Feasibility of Brain Atrophy Measurement in Clinical Routine without Prior Standardization of the MRI Protocol: Results from MS-MRIUS, a Longitudinal Observational, Multicenter Real-World Outcome Study in Patients with Relapsing-Remitting MS.

BACKGROUND AND PURPOSE: Feasibility of brain atrophy measurement in patients with MS in clinical routine, without prior standardization of the MRI protocol, is unknown. Our aim was to investigate the feasibility of brain atrophy measurement in patients with MS in clinical routine. MATERIALS AND METHODS: Multiple Sclerosis and Clinical Outcome and MR Imaging in the United States (MS-MRIUS) is a multicenter (33 sites), retrospective study that included patients with relapsing-remitting MS who began treatment with fingolimod. Brain MR imaging examinations previously acquired at the baseline and follow-up periods on 1.5T or 3T scanners with no prior standardization were used, to resemble a real-world situation. Brain atrophy outcomes included the percentage brain volume change measured by structural image evaluation with normalization of atrophy on 2D-T1-weighted imaging and 3D-T1WI and the percentage lateral ventricle volume change, measured by VIENA on 2D-T1WI and 3D-T1WI and NeuroSTREAM on T2-fluid-attenuated inversion recovery examinations. RESULTS: A total of 590 patients, followed for 16 months, were included. There were 585 (99.2%) T2-FLAIR, 425 (72%) 2D-T1WI, and 166 (28.2%) 3D-T1WI longitudinal pairs of examinations available. Excluding MR imaging examinations with scanner changes, the analyses were available on 388 (65.8%) patients on T2-FLAIR for the percentage lateral ventricle volume change, 259 and 257 (43.9% and 43.6%, respectively) on 2D-T1WI for the percentage brain volume change and the percentage lateral ventricle volume change, and 110 (18.6%) on 3D-T1WI for the percentage brain volume change and percentage lateral ventricle volume change. The median annualized percentage brain volume change was -0.31% on 2D-T1WI and -0.38% on 3D-T1WI. The median annualized percentage lateral ventricle volume change was 0.95% on 2D-T1WI, 1.47% on 3D-T1WI, and 0.90% on T2-FLAIR. CONCLUSIONS: Brain atrophy was more readily assessed by estimating the percentage lateral ventricle volume change on T2-FLAIR compared with the percentage brain volume change or percentage lateral ventricle volume change using 2D- or 3D-T1WI in this observational retrospective study. Although measurement of the percentage brain volume change on 3D-T1WI remains the criterion standard and should be encouraged in future prospective studies, T2-FLAIR-derived percentage lateral ventricle volume change may be a more feasible surrogate when historical or other practical constraints limit the availability of percentage brain volume change on 3D-T1WI.

Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2 inhibitors.

The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) is frequently dysregulated in cancers, and gain-of-function (GOF) EZH2 mutations have been identified in non-Hodgkin lymphomas. Small-molecule inhibitors against EZH2 demonstrated anti-tumor activity in EZH2-mutated lymphomas and entered clinical trials. Here, we developed models of acquired resistance to EZH2 inhibitor EI1 with EZH2-mutated lymphoma cells. Resistance was generated by secondary mutations in both wild-type (WT) and GOF Y641N EZH2 alleles. These EZH2 mutants retained the substrate specificity of their predecessor complexes but became refractory to biochemical inhibition by EZH2 inhibitors. Resistant cells were able to maintain a high level of H3K27Me3 in the presence of inhibitors. Interestingly, mutation of EZH2 WT alone generated an intermediate resistance phenotype, which is consistent with a previously proposed model of cooperation between EZH2 WT and Y641N mutants to promote tumorigenesis. In addition, the findings presented here have implications for the clinical translation of EZH2 inhibitors and underscore the need to develop novel EZH2 inhibitors to target potential resistance emerging in clinical settings.

TMPRSS2:ERG blocks neuroendocrine and luminal cell differentiation to maintain prostate cancer proliferation.

