Subcutaneous interferon ß-1a three times weekly and the natural evolution of gadolinium-enhancing lesions into chronic black holes in relapsing and progressive multiple sclerosis: Analysis of PRISMS and SPECTRIMS trials.

BACKGROUND: Evolution of gadolinium-enhancing lesions into chronic black holes (CBH) may be reduced by interferon (IFN) therapy. OBJECTIVE: The objective of this paper is to assess the effect of IFN ß-1a and placebo on CBH evolution and disability in patients with relapsing-remitting multiple sclerosis (RRMS), as well as CBH evolution in patients with secondary progressive multiple sclerosis (SPMS). METHODS: A post hoc, exploratory analysis of patients with RRMS and SPMS with monthly MRI scans (months -1 to 9) from two separate placebo-controlled clinical trials of IFN ß-1a was conducted. RESULTS: In RRMS patients, the risk of ≥1 evolved CBH was lower for IFN ß-1a versus placebo (odds ratio 0.42; p = 0.024); volume of newly evolved CBH was numerically reduced. A numerically higher proportion of patients with ≥1 evolving CBH vs no evolving CBH had confirmed three-month disability progression (four-year rate 55.8% vs 43.1%, respectively). Proportion of lesions evolving into CBH (patient level: 34.7% vs 12.6%, p < 0.0001; lesion level: 28.8% vs 11.0%, p < 0.0001) and evolved CBH volume (median 33.5 mm3 (Quartile 1, 0.0; Quartile 3, 173.4) vs 0.0 mm3 (0.0; 52.4); p = 0.0008) was higher for SPMS than RRMS patients treated with IFN ß-1a. CONCLUSION: In RRMS, IFN ß-1a significantly decreased the proportion of new T1 Gd+ lesions evolving into CBH and the risk of developing a CBH. In patients with SPMS, more lesions develop to CBH, indicating reduced repair capacity, and the natural history of lesion development appears to be unaffected by IFN ß-1a treatment.

Rebif(®) Quality of Life (RebiQoL): A randomized, multicenter, Phase IIIb study evaluating quality-of-life measures in patients receiving the serum-free formulation of subcutaneous interferon beta-1a for the treatment of relapsing forms of multiple sclerosis.

BACKGROUND: In clinical studies, treatment with subcutaneous interferon beta-1a (IFNß-1a) has been shown to reduce relapse rates and slow the progression of physical disability in patients with relapsing forms of multiple sclerosis (MS). A formulation of subcutaneous IFNß-1a has been developed that is free of fetal bovine serum and human serum albumin. OBJECTIVE: To evaluate (a) the impact on quality of life (QoL) and treatment satisfaction of transitioning from the original formulation of subcutaneous IFNß-1a to the serum-free formulation in patients with relapsing forms of MS; and (b) the impact of dose titration versus non-titration during the transition on tolerability and patterns of analgesic use. QoL was measured by the Multiple Sclerosis Treatment Concerns Questionnaire Global Side Effects (GSE) score. METHODS: Patients who had received the original formulation of IFNß-1a subcutaneously for ≥24 weeks were randomized to receive the serum-free formulation of IFNß-1a 44µg subcutaneously three times weekly for 12 weeks, with or without a dose titration over a 4-week period. After week 12, patients continued to receive serum-free subcutaneous IFNß-1a during a safety extension phase until they completed between 84 and 112 weeks of treatment. The primary endpoint was the percentage change from baseline to week 12 in GSE score in all patients. RESULTS: A total of 232 patients were randomized (titrated n=113; non-titrated n=119). The mean percent change (improvement) from baseline to week 12 in the GSE score was 5.0% (p<0.001 for mean change in GSE score from baseline); this change was similar between titrated and non-titrated patients and met criteria for non-inferiority to the original formulation. Adverse event (AE) incidence and use of analgesics for the treatment of flu-like symptoms (FLS) were less common in the titrated group. Few patients (<2%) discontinued due to AEs during weeks 0 to 12. CONCLUSION: Patients with relapsing forms of MS who transitioned from original-formulation subcutaneous IFNß-1a to serum-free subcutaneous IFNß-1a had overall improved QoL scores at 12 weeks of treatment. Titration during the transition resulted in a lower requirement for analgesic treatment of FLS and fewer AEs.

