HLA and amyotrophic lateral sclerosis: a systematic review and meta-analysis.

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with loss of upper and lower motor neurones. It leads to death by respiratory failure and has a typical prognosis of 2-3 years. The immune system has been shown to play a role in the pathophysiology of ALS. Some of the most important immune genes are within the human leukocyte antigen (HLA) region, and a recent genome-wide association study (GWAS) has identified a risk allele for ALS within the HLA region. Older studies have also suggested an HLA association with ALS, with certain HLA alleles showing differing expression between patients and controls. This systematic review and meta-analysis examines the previous studies performed in this field.Methods: We used established publication search engines. Findings were excluded if they did not meet the selection criteria. We then undertook statistical meta-analysis on the eligible papers, using a fixed effects model.Results: There were eight eligible papers. There were three statistically significant meta-analysis findings, although these would not be significant after correction for multiple comparisons. The frequencies of HLA-A9 and HLA-DR4 genotypes were lower in ALS subjects than controls, and HLA-B35 was higher in ALS subjects.Discussion: This systematic review and meta-analysis do not confirm all the previously reported associations of HLA with ALS, but shows three alleles of interest. However, there are limitations to the studies, which include the use of older serotyping methodology and the small numbers of subjects. Given the recent GWAS association with HLA, further modern HLA studies are warranted.

Cloning of a new form of EAAT2/GLT-1 from human and rodent brains.

Glutamate transporter 1 is the principal transporter that mediates glutamate clearance in the mammalian brain. In rodents, it is referred to as GLT-1, whereas in humans it is referred to as EAAT2. We have cloned a novel and abundantly expressed carboxyl-terminal splice variant of this transporter in both rodents and humans, which we denote as GLT-1d/EAAT2d. The novel splice variant results from usage of internal splice sites and the splicing event leads to novel extra sequence spliced in after exon 10. The open reading frames of GLT-1d and EAAT2d encode proteins of 572 and 566 amino acids respectively; both contain a C-terminal PDZ motif. When expressed in COS7 cells, the proteins function as glutamate transporters that are inhibited by dihydrokainate (a GLT-1/EAAT2 transporter inhibitor). RT-PCR amplification using GLT-1d specific primers confirmed expression of message in all brain regions examined (forebrain, midbrain, hindbrain and cerebellum) as well as spinal cord, astrocyte cultures, retina and peripheral tissues (liver, testis, small intestine and lung). Quantitative RT-PCR analysis showed that expression of GLT-1d is developmentally regulated. In adult human brain, EAAT2d message is âˆ¼ 30% of the level of EAAT2a message (the most abundant form), potentially making it the second most abundantly expressed form of EAAT2 in the brain. The amino terminal region of GLT-1d is also alternately spliced; the brain and testis forms contain a sequence corresponding to the amino acid sequence MASTEG whereas the corresponding liver sequence is MVS. In summary, we have cloned a novel EAAT2/GLT-1 splice variant from human and rodent brains. The splice variant is abundantly expressed in the brain, spinal cord, retina, liver and testis; it is a functional glutamate transporter; therefore, we conclude that it will likely have a functional role in glutamate homeostasis in the rodent and human nervous system, during development, adulthood, and plausibly in pathological states.

Monocyte CD14 and HLA-DR expression increases with disease duration and severity in amyotrophic lateral sclerosis.

Objective: To investigate changes in immune markers and frequencies throughout disease progression in patients with amyotrophic lateral sclerosis (ALS). Methods: In this longitudinal study, serial blood samples were collected from 21 patients with ALS over a time period of up to 16 months. Flow cytometry was used to quantitate CD14, HLA-DR, and CD16 marker expression on monocyte subpopulations and neutrophils, as well as their cell population frequencies. A Generalized Estimating Equation model was used to assess the association between changes in these immune parameters and disease duration and the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). Results: CD14 expression on monocyte subpopulations increased with both disease duration and a decrease in ALSFRS-R score in patients with ALS. HLA-DR expression on monocyte subpopulations also increased with disease severity and/or duration. The expression of CD16 did not change relative to disease duration or ALSFRS-R. Finally, patients had a reduction in non-classical monocytes and an increase in the classical to non-classical monocyte ratio throughout disease duration. Conclusion: The progressive immunological changes observed in this study provide further support that monocytes are implicated in ALS pathology. Monocytic CD14 and HLA-DR surface proteins may serve as a therapeutic target or criteria for the recruitment of patients with ALS into clinical trials for immunomodulatory therapies.

