Evaluation of IFNalpha bioavailability by MxA mRNA in HCV patients

Previously, we have reported the development of a new quantitative-competitive polymerase chain reaction (qc-PCR) method to evaluate interferon-beta (IFNbeta) bioavailability in multiple sclerosis (MS) patients, by measuring mRNA of mixovirus resistance protein A (MxA). Here we show that our assay is also able to assess IFNalpha bioavailability in hepatitis C virus (HCV) patients treated with different IFNalpha regimens. Indeed, our method was able to detect a slight constitutive expression of MxA mRNA in untreated HCV patients (median=70 fgMxA/pgGAPDH) and a significant induction 12 h after the first IFNalpha administration (median=750 fgMxA/pgGAPDH).     

Validating parameters of a luciferase reporter gene assay to measure neutralizing antibodies to IFNbeta in multiple sclerosis patients

Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon β (IFNβ) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INFβ. We assayed 163 sera from IFNβ treated MS patients with an optimized luciferase method and compared the results to those obtained with the reference cytopathic effect (CPE) method using A549 cells and an encephalomyocarditis virus (EMCV). Binding antibodies (BAbs) were measured using a capture ELISA as a screening test for NAbs in the CPE assay. NAb status measured by the luciferase and the ELISA/CPE method did not yield a significant difference. Log10 NAb titers obtained from the luciferase assay and the A549/EMCV CPE methods correlated very well. The inter-assay coefficient of variation for titers was between 17.8–29.3%, and the intra-assay coefficient of variation was between 6.3–15.2%. The luciferase assay is reliable, appropriately sensitive and requires less time than the currently available NAb methods.     

Cognitive impairment in three subtypes of multiple sclerosis

Patients with relapsing, primary progressive, and secondary progressive MS were administered the Tower of London and the Stroop Color-Word Interference Test, yielding several measures of executive function and speeded information processing. MS patients' performance was compared with healthy controls and with a clinical control group consisting of patients with rheumatoid arthritis. The MS patients performed the tests more slowly, but did not differ from either group of controls on measures of executive function. Slowing in the speed of information processing was characteristic of MS patients across two basic tasks differing in terms of controlled versus automatic processing and in terms of whether or not rapid responding was an explicit feature of successful performance. Although evident in all subtypes, this slowing was more pronounced in secondary progressive patients and somewhat less pronounced in primary progressive patients. Furthermore, the slowing was unrelated to patients' disability status or level of depression.     

Evaluation of the immunosuppressive effects of cyclophosphamide in patients with multiple sclerosis.

In a group of eight patients suffering from clinically definite multiple sclerosis, we studied the effects of treatment with cyclophosphamide on the immune reactivity in vitro and in vivo. The results are compared with those obtained in a control group consisting of eight patients who received no drug therapy and who were matched with the former group for age, sex and severity of disease. The results indicate that therapy with cyclophosphamide at a mean dose of 100 mg/day induces a profound lymphocytopenia in peripheral blood involving both T and B cells. Serum levels of immunoglobulins as well as primary and secondary antibody responses were depressed. In tests with standardized cell numbers, proliferative responses of lymphocytes in vitro and cytotoxic T cell function remained normal, whereas K and NK cell activities were diminished. Secondary cellular immune responses in vivo remained intact; however, the primary cellular immune response in vivo was markedly depressed. From these data, it is concluded that therapy with cyclophosphamide in man mainly affects humoral immune functions, but also cellular immunity, although to a lesser extent.     

The initiation of multiple sclerosis: a new infectious hypothesis

Both genetic and environmental factors cause multiple sclerosis (MS). Few genes have been identified, however, and environmental factors remain elusive. Some postulate an infectious cause, but no pathogens are reproducibly demonstrable in CNS lesions. I postulate that the CNS is not the infectious target in MS, but propose a two-hit infectious hypothesis focusing on nai;ve CD4 T-cells that initiate demyelination: (1) Various common viruses infect the thymus during childhood (first hit) and enhance nai;ve CD4 T-cell reactivity to CNS autoantigens; (2) Heterogeneous pathogens fully activate these T-cells during adulthood (second hit) to initiate myelin injury. The novel concept of thymic infection provides insight into the nature of some susceptibility genes, helps explain the high discordance rates in genetically susceptible individuals, and suggests it is futile to search for pathogens in MS lesions. Pathogen heterogeneity, i.e., the lack of a single infectious cause, implies there can be no simple therapies to prevent or treat MS.     

