm were selected to provide equal P(80-20) at a depth of 1.5 cm in water for energies of 6, 9, 12, 16 and 20 MeV, respectively, for a Varian 2100EX radiation therapy accelerator. A 15 x 15 cm(2) applicator was modified accordingly, and collimating inserts used in the variable-SCD applicator for segmented-field ECT were constructed with diverging edges using a computer-controlled hot-wire cutter, which resulted in 0.27 mm accuracy in the abutted edges. The resulting electron beams were commissioned for the pencil-beam algorithm (PBA) on the Pinnacle(3) treatment planning system. Four hypothetical planning target volumes (PTVs) and one patient were planned for segmented-field ECT using the new variable-SCD applicator, and the resulting dose distributions were compared with those calculated for the identical plans using the conventional 95 cm SCD applicator. Also, a method for quality assurance of segmented-field ECT dose plans using the variable-SCD applicator was evaluated by irradiating a polystyrene phantom using the treatment plans for the hypothetical PTVs. Treatment plans for all four of the hypothetical PTVs using the variable-SCD applicator showed significantly improved dose homogeneity in the abutment regions of the segmented-field ECT plans. This resulted in the dose spread (maximum dose-minimum dose), sigma, and D(90-10) in the PTV being reduced by an average of 32%, 29% and 32%, respectively. Reductions were most significant for abutted fields of nonadjacent energies. Planning segmented-field ECT using the variable-SCD applicator for a patient with recurrent squamous cell carcinoma of the left ear showed the dose spread, sigma, and D(90-10) of the dose distribution in the PTV being reduced by an average of 38%, 22% and 22%, respectively. The measured and calculated dose in a polystyrene phantom resulting from the variable-SCD, segmented-field ECT plans for the hypothetical PTVs showed good agreement; however, isolated differences between dose calculation and measurement indicated the need for a more accurate dose algorithm than the PBA for segmented-field ECT. These results confirmed our hypothesis that using the variable-SCD applicator for segmented-field ECT results in the PTV dose distribution becoming more homogenous and being within the range of 85-105% of the 'given dose'. Clinical implementation of this method requires variable-SCD applicators, and the design used in the present work should be acceptable, as should our methods for construction of the inserts. Dose verification measurements in a polystyrene phantom and the recommended improvements in dose calculation should be appropriate for quality assurance of segmented-field ECT.

Evaluating more naturalistic outcome measures: A 1-year smartphone study in multiple sclerosis.

OBJECTIVE: In this cohort of individuals with and without multiple sclerosis (MS), we illustrate some of the novel approaches that smartphones provide to monitor patients with chronic neurologic disorders in their natural setting. METHODS: Thirty-eight participant pairs (MS and cohabitant) aged 18-55 years participated in the study. Each participant received an Android HTC Sensation 4G smartphone containing a custom application suite of 19 tests capturing participant performance and patient-reported outcomes (PROs). Over 1 year, participants were prompted daily to complete one assigned test. RESULTS: A total of 22 patients with MS and 17 cohabitants completed the entire study. Among patients with MS, low scores on PROs relating to mental and visual function were associated with dropout (p < 0.05). We illustrate several novel features of a smartphone platform. First, fluctuations in MS outcomes (e.g., fatigue) were assessed against an individual's ambient environment by linking responses to meteorological data. Second, both response accuracy and speed for the Ishihara color vision test were captured, highlighting the benefits of both active and passive data collection. Third, a new trait, a person-specific learning curve in neuropsychological testing, was identified using spline analysis. Finally, averaging repeated measures over the study yielded the most robust correlation matrix of the different outcome measures. CONCLUSIONS: We report the feasibility of, and barriers to, deploying a smartphone platform to gather useful passive and active performance data at high frequency in an unstructured manner in the field. A smartphone platform may therefore enable large-scale naturalistic studies of patients with MS or other neurologic diseases.

Human transcription factor Sp3: genomic structure, identification of a processed pseudogene, and transcript analysis.