The biological outcome of TMPRSS2:ERG chromosomal translocations in prostate cancer (PC) remains poorly understood. To address this, we compared the transcriptional effects of TMPRSS2:ERG expression in a transgenic mouse model with those of ERG knockdown in a TMPRSS2:ERG-positive PC cell line. This reveals that ERG represses the expression of a previously unreported set of androgen receptor (AR)-independent neuronal genes that are indicative of neuroendocrine (NE) cell differentiation-in addition to previously reported AR-regulated luminal genes. Cell sorting and proliferation assays performed after sustained ERG knockdown indicate that ERG drives proliferation and blocks the differentiation of prostate cells to both NE and luminal cell types. Inhibition of ERG expression in TMPRSS2:ERG-positive PC cells through blockade of AR signaling is tracked with increased NE gene expression. We also provide evidence that these NE cells are resistant to pharmacological AR inhibition and can revert to the phenotype of parental cells upon restoration of AR/ERG signaling. Our findings highlight an ERG-regulated mechanism capable of repopulating the parent tumor through the transient generation of an anti-androgen therapy-resistant cell population, suggesting that ERG may have a direct role in preventing resistance to anti-androgen therapy.

Microdeletion/duplication at 15q13.2q13.3 among individuals with features of autism and other neuropsychiatric disorders.

BACKGROUND: Segmental duplications at breakpoints (BP4-BP5) of chromosome 15q13.2q13.3 mediate a recurrent genomic imbalance syndrome associated with mental retardation, epilepsy, and/or electroencephalogram (EEG) abnormalities. PATIENTS: DNA samples from 1445 unrelated patients submitted consecutively for clinical array comparative genomic hybridisation (CGH) testing at Children's Hospital Boston and DNA samples from 1441 individuals with autism from 751 families in the Autism Genetic Resource Exchange (AGRE) repository. RESULTS: We report the clinical features of five patients with a BP4-BP5 deletion, three with a BP4-BP5 duplication, and two with an overlapping but smaller duplication identified by whole genome high resolution oligonucleotide array CGH. These BP4-BP5 deletion cases exhibit minor dysmorphic features, significant expressive language deficits, and a spectrum of neuropsychiatric impairments that include autism spectrum disorder, attention deficit hyperactivity disorder, anxiety disorder, and mood disorder. Cognitive impairment varied from moderate mental retardation to normal IQ with learning disability. BP4-BP5 covers approximately 1.5 Mb (chr15:28.719-30.298 Mb) and includes six reference genes and 1 miRNA gene, while the smaller duplications cover approximately 500 kb (chr15:28.902-29.404 Mb) and contain three reference genes and one miRNA gene. The BP4-BP5 deletion and duplication events span CHRNA7, a candidate gene for seizures. However, none of these individuals reported here have epilepsy, although two have an abnormal EEG. CONCLUSIONS: The phenotype of chromosome 15q13.2q13.3 BP4-BP5 microdeletion/duplication syndrome may include features of autism spectrum disorder, a variety of neuropsychiatric disorders, and cognitive impairment. Recognition of this broader phenotype has implications for clinical diagnostic testing and efforts to understand the underlying aetiology of this syndrome.

Single-stage sacral coccygectomy and repair using human acellular dermal matrix (AlloDerm) with bilateral gluteus maximus flaps for hernia prophylaxis.

Sacrococcygectomy is often necessary for the ablation of malignancies involving the sacrum and coccyx, and can result in deep posterior peritoneal defects with disruption of the pelvic floor. Such a radical procedure is frequently associated with significant morbidity, including sacral herniation. Numerous techniques for the closure of the surgical defect have been described, with varying degrees of success in avoiding future herniation. We report the first single-stage coccygectomy and partial sacrectomy with closure utilizing human acellular dermal matrix (HADM) (AlloDerm; LifeCell Corporation, Branchburg, NJ) and bilateral gluteus maximus transposition flaps.

Digital ulcers in systemic sclerosis: prevention by treatment with bosentan, an oral endothelin receptor antagonist.