Intrathecal immune reset in multiple sclerosis: Exploring a new concept

Multiple sclerosis impairment is mainly driven by the progressive phase, whose pathology remains elusive. No drug has yet been able to halt this phase so therapeutic management remains challenging. It was recently demonstrated that late disability correlates with the spreading of cortical subpial lesions, and tertiary lymphoid organs (TLO) were identified in close apposition with these lesions. TLO are of crucial importance since they are able to mount a complete local immune response, as observed in the intrathecal compartment from the moment MS is diagnosed (i.e. oligoclonal bands). This article examines the consequences of this intrathecal response: giving a worst clinical prognostic value and bearing arguments for possible direct brain toxicity, intrathecal secretion should be targeted by drugs abating both B-lymphocytes and plasma cells. Another consequence is that intrathecal secretion has value as a surrogate marker of the persistence of an ongoing intrathecal immune reaction after treatment. Although it is still unsure which mechanism or byproduct secreted by TLO triggers cortical lesions, we propose to target TLO components as a new therapeutic avenue in progressive MS.     

Hormonal influences in multiple sclerosis: new therapeutic benefits for steroids

Multiple sclerosis (MS) is one of the most common neurological disorders. It affects mainly women. This autoimmune disease of the central nervous system (CNS) is characterized by intermittent or chronic damage to the myelin sheaths (demyelination), focal inflammation and axonal degeneration. During the early relapsing/remitting stages of MS, myelin can regenerate, but as the disease progresses the remyelination of axons becomes insufficient, leading to impaired axon conduction, neurodegeneration and the worsening of symptoms. The present pharmacological treatment of MS is limited to the administration of immunomodulatory and anti-inflammatory drugs, which are only palliative and do not significantly slow progress of the disease. What are needed are agents that target different cell types in the CNS to protect axonal networks and stimulate the endogenous capacity of myelin repair. Estrogens and progestins may be the basis for such a new therapeutic approach. Although clinical observations provide only indirect or insufficient evidence for an influence of sex steroids on the progress of MS, experimental studies have shown that estrogens and progestins exert multiple beneficial effects in experimental autoimmune encephalomyelitis (EAE), a widely used MS disease model. Moreover, both types of hormones have been shown to promote the viability of neurons and the formation of myelin. These promising experimental results should encourage the launch of prospective clinical studies to clarify the influence of hormones on the course of MS and the effect of hormone treatments, in particular those presently used in contraception and hormone replacement therapy (HRT).     

Analysis of neutralizing antibodies to therapeutic interferon-beta in multiple sclerosis patients: a comparison of three methods in a large Australasian cohort

Persistent high-titre neutralizing antibodies (NAB) to therapeutic interferon-β (IFNβ) in multiple sclerosis patients reduce therapeutic efficacy. Difficulties in standardization of cell-based bioactivity assays have hindered interlaboratory comparison of NAB titres and the determination of a clinically relevant definition of seropositivity. We determined NAB status in Australasian multiple sclerosis patients receiving IFNβ using both the antiviral cytopathic effect (CPE) assay (n = 227) and the more specific ELISA for the type I interferon-inducible MxA protein (n = 350). While the log₁₀ titres determined in the two assays were highly correlated (p < 0.0001; r = 0.967) with similar distributions, the MxA assay was more sensitive, detecting lower concentrations of NAB than the CPE assay. The range of titres determined in the CPE assay was 10 to > 7290; and 9 to 53,700 in the MxA assay, with ranked titre distribution highlighting the arbitrary nature of currently accepted definitions of NAB seropositivity. Bioactivity of injected IFNβ  was significantly reduced in NAB-positive patients (p = 0.006; NAB MxA titres = 184 to 5340) compared to NAB-negative patients as assessed ex vivo using real-time RT-PCR analysis of MxA gene induction. The range of MxA mRNA levels in healthy controls was remarkably consistent with previously published results, regardless of the assay standardization method [Gilli, F., Sala, A., Marnetto, F., Lindberg, R.L., Leppert, D. and Bertolotto, A. (2003) Comparison of IFNbeta bioavailability evaluations by MxA mRNA using two independent quantification methods. Abstract, ECTRIMS Meeting, Milan, Italy; Pachner, A., Narayan, K., Price, N., Hurd, M. and Dail, D. (2003a) MxA Gene Expression Analysis as an Interferon-beta Bioactivity Measurement in Patients with Multiple Sclerosis and the Identification of Antibody-Mediated Decreased Bioactivity. Mol. Diagn. 7, 17–25]. Assessment of IFNβ response ex vivo accounts for both circulating factors and the cellular response to IFNβ, and the data support the development of the MxA gene induction assay for the routine screening of patients receiving IFNβ.     