The human transcription factor Sp3 has been widely studied at the translational level and has been described as a regulatory factor for a number of genes. Sp3 is currently characterized as a bifunctional transcription factor having the ability to behave as both an activator and/or a repressor in various promoter regions. Previous translational studies have attempted to determine the basis for these diverse functions with mostly contradictory evidence to date. Little data are available, however, concerning genomic structure, full-length cDNA, potential transcript variants, or location of translation initiation sites for the large isoform of the Sp3 gene. In this study, bacterial artificial chromosome (BAC) sequencing, reverse transcription-polymerase chain reaction (RT-PCR), genomic PCR, and database mining indicate that the Sp3 gene encompasses seven exons spanning more than 55 kb of genomic DNA on Chromosome 2. The 5' end of this sequence contains a large CpG island. This work also detected a processed pseudogene, psiSp3, located on Chromosome 13, spanning approximately 4.0 kb. Northern blot analysis detected three predominant transcripts at 4.0, 6.0 and 2.5 kb. Sequence analysis indicated that alternative splicing of exon 3 allows for multiple transcripts of Sp3. Each sequenced transcript possesses three to five potential translation initiation sites. This diversity at the level of gene expression will likely be key to understanding the diverse functions of Sp3.

Structure-based discovery of nonpeptidic small organic compounds to block the T cell response to myelin basic protein.

We have utilized a computational structure-based approach to identify nonpeptidic small organic compounds that bind to a human leukocyte antigen (HLA) DR1301 molecule (HLA-DR1301 or DR1301) and block the presentation of myelin basic protein peptide 152-165 (MBP 152-165) to T cells. A three-dimensional (3D) structure of DR1301 was derived by homology modeling followed by extensive molecular dynamics simulation for structural refinement. Computational structure-based database searching was performed to identify nonpeptidic small-molecule candidates from the National Cancer Institute (NCI) database containing over 150 000 compounds that can effectively interact with the peptide-binding groove of the HLA molecule. By in vitro testing of 106 candidate small molecules, two lead compounds were confirmed to specifically block IL-2 secretion by DR1301-restricted T cells in a dose-dependent and reversible manner. The specificity of blocking DR1301-restricted MBP presentation was further validated in a binding assay using an analogue of the most potent lead compound. Computational docking was performed to predict the three-dimensional binding model of these confirmed small molecule blockers to the DR1301 molecule and to gain structural insight into their interactions. Our results suggest that computational structure-based searching is an effective approach to discover nonpeptidic small organic compounds to block the interaction between DR1301 and T cells. The nonpeptidic small organic compounds identified in this study are useful pharmacological tools to study the interactions between HLA molecules and T cells and a starting point for the development of a novel therapeutic strategy for the treatment of multiple sclerosis (MS) or other immune-related disorders.

Role of an internal ribosome entry site in the translational control of the human transcription factor Sp3.

Sp1 and Sp3 are transcription factors involved in the regulation of numerous genes involved in oncogenesis. Sp3 is a bi-functional transcription factor with three different isoforms. Its bi-functional activity may in part be regulated by the relative expression of these isoforms. Northern blot analysis of Sp3 detects only a single transcript. Analysis of the known Sp3 cDNA sequence shows a high GC content and seven out-of-frame AUG codons located between the 5'-end of the mRNA and the two internal AUG initiation sites. This makes it highly unlikely that cap-recruited, translation initiation competent ribosomes could reach the internal start sites. A full human Sp3 expression construct was cloned. A bicistronic vector using Renilla and firefly luciferase showed internal ribosome entry site (IRES) activity in Sp3 RNA immediately 5' to the internal AUG sites. Also, the two smaller isoforms were translated more efficiently when full-length, uncapped transcripts were used, while the larger isoform was not translated. Mutants of Sp3 with AUG codons introduced 5' of the two internal start sites were generated. Results showed that they were unable to suppress the smaller isoforms in vitro. Furthermore, dual non-AUG to AUG mutations showed occlusion of the second introduced isoform (i.e., the isoform situated more 3') but not of the internally initiated isoforms. These experiments are consistent with IRES-mediated translation of the two smaller isoforms of Sp3. The presence of an IRES allows the possibility that Sp3 isoform ratios and activity are controlled at the translational level. This mechanism may allow cells to control the expression of numerous genes during mitosis and, thus, have profound effects on cell cycle regulation and tumorigenesis.

AUA as a translation initiation site in vitro for the human transcription factor Sp3.

Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Although the size of Sp3 mRNA is 4.0 kb, the size of the known Sp3 cDNA sequence is 3.6 kb. Thus, Sp3 functional studies have been performed with an artificially introduced start codon, and thus an aminoterminus that differs from the wild-type. Ideally, full-length cDNA expression vectors with the appropriate start codon should be utilized for these studies. Using 5'rapid amplification of cDNA ends, a full-length Sp3 cDNA clone was generated and the sequence verified in nine cell lines. No AUG initiation codon was present. However, stop codons were present in all three frames 5' to the known coding sequence. In vitro translation of this full-length cDNA clone produced the expected three isoforms-one at 100 kDa and two in the mid 60 kDa range. Electrophoretic mobility shift assays showed that the protein products had the ability to bind to the Sp1/3 consensus sequence. In vitro studies, using our Sp3 clone and site directed mutagenesis, identified the translation initiation site for the larger isoform as AUA. AUA has not been previously described as an endogenous initiation codon in eukaryotes.