OBJECTIVE: Recurrent digital ulcers are a manifestation of vascular disease in patients with systemic sclerosis (SSc; scleroderma) and lead to pain, impaired function, and tissue loss. We investigated whether treatment with the endothelin receptor antagonist, bosentan, decreased the development of new digital ulcers in patients with SSc. METHODS: This was a randomized, prospective, placebo-controlled, double-blind study of 122 patients at 17 centers in Europe and North America, evaluating the effect of treatment on prevention of digital ulcers. The primary outcome variable was the number of new digital ulcers developing during the 16-week study period. Secondary assessments included healing of existing digital ulcers and evaluation of hand function using the Scleroderma Health Assessment Questionnaire. RESULTS: Patients receiving bosentan had a 48% reduction in the mean number of new ulcers during the treatment period (1.4 versus 2.7 new ulcers; P = 0.0083). Patients who had digital ulcers at the time of entry in the study were at higher risk for the development of new ulcers; in this subgroup the mean number of new ulcers was reduced from 3.6 to 1.8 (P = 0.0075). In patients receiving bosentan, a statistically significant improvement in hand function was observed. There was no difference between treatment groups in the healing of existing ulcers. Serum transaminase levels were elevated to >3-fold the upper limit of normal in bosentan-treated patients; this elevation is comparable with that observed in previous studies of this agent. Other side effects were similar in the 2 treatment groups. CONCLUSION: Endothelins may play an important role in the pathogenesis of vascular disease in patients with SSc. Treatment with the endothelin receptor antagonist bosentan may be effective in preventing new digital ulcers and improving hand function in patients with SSc.

Global expression analysis of the fibroblast transcriptional response to TGFbeta.

OBJECTIVES: Transforming Growth Factor-beta (TGFbeta) is the predominant cytokine in all forms of fibrotic reactions. As well as being secreted by immune modulators of fibrosis such as macrophages, it is involved in an autocrine feedback loop of fibroblast stimulation whose regulation is still poorly understood. We wished to gain some insight into the mechanisms of the fibroblast response to TGFbeta. METHODS: We undertook an exhaustive transcript profiling experiment using a widely validated restriction enzyme based method for identifying differentially expressed genes (GeneCalling). Transcriptional responses throughout a 24-hour time course were examined at multiple time points and classified. RESULTS: By 24 hours of TGF treatment over 1000 bands, representing a large number of transcripts, were down- or upregulated greater than 2-fold. All of the known genes responsive to TGFbeta, such as collagen and connective tissue growth factor, were upregulated. CONCLUSIONS: This encyclopedic method revealed many unknown transcriptional responses to TGFbeta including the upregulation of a variety of less expected cytoskeletal and matrix components, as well as interactions between the TGFbeta and tumor necrosis factor (TNF) pathways and alterations in cell death-related pathways. These may in part explain the idiosyncratic responses of mesenchymal cells to TGFbeta.

The hcKrox gene family regulates multiple extracellular matrix genes.

The transcription factor cKrox was originally identified as a protein that bound to a negative transcription regulatory element in the murine alpha1(I) collagen promoter. We recently reported the cloning and characterization of human cKrox (hcKrox). Overexpression of hcKrox in NIH3T3 fibroblasts efficiently repressed the promoters of the fibronectin and alpha1(I) collagen genes (70-90%) in transient transfection assays and suppressed the endogenous genes in hcKrox expressing permanent cell lines. We have now isolated genomic clones and cDNAs encoding two novel transcription factors related to hcKrox termed hcKrox-beta and hcKrox-gamma (the original clone is now referred to as hcKrox-alpha). Both contain three kruppel-like zinc-finger DNA binding motifs that are 71-78% identical to those of hcKrox-alpha. The NH(2)-terminus of all three proteins contains a POZ domain, a conserved 120 amino acid motif involved in transcriptional repression and protein dimerization. RT-PCR experiments demonstrate that all three hcKrox family members are expressed in foreskin and dermal fibroblasts. Transient transfection studies in NIH3T3 fibroblasts demonstrate that hcKrox-alpha -beta and -gamma, as well as the murine cKrox-beta homologue, LRF, suppress transcription driven by promoters for the alpha1(I) and alpha2(I) collagen, fibronectin and elastin genes. Electrophoretic mobility shift assays and coimmunoprecipitation studies suggest that homo- and heterodimerization occurs between cKrox family members. Dimer formation is influenced by amino acids in the NH(2)-terminal POZ domain and the Zn(+2)-finger region. Immunoprecipitation studies indicate that cKrox can form heterodimers in solution in the absence of DNA. Thus, a multi-gene family exists that can coordinately regulate several extracellular matrix genes and has the potential to form many heterodimeric transcription factors.