Patient-rated ease of use and functional reliability of an electronic autoinjector for self-injection of subcutaneous interferon beta-1a for relapsing multiple sclerosis.

BACKGROUND: For patients with multiple sclerosis (MS), electronic autoinjectors may improve convenience and reduce discomfort associated with chronic injections. OBJECTIVE: To assess ease of use, patient satisfaction, and functional reliability of an investigational electronic autoinjector for self-injection of subcutaneous interferon beta-1a (IFNß-1a). METHODS: This prospective, multicenter, open-label, single-arm, 12-week, Phase IIIb study enrolled patients aged 18-65 years with relapsing MS receiving IFNß-1a 44µg subcutaneously 3 times weekly for ≥12 weeks before enrollment. Thereafter, patients continued their regimen using an electronic autoinjector. The primary endpoint was the proportion of patients rating the autoinjector 'easy to use' or 'very easy to use' on a User Trial Questionnaire at week 12. Secondary endpoints included patient responses to questions regarding device reliability, patient satisfaction, and convenience. RESULTS: Of 103 patients enrolled, 88 completed the study. The primary objective was met, with most patients (78%) indicating the device was 'easy to use' or 'very easy to use' at week 12 (worst-case imputation). In an analysis of secondary endpoints, over 60% of patients responded favorably to each of the User Trial questions regarding device ease-of-use and their satisfaction with the device. Overall convenience was judged the most important benefit of the device. Adverse events reported were consistent with the known safety profile of IFNß-1a, with injection site reactions the most frequently reported. CONCLUSION: These data show that patients found the electronic autoinjector for delivery of subcutaneous IFNß-1a reliable and easy to use, suggesting the device may help patients with relapsing MS to administer self-injections.

Modulation of splicing events in histone deacetylase 3 by various extracellular and signal transduction pathways.

Within the context of the chromatin environment histone deacetylases are important transcriptional regulators. Three classes of human histone deacetylases have currently been identified on the basis of their similarity to yeast proteins. The class I enzymes contain four members: HDACs 1-3 and HDAC8. Of these, HDAC3 is known to generate transcript variants with altered amino-terminal regions. Here we describe the identification of a novel splice variant of HDAC3, in which exon 3 is alternatively spliced from the messenger RNA transcript. We show that this human HDAC3 splice transcript is upregulated by treatments with histone deacetylase inhibitors. We also demonstrate evidence of splicing events in murine HDAC3 as a response to various signals, including switching between splice transcript isoforms following treatments with kinase inhibitors or by osmotic shock. In contrast, such switching events were not observed in human cells. These results indicate that differential pathways in mouse and human may control the regulation of HDAC3, and that splice variants may play important roles in responding to exogenous stimuli that act via signal transduction pathways.

Differential expression of class I HDACs: roles of cell density and cell cycle.

Enzymes which affect histone acetylation status have been shown to play an important role in determining transcriptional activity in chromatin through conformational modification of its structure. Since the timely presence of such enzymes may be of critical importance, our experiments were designed to determine whether the level of expression of HDAC1 is cell cycle dependent and/or affected by a high cell density. Our results show that in mouse fibroblasts the expression of mHDAC1 is neither affected by cell cycle phases nor by cell density. In contrast, the expression of several hHDACs including hHDAC1 were affected in a cell density dependent fashion in the human prostate adenocarcinoma cell line PC3, paralleling our previously published findings in the hepatocellular carcinoma derived cell line Hep3B. Differential recruitment of HDAC mRNAs suggests that these enzymes may play unique roles in different cell types and under different environmental conditions (i.e., exposure to various cell densities and cell-cell contacts). Our study has implications for the proposed use of HDAC inhibitors in the treatment of human malignancy, highlighting issues of drug action selectivity in tissues and potential secondary effects.

Cell type-specific roles of histone deacetylase in TR ligand-independent transcriptional repression.

