Ralf Giess

CNTF is a major protective factor in demyelinating CNS disease: A neurotrophic cytokine as modulator in neuroinflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-α, suggesting that endogenous CNTF may counterbalance this effect of TNF-α (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.

A phase I/II trial of recombinant methionyl human brain derived neurotrophic factor administered by intrathecal infusion to patients with amyotrophic lateral sclerosis.

BACKGROUND: Brain derived neurotrophic factor (BDNF) is a potent survival factor for motoneurons. This study investigated the safety and tolerability of recombinant methionyl ( r -metHuBDNF) infused intrathecally by means of an implanted pump in patients with ALS. METHODS: Twenty-five probable or definite ALS were treated with either r -metHuBDNF (25, 60, 150, 400 or 1000 mug/day) or placebo in a 12- week, randomized, double-blinded, sequential, dose-escalation study. Test treatment was interrupted by a washout period from days 11 to 25 to allow the evaluation of laboratory safety measures. In each dose cohort four patients received r -metHuBDNF and one received placebo. On completion of the double-blind period of the study all patients continued to receive r -metHuBDNF in an open-label extension for up to 60 weeks. Lumbar cerebrospinal fluid (CSF) samples were taken periodically from all patients for the measurement of r -metHuBDNF levels and in a minority of patients these were supplemented by cisternal samples. RESULTS: Within days after the initiation of infusion the majority of patients receiving r -metHuBDNF reported mild sensory symptoms, including paraesthesias or a human BDNF sense of warmth, which were usually confined to the lower limbs and were frequently exacerbated by neck flexion. In most instances these symptoms patients with decreased or even disappeared over several weeks. Sleep disturbance, dry mouth, agitation and other behavioural effects were encountered at higher doses (>150 mug/day) and necessitated dose reductions. The spinal CSF levels of r -metHuBDNF were directly related to dose, with a lumbar to cervical ratio of approximately 4:1. CONCLUSIONS: The intrathecal delivery of r -metHuBDNF in doses of up to 150 mug/day was well tolerated and appears feasible. The reversible CNS effects with higher dose indicate that BDNF can be delivered cranially against CSF flow. The small number of patients and the design of the study did not permit conclusions to be drawn about the efficacy of the treatment. (ALS 2000; 1:201-206)

Disease progression in amyotrophic lateral sclerosis: Predictors of survival

Predicting the rate of disease progression has become important as trials of new medical treatments for amyotrophic lateral sclerosis (ALS) are planned. Bulbar onset, early impairment of forced vital capacity, and older age have all been associated with shorter survival. We performed a retrospective study to compare survival factors with disease progression in a German ALS population. We analyzed disease progression in 155 patients at intervals of 4 months over a period of 3 years. To evaluate disease progression, the ALS functional rating scale (ALS‐FRS), forced vital capacity (FVC%), and a Medical Research Council (MRC) compound score based on a nine‐step modified MRC scale were used. We compared age (<55 years vs. ≥55 years), different sites of disease onset (bulbar vs. limb), and gender to the rate of disease progression and performed survival analyses. No overall significant difference could be detected when analyzing these subgroups with regard to disease progression. By contrast, significantly longer survival was observed in the younger age group (56 months vs. 38 months, P < 0.0001) and in patients with limb‐onset disease (51 months vs. 37 months, P = 0.0002). Using Cox analyses values we found that the declines of ALS‐FRS, FVC%, and MRC compound score were predictive of survival (P < 0.0001, P = 0.002, and P = 0.003, respectively). Future studies are needed to clarify whether nonspecific factors including muscle atrophy, dysphagia, and coexisting diseases influence prediction of survival in ALS patients. A more precise set of predictors may help to better stratify patient subgroups for future treatment trials.

Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: Potential impact of CNTF as a candidate modifier gene

Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene are found in approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS), or amyotrophic lateral sclerosis 1. Here we describe a 25-year-old male patient who died from FALS after a rapid disease course of 11 mo. Sequencing of the SOD-1 gene revealed a heterozygous T-->G exchange at position 1513 within exon 5, coding for a V-->G substitution at position 148 of the mature protein. Genetic analysis of this family revealed the same mutation in both his healthy 35-year-old sister and his mother, who did not develop the disease before age 54 years. Screening for candidate modifier genes that might be responsible for the early onset and severe course of the disease in the 25-year-old patient revealed an additional homozygous mutation of the CNTF gene not found in his yet unaffected sister. hSOD-1G93A mice were crossbred with CNTF(-/-) mice and were investigated with respect to disease onset and duration, to test the hypothesis that CNTF acts as a candidate modifier gene in FALS with mutations in the SOD-1 gene. Such hSOD-1G93A/CNTF-deficient mice develop motoneuron disease at a significantly earlier stage than hSOD-1G93A/CNTF-wild-type mice. Linkage analysis revealed that the SOD-1 gene was solely responsible for the disease. However, disease onset as a quantitative trait was regulated by the allelic constitution at the CNTF locus. In addition, patients with sporadic amyotrophic lateral sclerosis who had a homozygous CNTF gene defect showed significantly earlier disease onset but did not show a significant difference in disease duration. Thus, we conclude that CNTF acts as a modifier gene that leads to early onset of disease in patients with FALS who have SOD-1 mutations, in patients with sporadic amyotrophic lateral sclerosis, and in the hSOD-1G93A mouse model.

Autonomic dysfunction in ALS: A preliminary study on the effects of intrathecal BDNF

This pilot study aimed at exploring the effects of intrathecally administered brain derived neurotrophic factor (BDNF) on autonomic functions in patients with ALS. A battery of autonomic sympathetic and parasympathetic tests was performed at baseline and after nine months of treatment in 10 ALS patients participating in a double‐blind placebo‐controlled phase II/III study of intrathecally administered BDNF. Results of patients treated with BDNF (25 or 150 µg/day) were compared to those receiving placebo. Sudomotor function and blood pressure response to handgrip significantly worsened during the treatment period (55.4+/−26.1 vs. 38.9+/−23.9 g/m2h, p<0.05; 20+/−6 vs. 13+/−4 µHg, p<0.05) whereas other sympathetic and all parasympathetic function tests only tended to be more abnormal at follow‐up. Serum norepinephrine levels increased significantly during the nine‐months observation period. The results of autonomic function tests were not different between patients treated with BDNF and placebo, but norepinephrine levels were higher in the BDNF group. We conclude that autonomic nervous system function deteriorates along with poorer motor performance independently from treatment with BDNF. The elevation of norepinephrine levels might reflect a non‐specific up‐regulation, and its association with BDNF an autocrine effect.

Leukaemia inhibitory factor (LIF) gene mutations in women with unexplained infertility and recurrent failure of implantation after IVF and embryo transfer.

OBJECTIVE Leukaemia inhibitory factor (LIF) plays a central role in the control of implantation. We undertook this study to investigate the prevalence of LIF gene alterations in women with unexplained infertility and with recurrent failure of implantation after in vitro fertilisation (IVF) and embryo transfer. PATIENTS AND METHODS Forty five women with recurrent failure of implantation after IVF (group A), 50 with unexplained infertility (group B) and 105 fertile women (controls) were screened for LIF gene mutations. Standard genomic DNA extraction, PCR amplification of the LIF gene and single-strand conformation polymorphism (SSCP) analysis were used to search for mutations which were subsequently confirmed by DNA sequencing. RESULTS In group A, one woman was identified as having a neutral LIF gene polymorphism in exon 3 without affecting protein conformation. In group B, one woman with a heterozygous mutation and one with a neutral polymorphism were detected. In controls, only one woman with a neutral polymorphism in the intron between exons 2 and 3 was found. The woman with a potentially functional LIF gene mutation in group B achieved an ongoing clinical pregnancy after ovarian superovulation. DISCUSSION Potentially functional mutations in the LIF gene do infrequently occur in women with unexplained infertility and may play a role in the etiology of infertility. However, routine screening for LIF mutations or polymorphisms in these women is not justified for the low prevalence of gene alterations.

Genetic variation at position -1082 of the interleukin 10 (IL10) promotor and the outcome of multiple sclerosis.

Interleukin-10 is a potent immunomodulatory cytokine with possible implications for the pathogenesis of multiple sclerosis. Increased IL10 mRNA expression is associated with stable disease. The interleukin 10 gene is highly polymorphic and certain haplotypes result in differential interleukin-10 expression. The presence of guanine instead of adenine at position -1082 in the IL10 promotor was shown to result in a higher IL10 production. We analysed this diallelic polymorphism in patients with multiple sclerosis but did not find any association between a certain -1082 IL10 genotype and susceptibility to or severity of multiple sclerosis.