Deletion of the SSK1 response regulator gene in Candida albicans contributes to enhanced killing by human polymorphonuclear neutrophils.

The isolation and partial functional characterization of the two-component response regulator SSK1 gene of Candida albicans was previously reported. Compared to wild-type (CAF2-1) and gene-reconstituted (SSK23) strains, the ssk1 null strain (SSK21) was avirulent in a murine model of hematogenously disseminated candidiasis and less able to adhere to human esophageal cells. More recent data indicate that SSK21 is sensitive to 4 to 8 mM H(2)O(2) in vitro than CAF2-1 and SSK23. Furthermore, microarray studies indicate that the regulation of two classes of genes, those encoding cell wall functions and stress adaptation, are altered in the ssk1 mutant. In the present study, the susceptibility of strains CAF2-1, SSK21, and SSK23 to killing by human polymorphonuclear neutrophils (PMNs) was assessed. Results are also described for a newly constructed ssk1 mutant (SSK24) in which the URA3 gene is integrated into its native locus. Our results indicate that killing of SSK21 and SSK24 was significantly greater than that of CAF2-1 and SSK23 (P < 0.01). In order to determine why Ssk1p at least partially protects the organism against the killing activity of human PMNs, we compared the signal transduction activity and the inflammatory response gene profiles of PMNs infected with either the wild type or the ssk1 mutant. Phosphorylation of the mitogen-activated protein kinases p42/44 and p38 from neutrophils infected with either CAF2-1 (wild type) or SSK21 (ssk1/ssk1) was similar, while expression and phosphorylation of the JNK mitogen-activated protein kinase was not observed following infection with either strain. On the other hand, we observed an upregulation of seven inflammatory response genes in PMNs infected with the SSK21 mutant only, while an increase in interleukin-10 expression was measured in PMNs infected with either strain. Downregulation of interleukin-2 was observed in PMNs infected with either strain. Verification of the transcriptional profiling was obtained by reverse transcription-PCR for three of the genes that were upregulated in neutrophils infected with the ssk1 mutant. Also, the sensitivity of strain SSK21 to human defensin-1, one of the nonoxidative, antimicrobial peptides of PMNs, was greater than that of CAF2-1, demonstrating that nonoxidative killing in PMNs may contribute to the increased susceptibility of the ssk1 mutant. Our results indicate that the Ssk1p response regulator protein may provide at least partial adaptive functions for the survival of C. albicans following its encounter with human neutrophils.

A phase I study of the safety of honeybee venom extract as a possible treatment for patients with progressive forms of multiple sclerosis.

Although several reports suggest that bee venom may be an effective treatment for patients with multiple sclerosis (MS), patients may be subjected to real risks of serious allergic reactions as well as emotional and economic costs. This study was conducted to evaluate the safety of bee venom extract as a possible treatment for patients with progressive forms of MS. A total of nine bee venom nonallergic patients with progressive forms of MS, who were 21-55 years of age with no other illnesses, were entered into four groups (A, B, C, and D) on a structured 1-year immunization schedule. Hyperreactivity to bee venom was evaluated by questionnaire, physical examination, and a battery of hematologic, metabolic, and immunologic tests. Responses to therapy were evaluated by questionnaire, functional neurological tests, and changes in measurement of somatosensory-evoked potentials. Although no serious adverse allergic reactions were observed in any of the nine subjects, four experienced worsening of neurological symptoms, requiring termination in the study; this could not be ascribed to side effects of the therapy. Of the remaining five subjects, three felt that the therapy had subjective amelioration of symptoms and two showed objective improvement. Although this preliminary study suggests safety, because of the small numbers studied, there were no definite conclusions regarding efficacy and therefore there was little evidence to support the use of honeybee venom in the treatment of MS. Larger and more carefully conducted multicenter studies will be required to establish efficacy.

Eight-year immunogenicity and safety of interferon beta-1a-Avonex treatment in patients with multiple sclerosis.