Recent evidence indicates that corepressor protein with histone deacetylase (HDAC) activity mediates thyroid hormone receptor (TR) transcriptional repression. In order to examine the physiological relevance of HDAC in ligand-independent TR-mediated repression, we studied the effect of trichostatin A (TSA), a specific HDAC inhibitor, in transient transfection studies with natural reporters, and assessed the expression of TR-regulated endogenous genes. Luciferase-coupled DR4-, F2-, PAL- or GH-TREs and TRbeta1 expression vectors were cotransfected in CV-1 and GH(3) cells. We did not observe any effect of TSA on TR-induced basal repression in CV-1 cells. Instead, TSA was able to induce an increase in transcription without T(3) on all TREs tested in GH(3) cells. This increase was >7-fold on F2-, >4-fold on DR4-, and 3-fold on GH-TREs. The cotransfection of a TRbeta1 mutant that exhibits decreased affinity with N-CoR (AHT) reduced the TSA effect in GH(3) cells, demonstrating a primary role for TR/N-CoR/Sin3/HDAC complex. Next, we examined the effects of TSA on endogenous GH mRNA production in GH(3) cells by Northern blot analysis. We observed an increase of 50-70% of GH mRNA in cells treated with TSA in hypothyroid medium, and an increase of GH mRNA in T(3)-treated cells after TSA treatment. Our results show that TSA can increase the expression of endogenous genes that are susceptible to TR regulation. These results support an active role of HDAC in transcriptional repression by ligand-independent TR.

A compendium of gene expression in normal human tissues.

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Enhanced B7 costimulatory molecule expression in inflammatory human sural nerve biopsies.

OBJECTIVES: To define the role of the costimulatory molecules B7-1 and B7-2 in inflammatory disorders of the peripheral nervous system. B7 molecules are essential for effective antigen presentation and may determine the differentiation of T cells into a Th-1 or Th-2 phenotype, thus modulating immune response and disease course. METHODS: Forty nine sural nerve biopsies from patients with neuroborreliosis, Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), CIDP variants and hereditary neuropathies, and those with no detectable abnormality were investigated. The expression of B7-1 and B7-2 mRNA and protein was investigated by polymerase chain reaction (PCR) and immunocytochemistry. RESULTS: B7-1 mRNA was strongly upregulated in both cases of neuroborreliosis, in two cases of GBS and one case of variant CIDP. Moderate to low levels were detected in the remaining GBS and CIDP biopsies and were rarely found in a non-inflammatory control group consisting of hereditary neuropathy and normal nerves. At the immunocytochemical level, strong expression of B7-1 protein was found in both neuroborreliosis cases, and moderate or low expression in six of eight GBS cases and seven of 17 CIDP cases investigated, whereas only one of five non-inflammatory control nerves showed staining, which was very weak. In neuroborreliosis, B7-1 protein was found very pronounced in epineurial infiltrates, whereas in GBS and CIDP, labelling was predominantly endoneurial and localised to putative macrophages. B7-2 mRNA and protein were expressed only at low levels in neuroborreliosis and selected autoimmune neuropathy cases, and were essentially absent from non-inflammatory controls. CONCLUSIONS: B7 molecules are expressed in the peripheral nervous system and regulated during disease, and their presence in macrophages underlines the putative function of endoneurial macrophages as local antigen presenting cells in the immunopathology of peripheral nerve. B7-1 rather than B7-2 is preferentially upregulated, possibly promoting the induction of a Th-1-type T cell response within the nerve.

Cloning and expression of a murine histone deacetylase 3 (mHdac3) cDNA and mapping to a region of conserved synteny between murine chromosome 18 and human chromosome 5.

Histone deacetylases (HDACs) are enzymes that play a pivotal role in transcription, differentiation, and cell cycle progression. We previously cloned human HDAC3 cDNA and showed that its transfection into THP-1 cells results in G2/M cell cycle accumulation. Using bioinformatic screening and PCR, we have now cloned the murine Hdac3 cDNA, which encodes a 428-amino-acid protein with near complete identity to its human ortholog. To establish a link to a potential disease locus, we performed PCR-based chromosomal mapping for the mHdac3 gene and chromosomal fluorescence in situ hybridization (FISH) for the human gene. mHdac3 localizes to chromosome 18 and human HDAC3 gene localizes to a syntenic region in chromosome 5 at band q31.3-q32 telomeric to the cytokine gene cluster. Transfection of mHdac3 into HeLa cells led to accumulation in G2/M. Our results suggest a cell cycle function for murine Hdac3, reflecting the complex regulatory roles of this gene family.

Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate.

The reversible acetylation of histones by histone deacetylases (HDACs) and acetyltransferases (HATs) plays a fundamental role in gene transcription. We previously showed that HDAC mRNA is upregulated in immune cells upon PHA-induced activation. Little is known, however, about the differential regulation of HDAC mRNAs by the HDAC inhibitors Trichostatin A (TSA) and butyrate, agents known to block proliferation and induce apoptosis. We report that apoptosis-inducing concentrations of TSA and butyrate upregulate the expression of HDAC mRNAs in a differential manner and act synergistically with PHA to induce HDAC expression, suggesting the presence of independent HDAC regulatory mechanisms. Moreover, we show that HDAC inhibitor-induced apoptosis is associated with early abrogation of gamma-IFN production by Th1 lymphocytes and with p53 mRNA downregulation. Our findings highlight the dynamic interplay of cell cycle-, activation- and apoptosis-related proteins in association with time-dependent expression of HDACs and are suggestive of different specific roles for these enzymes.

Differential display cloning of a novel human histone deacetylase (HDAC3) cDNA from PHA-activated immune cells.

The nucleosomal histones can be modified through reversible acetylation by histone acetyltransferases (HATs) and deacetylases (HDACs). HATs induce nucleosomal relaxation and allow DNA-binding by transcriptional activators. HDACs from corepressor complexes which negatively regulate cell growth. However, the HDAC inhibitors butyrate and Trichostatin A block T cell proliferation, suggesting that not all effects of HDACs lead to repression. Using mRNA differential display and 5'RACE we isolated human HDAC3, a novel gene that is upregulated in PHA-activated T cell clones. HDAC3 is homologous to other human HDACs and yeast RPD3. In peripheral blood mononuclear cells (PBMCs), activation by PHA, PMA and alpha-CD3 increased HDAC mRNA but no effect was seen with IFN-gamma, LPS, or IL-4. In contrast, GMCSF downregulated PBMC levels of HDAC3 mRNA. All HDACs were found to be ubiquitously expressed in immune and non-immune tissues. In human myeloid leukemia THP-1 cells, HDAC3 transfection resulted in increased size, aberrant nuclear morphology and cell cycle G2/M cell accumulation. Functional activity of the expressed HDAC3 protein was confirmed in alpha-HDAC3 antibody immunoprecipitates by a histone deacetylase assay. Our study suggests the participation of HDACs in cell cycle progression and activation.

Constitutive expression of costimulatory molecules by human microglia and its relevance to CNS autoimmunity.

Human microglia constitute the primary residential antigen presenting cells (APCs) in the central nervous system (CNS) and have the capacity of activating myelin reactive T-cells. T-cell activation requires two signals: first is the interaction of the T-cell receptor with the MHC-antigen complex and, secondly, contact of the CD28/CTLA4 T-cell surface molecules with the B7 family of costimulatory molecules on the APCs. We have previously shown high expression of B7.1 in early multiple sclerosis (MS) plaques, suggesting that acute T-cell-mediated CNS inflammation may require local B7.1 upregulation. We have now examined the expression of B7.1 and B7.2 costimulatory molecules on resting ex-vivo human microglia isolated directly from biopsy specimens. We found constitutive expression of B7.2 but not B7.1 on resting microglia, suggesting that B7.2 expression may lead to downregulation of pro-inflammatory Th1 T-cell responses in the normal brain.

Focal brain dysfunction in a 41-year old man with familial alternating hemiplegia.