The plasticity of dendritic cell responses to pathogens and their components.

Dendritic cells are involved in the initiation of both innate and adaptive immunity. To systematically explore how dendritic cells modulate the immune system in response to different pathogens, we used oligonucleotide microarrays to measure gene expression profiles of dendritic cells in response to Escherichia coli, Candida albicans, and influenza virus as well as to their molecular components. Both a shared core response and pathogen-specific programs of gene expression were observed upon exposure to each of these pathogens. These results reveal that dendritic cells sense diverse pathogens and elicit tailored pathogen-specific immune responses.

Suprathreshold repetitive transcranial magnetic stimulation elevates thyroid-stimulating hormone in healthy male subjects.

Repetitive transcranial magnetic stimulation (rTMS) has been introduced as a new antidepressive treatment strategy. The mode of action by which the antidepressive effect is brought about is not yet clear. Other antidepressive treatment strategies such as sleep deprivation are associated with an increase of plasma thyroid-stimulating hormone (TSH) levels that correlate with clinical improvement. In the present study, the effect of left prefrontal suprathreshold (120% of motor threshold) rTMS on TSH plasma levels of 19 healthy male subjects was investigated in comparison with subthreshold (80% of motor threshold) and sham stimulation. Suprathreshold rTMS was followed by a significant relative increase of TSH levels 10 and 60 minutes after stimulation in comparison with subthreshold and sham stimulation. The more pronounced effect of suprathreshold rTMS on TSH plasma levels might be important for the determination of optimal stimulation parameters in the treatment of depressed patients.

Results of a phase-I/II randomized, masked, placebo-controlled trial of recombinant human interleukin-11 (rhIL-11) in the treatment of subjects with active rheumatoid arthritis.

Interleukin-11 (IL-11) is a pleiotropic cytokine that regulates the growth and development of hematopoietic stem cells and decreases the proinflammatory mediators of cytokine and nitric oxide production. In animal models of arthritis, treatment with recombinant human IL-11 (rhIL-11) reduces both the level of synovitis and the histologic lesion scores in the joints. The goal of this phase-I/II study in adults with rheumatoid arthritis (RA) was to evaluate the safety and clinical activity of different doses and schedules of rhIL-11 in patients with active RA for whom treatment with at least one disease-modifying antirheumatic drug had failed. This was a multicenter, randomized, placebo-controlled trial that evaluated the safety and tolerability of rhIL-11 in 91 patients with active RA. rhIL-11 was administered subcutaneously; patients were randomized into one of five treatment groups (ratio of rhIL-11 to placebo, 4:1). Patients were treated for 12 weeks with either 2.5 or 7.5 microg/kg of rhIL-11 or placebo twice per week or 5 or 15 microg/kg of rhIL-11 or placebo once per week. The status of each subject's disease activity in accordance with the American College of Rheumatology (ACR) criteria was assessed before, during, and after completion of administration of the study drug. Administration of rhIL-11 was well tolerated at all doses and schedules. The most frequent adverse event was a reaction at the injection site. The data suggest a statistically significant reduction in the number of tender joints (P < 0.008) at the 15 microg/kg once-weekly dose schedule but showed no overall significant benefit at the ACR criterion of a 20% response. The trial showed rhIL-11 to be safe and well tolerated at a variety of doses and schedules over a 12-week treatment period in patients with active RA. The only adverse event clearly associated with rhIL-11 administration was reaction at the injection site.