Comparison of maximal voluntary isometric contraction and Drachman's hand‐held dynamometry in evaluating patients with amyotrophic lateral sclerosis

Maximal voluntary isometric contraction (MVIC) is a standard tool for assessment of muscle strength in treatment trials for amyotrophic lateral sclerosis (ALS). There is need for more practical bedside techniques especially for severely disabled patients. Hand‐held dynamometry (HH‐Dyn) is an inexpensive and easy‐to‐handle device. MVIC was measured in five proximal muscle groups bilaterally and compared with HH‐Dyn in 43 ALS patients. After a training period we found good intrarater correlation for HH‐Dyn (r = 0.99), with a low coefficient of variation. Measurements tended to become more accurate after repeated testing due to practice effects in examiners and patients. Overall correlation between HH‐Dyn and MVIC was good [r = 0.85 (P < 0.01)]. Strength‐range‐specific analysis showed a significant linear correlation up to 20 kg (44 lbs.) [r = 0.57 (P < 0.01)]. However, we found a tendency to underestimate muscle strength above 10 kg by HH‐Dyn as compared with MVIC, but this became meaningful only above a force of 20 kg. HH‐Dyn provides a strength estimate with a precision close to MVIC in weak muscle groups (MRC grade 4). With standardization and appropriate training, HH‐Dyn is a useful bedside test, providing an alternative to MVIC for follow‐up assessment in ALS.

Gene polymorphism at position –308 of the tumor necrosis factor α promotor is not associated with disease progression in multiple sclerosis patients

Tumor necrosis factor-α (TNFα) is a pluripotent proinflammatory cytokine and is thought to play an important role in the inflammatory process of multiple sclerosis (MS). A G→A transition in the TNFα promotor at position –308 (TNF2 allele) has been shown to be associated with increased TNFα production. This study was designed to detect wether the TNF2 allele is associated with disease progression in MS. We examined the TNFα–308 polymorphism with an allelic discrimination PCR to detect the G→A transition in the genomic DNA of 283 MS patients from Germany and in 72 patients with amyotrophic lateral sclerosis (ALS) and 66 with stroke from the same genetic background who served as controls. Disease severity was defined by the progression index (PI) and by progression to the important clinical landmarks of Extended Disability Status Score (EDSS) 3.5 and 6. In addition, we evaluated the TNFα mRNA expression in whole blood with quantitative PCR. No differences were found between the presence of the TNF2 allele in MS, ALS, or stroke patients. Among the MS patients the TNF2 allele was not associated with a certain disease course. No association was found between the accumulation of neurological deficits and progression to clinical landmarks. Although MS patients with the TNF2 allele tended to progress more rapidly from EDSS 3.5 to EDSS 6 this difference was nonsignificant (P = 0.2). Nevertheless, we observed significantly higher TNFα mRNA expression in blood cells of stable patients carrying the TNF2-allele in comparison to the group with the wild type (P = 0.024). To examine the effect of genetic background we examined the DNA of 60 MS patients and 20 healthy controls in a Cypriot population of Greek origin. There was a significantly lower frequency of the TNF2 allele in the Cyprus population than in Germans (P = 0.01). No significant differences were found between the frequencies of the TNF2 allele in Cypriot MS patients and controls. Although the TNF2 allele is associated with higher TNFα mRNA baseline levels, our data indicate that this allele appears not to contribute to MS susceptibility or severity. In addition our data demonstrate that the TNFα–308 polymorphism is segregated differentially in two European populations of different genetic origin.

Potential role of LIF as a modifier gene in the pathogenesis of amyotrophic lateral sclerosis

Article abstract Leukemia inhibitory factor (lif) is a potent survival factor for motoneurons in cell culture and in vivo. The authors screened 104 patients with ALS and 338 control subjects for mutations in the LIF gene. In four ALS patients, but in no control subject, a G-to-A point mutation at position 3400 was identified, which leads to an amino acid exchange of valine to methionine at position 64 of the mature lif protein. This region of the lif protein (AB loop) interacts with the lif receptor. The authors suggest that LIF could act as a modifier gene which, in combination with other genetic predispositions, might lead to motoneuron disease.