A new primate model for multiple sclerosis in the common marmoset

Experimental autoimmune encephalomyelitis (EAE) in outbred marmoset monkeys (Callithrix jacchus) is a recently developed nonhuman primate model of multiple sclerosis. Here, Bert 't Hart and colleagues compare this model to EAE in rhesus monkeys, highlighting autoimmune mechanisms in CNS inflammation and demyelination, including the role of major histocompatibility complex restriction and preclinical evaluation of innovative immunotherapies.     

Brain damage assessment in patients with multiple sclerosis by means of MRI

Brain magnetic resonance imaging (MRI) findings in 32 patients recently clinically diagnosed with multiple sclerosis (MS) by McDonald's criteria are presented. Demyelinization plaques were examined according to their number, size and location in brain tissue. Classification was done by Frederiksen's quantification. The study included 18 (56.3%) female and 1-4 (43.7%) male patients aged 17-44, mean 32 +/- 2.4 years. The predominant plaque location was periventricular (100%), involving lateral brain chambers, followed by subcortical, pontine, corpus callosum, cerebellar and other locations (medulla oblongata, spinal marrow). According to plaque number, patients were divided into six groups, from 2-6 plaques (group 1), to 26 lesions (divided into four subgroups). Patients with at least one plaque 15 mm in diameter were allocated to a separate group. The largest was the group with 7-15 plaques (37%). According to plaque size, patients were divided into three groups: plaque of up to 6 mm in diameter, 6-15 mm in diameter, and at least one plaque 15 mm in diameter. Patients with plaque size 6-15 mm were found to predominate (64%). There was a surprisingly high incidence of severe brain damage, i. e. higher degrees by Frederiksen's quantification. The third and fourth quantification degrees were most common, with a significant number of plaques not exceeding 15 mm in diameter; however, there also were been patients with plaques of 15 mm in diameter, which is quite surprising at this early stage of the disease. This pilot study indicated that research should be extended to patients newly clinically diagnosed with MS, comparing their clinical symptoms and Frederiksen's quantification.     

CT quantification of hemoperitoneum volume in abdominal haemorrhage: a new method

Introduction

In emergency departments, focused assessment for sonographic examination of trauma patients (FAST) accurately detects hemoperitoneum in unstable patients. Currently, only an approximation of the volume of free intraperitoneal fluid (FIPF) can be done using ultrasound (US) and CT scans. We previously reported a new method developed on an experimental cadaveric model using US examination of the abdomen and applying a mathematic formula to effusion measurements to evaluate the exact volume of FIPF. The aim of this prospective study is to extrapolate this method in a clinical practice and apply it to CT measurements of the same area.

Patients and methods

We included prospectively eleven patients admitted with acute intraperitoneal haemorrhage: 10 patients with post-traumatic hemoperitoneum and 1 patient with a ruptured extra-uterine pregnancy. The mean age was 43.2 years (extremes: 21–82). There were six males and five females. All of these patients had to undergo emergency surgery by laparotomy or laparoscopy. The amount of FIPF was assessed preoperatively on axial sections of CT scan, by measuring fluid thickness in millimetres in the hepatorenal pouch (Morrison’s pouch), between the inferior aspect of the liver and the anterior aspect of the right kidney. During the emergency surgical procedure, we collected and quantified FIPF volume by direct measure in all cases.

Results

The correlation between fluid thickness x (mm) on the CT scan and the estimated amount of FIPF was established by the following linear function: volume (mL) = 81.068x + 263.2. The Spearman’s R obtained is 0.779 and the significance level is 0.005. We found a constant correlation between FIPF measured by radiologic procedure and direct per-operative measurement of FIPF.

Conclusion

This new linear function can be used to measure the exact volume of FIPF. This evaluation can help surgical decisions, especially when abdominal trauma is associated with other haemorrhagic lesions.     