Embryonic central nervous system angiogenesis does not involve blood-borne endothelial progenitors.

We asked, whether, in the blood of avian embryos, endothelial precursor cells circulate that actually contribute to the growing vascular system in and around the central nervous system (CNS). We compared the morphology and distribution of QH1-positive cells after transplantation of quail paraxial mesoderm, after blood transfusion, in quail-chick parabiosis, or after quail bone-marrow transplantation. After head mesoderm transplantation from quail to chick, we observed sprouting endothelial cells (ECs), capillary tube formation, and chimeric endothelial lining of large arteries in the host brain. These QH1-positive quail cells showed EC morphologies that demonstrated three different aspects of CNS angiogenesis: invasion by means of filopodia, clonal proliferation and tube formation, and integration into preexisting EC layers. After blood transfusion or in chick-quail parabiosis, blood-borne QH1+ cells were found in the lumen of but not integrated into the wall of the host vascular system. Neither were QH1+ cells observed in the capillary walls of parabiotic chick chorioallantoic membranes. In both cases, the quail cells showed typical macrophage morphology. In chicks that had received quail bone marrow transplants onto their chorioallantoic membranes, QH1+ cells with macrophage, but not EC shape were occasionally seen near the inoculation site. We conclude that (1) blood-borne cells do not become ECs or directly contribute to angiogenesis inside, or in vascular plexuses around the CNS during embryonic development; (2) blood-borne cells do not contribute to the intraneural macrophage population of the embryonic CNS.

Role of apoptosis and transforming growth factor beta1 in fibroblast selection and activation in systemic sclerosis.

OBJECTIVE: We hypothesized that pathophysiologic events during the development of systemic sclerosis (SSc) may lead to selection and propagation of certain apoptosis-resistant fibroblast subpopulations. The aim of this study was to examine a possible role for apoptosis in fibroblast selection in SSc and the role of transforming growth factor beta1 (TGFbeta1). METHODS: We compared SSc and normal fibroblasts for their susceptibility to anti-Fas-induced apoptosis and analyzed 2 models that might lead to fibroblast resistance to apoptosis in this process: long-term exposure to either anti-Fas or TGFbeta1. RESULTS: SSc-derived fibroblasts were resistant to anti-Fas-induced apoptosis, showing 5.5 +/- 17.2% (mean +/- SD) apoptosis, compared with 32.1 +/- 14.0% among normal fibroblasts (P < 0.05). Anti-Fas-selected normal fibroblasts showed 9.0 +/- 3.7% apoptosis, compared with 21.6 +/- 5.9% for sham-treated cells, which is consistent with the elimination of apoptosis-susceptible subpopulations. Normal fibroblasts subjected to 6 weeks of TGFbeta1 treatment showed not only resistance to apoptosis, but also proliferation (118.5 +/- 35.4%), after anti-Fas treatment, compared with sham-treated cells (35.1 +/- 11.1% apoptotic cell death). TGFbeta1 treatment also increased the proportion of myofibroblasts (47% versus 28% in controls). Cultured SSc fibroblasts had a greater proportion of myofibroblasts (32-83%) than did normal fibroblasts (4-25%). We also examined the relationship between collagen gene expression and the myofibroblast phenotype in normal and SSc skin sections. Only 2 of 7 normal sections had alpha-smooth muscle actin (a-SMA)-positive cells (mean +/- SD score 0.29 +/- 0.49 on a scale of 0-3), but all SSc sections were positive for alpha-SMA, with a mean score of 1.90 +/- 0.88 for lesional and 1.50 +/- 0.71 for nonlesional sections. Scores for alpha1(I) procollagen messenger RNA (mRNA) in lesional skin (mean +/- SD 3.30 +/- 0.82 on a scale of 1-4) were significantly higher than in normal (1.43 +/- 0.79) or nonlesional (1.40 +/- 0.52) skin, but scores varied, and there was no correlation between collagen mRNA and alpha-SMA levels. CONCLUSION: Our results show that resistance to apoptosis is an important part of the SSc phenotype. TGFbeta1 may play a role by inducing apoptosis-resistant fibroblast populations, and also by inducing myofibroblasts and by enhancing extracellular matrix synthesis.