The acute pathophysiologic changes during hemiplegic spells and the long-term outcome of alternating hemiplegia remain obscure. In a 41-year-old male with familial alternating hemiplegia we found an increase in right frontal cerebral blood flow 3 h into a 5-h left hemiplegic episode. A repeat high-resolution brain SPECT study performed 26 h after the resolution of the left hemiplegia revealed normalization of the frontal blood flow accompanied by hyperperfusion in the right parietal lobe. An interictal SPECT scan several weeks later showed no asymmetries. Head CT and MRI scans were negative. Neuropsychologic assessment and neurologic examination revealed evidence of a diffuse disorder which predominantly involved the right hemisphere. To our knowledge, there are no previous correlative studies of serial high-resolution brain SPECT with MRI, or of detailed neuropsychologic assessment, in adult patients with such an advanced course of alternating hemiplegia of childhood.

Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin 12 cytokine in multiple sclerosis lesions.

Resting autoreactive T cells are present in the circulation of normal individuals without pathologic consequences. In autoimmune animal models, stimulation of these self-reactive T cells in the presence of costimulatory molecules B7-1 results in T cell-mediated autoimmune disease, whereas B7-2 stimulation generates regulatory autoreactive T cells that abrogate disease severity. Thus, reactivation in the brain of myelin-autoreactive T cells by antigen with costimulatory molecules may be a critical event in the pathophysiology of multiple sclerosis (MS), a putative autoimmune disease of central nervous system (CNS) myelin. We investigated the expression of cytokines and costimulatory molecules in a panel of 41 histologically characterized CNS specimens from 15 MS and 10 control cases using semiquantitative reverse transcriptase-polymerase chain reaction and immunocytochemistry. In four cases, vascular CNS infarcts with inflammation were compared with MS plaques from the same brain. We observed increased expression of B7-1 and interleukin (IL) 12p40 in acute MS plaques, particularly from early disease cases but not in inflammatory infarcts. B7-1 staining was localized predominantly to the lymphocytes in perivenular inflammatory cuffs but not the parenchyma. In contrast, B7-2 was expressed predominantly on macrophages both in MS lesions of varied time duration and in inflammatory infarcts. These findings indicate that an early event in the initiation of MS involves upregulation of B7-1 and IL-12, resulting in conditions that maximally stimulate T cell activation and induction of T helper 1-type immune responses.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.

Tropical spastic paraparesis on the Caribbean coast of Colombia.

Tropical spastic paraparesis (TSP) is a retroviral disease characterized predominantly by a chronic myelopathy and progressive leg weakness. Four patients from the northern coast of Columbia with chronic spastic paraparesis and serum positivity for antibodies to human T cell lymphotropic virus type 1 by enzyme-linked immunosorbent assay and Western blot are reported. All patients had mixed ethnic origins (white, black, and amerindian). This is the first report of TSP on the Caribbean coast of Colombia. This study extends the geographic boundaries of TSP in the Caribbean basin.

Paraneoplastic opsoclonus-myoclonus associated with anti-Hu antibody.

We report a 74-year-old woman with opsoclonus, myoclonus, ataxia, and encephalopathy who had small-cell lung cancer and high titers of anti-Hu antibody in her serum. At autopsy, there were perivascular inflammatory infiltrates in the brainstem, putamen, and meninges overlying the orbital frontal cortex. Immunohistochemical studies showed the expression of the Hu antigens by the tumor and the presence of deposits of anti-Hu IgG in the patient's cortex, brainstem, and cerebellum, suggesting that anti-Hu immune response was related to the patient's clinical syndrome. This case of paraneoplastic opsoclonus, myoclonus, ataxia, and encephalopathy expands the spectrum of neurologic dysfunction associated with the anti-Hu antibody.

Spontaneous calcific embolization to the supraclinoid internal carotid artery from a regurgitant bicuspid aortic valve.

Calcific brain embolization leading to stroke is rarely recognized. We report a case of spontaneous embolization to the right cerebral circulation from a severely regurgitant, calcified bicuspid valve. Bicuspid aortic valves constitute the most common congenital heart abnormality, and have a tendency to become stenosed, regurgitant, calcified, or infected. The presence of heavy calcification in a noninfected bicuspid valve may lead to dislodgement of calcific embolic material which is not necessarily heralded by acute valvular rupture. Accurate localization of the calcific embolus to the right supraclinoid internal carotid artery was provided by spiral CT imaging in this case.