An open-label extension study of the phase III trial of intramuscular interferon beta-1a (IFNbeta-1a-Avonex) was conducted to evaluate the immunogenicity and safety of IFNbeta-1a-Avonex over six years in patients with relapsing multiple sclerosis (MS). Patients who participated in the pivotal phase III study were offered enrolment; entry was also open to patients who had not participated. All patients received IFNbeta-1a-Avonex 30 microg intramuscularly once weekly for six years, for a treatment duration of up to eight years in patients who received IFNbeta-1a-Avonex in the phase III trial. Serum levels of IFNbeta antibodies were measured every six months using a screening enzyme-linked immunosorbent assay (ELISA) followed by an antiviral cytopathic effect assay to detect neutralizing antibodies (NAbs) in serum samples positive on ELISA. The incidence of adverse events and laboratory test results assessed safety. Of 382 total patients, 218 had participated in the phase III study (103 placebo, 115 IFNbeta-1a-Avonex) and 164 had not participated; 24 of the 164 were IFNbeta-naïve. At baseline, 281 patients were negative for IFNbeta antibodies (NAb-). NAbs (titre > or = 20) developed at any time over six years in 5% of these patients. Of 140 patients who had been on IFNbeta-1b-Betaseron, 49 were positive for NAbs (NAb+) at baseline; 11 of 115 who had been on IFNbeta-1a-Avonex were NAb+ at baseline. Thirty-nine of 49 patients who had been on Betaseron and were NAb+ had titres < 100; 36 of these 39 seroconverted to NAb- while on IFNbeta-1a-Avonex, with a median time of approximately six months. Ten patients who had been on Betaseron had NAb titres > or = 100; five remained NAb+ during six years on IFNbeta-1a-Avonex and five seroconverted to NAb-, but only after at least two years. Five patients who had been on IFNbeta-1a-Avonex during the clinical trial were NAb+ with titres < 100 at baseline; four seroconverted to NAb-, with a median time of two to three years. Six patients who had been on IFNbeta-1a-Avonex had NAb titres > or = 100; five of these remained NAb+ at six years. No patient with a NAb titre > 1000 seroconverted to NAb-, whether initially treated with IFNbeta-1a-Avonex or -Betaseron. Adverse events were similar to those observed in the pivotal phase III trial. Results from this trial indicated that IFNbeta-1a-Avonex was associated with a low incidence of NAbs and was well tolerated for up to eight years. Further, the results indicate that persistence of NAbs is dependent on titre and IFNbeta product.

Selecting a disease-modifying agent as platform therapy in the long-term management of multiple sclerosis.

Multiple sclerosis (MS) is a complex, incurable disease. Treatment consists of lifelong disease and symptom management. FDA-approved therapies for relapsing MS include subcutaneous (SC) interferon beta-1b (IFNbeta1b, Betaseron), IM interferon-beta-1a (Avonex), SC interferon-beta-1a (Rebif), glatiramer acetate (Copaxone), and mitoxantrone (Novantrone), all of which are known as disease-modifying agents (DMAs). DMAs that can be initiated and continued on a long-term basis can be referred to as platform therapies. During periods of disease instability with increased disease activity, corticosteroids or immunosuppressive agents can be used in combination with appropriate DMA platform therapy to help control symptoms. To date, long-term comparative studies of DMAs are not available. However, based on the effects of these agents on disability progression, relapse rates, MRI outcomes, and neutralizing antibodies observed in phase III randomized clinical trials, IFNbeta1a products are the DMAs of choice for platform therapy for MS. Evidence indicates that IFNbeta1a may also be beneficial in the early stages of the disease. Research is ongoing to identify other appropriate add-on agents (e.g., antigen-specific therapies) to be used in combination with existing DMAs to effectively manage MS.

Sp3 expression in immune cells: a quantitative study.

We previously reported the lack of expression of the bifunctional transcription factor Sp3 in peripheral blood mononuclear cells from most patients with multiple sclerosis (MS) (Grekova et al, 1996). An RT-PCR technique was developed to evaluate Sp3 mRNA levels in peripheral blood mononuclear cell subsets. Semi-quantitative and quantitative competitive RT-PCR assays were used to compare the level of Sp3 expression among subjects and among immune cell subsets. The competitor DNA fragment contained a deletion from the normal Sp3 cDNA sequence. The wild-type Sp3 cDNA and the competitor DNA fragment amplified with equal efficiency, and the two PCR products were distinguished by size. These studies demonstrated that normal CD4(+) and CD8(+) T cells, B cells, and macrophages expressed comparable amounts of Sp3 mRNA. No Sp3 expression could be detected in normal natural killer cells nor in any of these cell types from Sp3-negative MS patients. We propose that transcription of the Sp3 gene is blocked in immune cells from most patients with MS and that this contributes to the development of central nervous system inflammation in the disease.