Recombinant human relaxin in the treatment of scleroderma. A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Relaxin is a pregnancy-related hormone that has tissue remodeling and antifibrotic effects. Systemic sclerosis (scleroderma) is characterized by fibrosis of the skin, vasculature, and internal organs. OBJECTIVE: To assess the efficacy, safety, and dose-response effect of recombinant human relaxin in patients with scleroderma. DESIGN: Multicenter, parallel-group, randomized, double-blind, placebo-controlled trial. SETTING: Academic referral centers. PATIENTS: 68 patients who had had stable, diffuse scleroderma (moderate to severe) for less than 5 years. INTERVENTION: Recombinant human relaxin, 25 or 100 microg/kg of body weight per day, or placebo administered by continuous subcutaneous infusion over 24 weeks. MEASUREMENTS: Modified Rodnan skin score was the primary efficacy measure. Secondary measurements were pulmonary function, the Health Assessment Questionnaire, and other measures of scleroderma that reflected fibrosis. RESULTS: Patients who received 25 microg/kg of recombinant human relaxin per day had significantly lower skin scores than those who received placebo (mean change, -3.6 at 4 weeks [P = 0.021], -7.5 at 12 weeks [P < 0.001], and -8.7 at 24 weeks [P = 0.040]). Similar trends were noted in other outcome measures, including forced vital capacity, measures of oral aperture and hand extension, functional status, and global assessment. Patients who received 100 microg/kg of relaxin per day did not differ from those who received placebo. Drug-related adverse events included menometrorrhagia, reversible anemia, and complications of the subcutaneous drug administration system (site irritation and local infection). CONCLUSIONS: Twenty-four weeks of recombinant human relaxin, 25 microg/kg per day, is associated with reduced skin thickening, improved mobility, and improved function in patients with moderate to severe diffuse scleroderma.

Induction of heme oxygenase-1 by hypoxia and free radicals in human dermal fibroblasts.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme catabolism and presumably is involved in cellular iron homeostasis. It is induced by a variety of cellular stresses, including oxygen deprivation and free radical-mediated stress. We examined induction of HO-1 mRNA in skin fibroblasts and investigated the mechanism by which it occurs. Hypoxia did not appear to act via induction of oxygen free radicals: induction of HO-1 was not sensitive to the free radical scavenger GSH or other antioxidants. Moreover, hypoxia did not increase steady-state levels of free radicals generated by fibroblasts. In contrast, HO-1 induction by the oxidants, H(2)O(2) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) was significantly attenuated in the presence of free radical scavengers. This correlated with increased levels of free radical production in fibroblasts treated with these oxidants. Iron depletion by desferrioxamine mesylate, a specific iron complexon, completely inhibited hypoxic stimulation of HO-1 but did not attenuate the effect of H(2)O(2) and CCCP on HO-1 mRNA. Addition of Fe(2+), Fe(3+), or holo-transferrin to fibroblasts increased levels of HO-1 mRNA. Treatment of cells with hypoxia, but not H(2)O(2) or an exogenous source of iron, significantly increased the half-life of HO-1 mRNA. The data suggest hypoxia regulates HO-1 gene expression by a specific posttranscriptional mechanism: stabilization of mRNA. Hypoxia has previously been shown to increase fibroblast collagen synthesis and is thought to play a role in pathogenesis of systemic sclerosis (SSc). Skin fibroblasts isolated from patients with SSc demonstrated significantly stronger induction of HO-1 by hypoxia than did fibroblasts from normal controls. We hypothesize that exposure of SSc fibroblasts to hypoxic conditions leads to in vivo selective proliferation of cells that adapt to hypoxia.