No evidence for transmission disequilibrium between a new marker at the myelin basic protein locus and multiple sclerosis in French patients

The myelin basic protein (MBP) gene is a candidate locus for susceptibility to multiple sclerosis. Several groups have tested a complex (TGGA)n repeat in the 5' region of this gene for association/linkage with multiple sclerosis, with divergent results. This region of tandem repetitive sequence has been subjected to complex rearrangements, and there is a possibility that alleles of the same size have different internal structures, which reduces the interest of this marker for linkage disequilibrium studies and may at least partly explain the conflicting results obtained so far. To overcome this problem, we isolated a new polymorphic (CA)n repeat within the Golli-MBP locus. The limited number of alleles identified makes this other marker suitable for transmission disequilibrium studies. We tested this marker for linkage with multiple sclerosis, using the transmission-disequilibrium test (TDT) on a sample of 196 nuclear families in which the genotypes of both parents could be unambiguously defined. We found no evidence of transmission disequilibrium between multiple sclerosis and any of the three alleles of this marker, even when the patients were subdivided according to their HLA-DRB1*1501 status. The present data thus provide no evidence for a contribution of the MBP gene to multiple sclerosis susceptibility in French patients.     

A new method of wet scanning electron microscopy for the analysis of myelination in EAE mouse model of multiple sclerosis

Development of effective therapies for multiple sclerosis (MS) is dependent on the advancement of improved tools for evaluation of progression of this disease in animal models. We present a novel technique utilizing scanning electron microscopy (SEM) for imaging wet biological specimens thus enabling rapid and high-resolution imaging of myelin in mouse spinal cord (SC). We demonstrate the advantages of using the wet SEM technique to image myelin in a murine model of MS, experimental autoimmune encephalomyelitis (EAE) induced in the Biozzi (antibody-high) mouse, by sensitization with spinal cord homogenate (SCH) in adjuvant. Our studies show that the methodology allows easy identification of normal and pathological components with great clarity, which is then correlated with light microscopy (LM) and validated thereby. Furthermore, we demonstrate gold immunolabeling of specific epitopes. We conclude that the new technique provides a quick, accurate, and detailed structural evaluation of the SC that can be applied to advance the research of MS.     

The etiology of multiple sclerosis: a new and extended vascular-ischemic model

It is hypothesized that multiple sclerosis is a disease of the cerebro-vascular system. The basic defect is visualized as a wound in the CNS due to a focal hypertension of genetically susceptible vessels which results in vascular injury and the initiation of a series of biochemical and physiological events culminating in an ischemic hypoxia leading to demyelination and a secondary damaging process associated with the immune system.     

Hemolytic reaction of sheep erythrocytes as a method of diagnosing multiple sclerosis

The ability of the blood sera from patients with disseminated sclerosis (DS) to lyse native sheep red cells but not any other red cells was revealed. A method for the diagnosis of DS was developed: sheep red cells hemolysis test (SRCHT). The test was positive in all DS patients (altogether 96 cases) and negative in the controls. The hemolysin titer correlated with the disease severity, SRCHT permitted the diagnosis of DS long before manifestation of clinical symptoms.     

A review of the evidence for a natalizumab exit strategy for patients with multiple sclerosis

Natalizumab is an effective treatment for relapsing-remitting multiple sclerosis (RRMS), but treatment for >2?years is associated with an increased risk of opportunistic infection and progressive multifocal leukoencephalopathy (PML). For this reason, patients and physicians may consider discontinuing natalizumab therapy. This article reviews the evidence for the various therapeutic approaches that may be taken in such patients. Stopping therapy altogether is unlikely to be appropriate for most patients, as it is associated with a high rate of relapse or rebound. Continuing natalizumab therapy with increased monitoring and vigilance for PML may be an acceptable option for some patients, while the data on extending the dosing interval of natalizumab look promising. In some patients whose pre-natalizumab disease activity was not very high and who did not relapse while on natalizumab, switching to a first-line treatment may be an option. In this case, dimethyl fumarate may carry a lower risk of relapse than interferon beta or glatiramer acetate. Fingolimod is the most studied post-natalizumab therapy, and the relapse rate appears to be higher than on natalizumab but lower than was seen before initiation of natalizumab. The evidence suggests that the washout period between natalizumab and fingolimod should not exceed 12?weeks. Finally, the limited evidence available for rituximab and alemtuzumab is promising, and further data on these and other newer therapies for RRMS are awaited.     

Management of urinary incontinence in patients with multiple sclerosis

We investigated the management of urinary incontinence in 50 patients with multiple sclerosis (MS) in two London boroughs. Only seven appeared to be satisfied with the management of their bladder problems. A total of 51 suggestions was made for improving management in 33 of the patients. Most of these suggestions involved services which were available though not being used. The management of urinary incontinence in patients with MS should be tailored to the requirements of the individual. Alternative forms of management may often not be reaching patients who might benefit from them.