Extra-motor abnormalities in amyotrophic lateral sclerosis: another layer of heterogeneity.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease defined by the presence of muscle weakness. The motor features of disease are heterogeneous in site of onset and progression. There are also extra-motor features in some patients. The genetic basis for extra-motor features is uncertain. The heterogeneity of ALS is an issue for clinical trials. Areas covered: This paper reviews the range and prevalence of extra-motor features associated with ALS, and highlights the current information about genetic associations with extra-motor features. Expert commentary: There are extra-motor features of ALS, but these are not found in all patients. The most common is cognitive abnormality. More data is required to ascertain whether extra-motor features arise with progression of disease. Extra-motor features are reported in patients with a range of causative genetic mutations, but are not found in all patients with these mutations. Further studies are required of the heterogeneity of ALS, and genotype/phenotype correlations are required, taking note of extra-motor features.

Altered expression of metabolic proteins and adipokines in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the loss of upper cortical and lower motor neurons. ALS causes death within 2-5years of diagnosis. Diet and body mass index influence the clinical course of disease, however there is limited information about the expression of metabolic proteins and fat-derived cytokines (adipokines) in ALS. In healthy controls and subjects with ALS, we have measured levels of proteins and adipokines that influence metabolism. We find altered levels of active ghrelin, gastric inhibitory peptide (GIP), pancreatic polypeptide (PP), lipocalin-2, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6) and 8 (IL-8), and tumor necrosis factor alpha (TNFα) in the plasma of ALS patients relative to controls. We also observe a positive correlation between the expression of plasma nerve growth factor (NGF) relative to disease duration, and an inverse correlation between plasma glucagon and the ALS functional rating scale-revised (ALSFRS-R). Further studies are required to determine whether altered expression of metabolic proteins and adipokines contribute to motor neuron vulnerability and how these factors act to modify the course of disease.

Serial measurements of phosphorylated neurofilament-heavy in the serum of subjects with amyotrophic lateral sclerosis.

There is a need for a blood biomarker of disease activity in ALS. This marker needs to measure the loss of motor neurones. Phosphorylated neurofilament heavy chain (pNfH) in the serum is a biomarker of axonal injury. Previous studies have found that levels of pNfH are elevated in ALS. We have performed a serial study of pNfH levels in 98 subjects from our ALS clinic. There was significant elevation of levels of pNfH in subjects with ALS compared to controls, although there was considerable variability. In studies of individuals who had two or more serial samples, we found that the levels of pNfH increased over time in the early stage of disease. Levels were low in subjects with long survival. The rate of rise of pNfH was inversely correlated with survival. We suggest that the initial level of pNfH is a marker of disease severity and that changes in pNfH levels are markers of disease progression.

Elevation of the terminal complement activation products C5a and C5b-9 in ALS patient blood.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons within the central nervous system. Neural degeneration and inflammatory processes, including activation of the complement system are hallmarks of this pathology. Our past work in ALS animal models (hSOD1 G93A rodents) has revealed that blockade of the receptor for complement activation fragment C5a (C5aR), improves ALS-like symptoms and extends survival. We now show that the levels of C5a and C5b-9, but not C3a nor C4a, are significantly elevated in plasma from ALS patients compared to healthy controls. C5a was also elevated within leukocytes from ALS patients suggesting heightened C5a receptor interaction. Overall, these findings indicate that there is enhanced peripheral immune complement terminal pathway activation in ALS, which may have relevance in the disease process.

Gender differences in autoimmune disease.

Autoimmune diseases are a range of diseases in which the immune response to self-antigens results in damage or dysfunction of tissues. Autoimmune diseases can be systemic or can affect specific organs or body systems. For most autoimmune diseases there is a clear sex difference in prevalence, whereby females are generally more frequently affected than males. In this review, we consider gender differences in systemic and organ-specific autoimmune diseases, and we summarize human data that outlines the prevalence of common autoimmune diseases specific to adult males and females in countries commonly surveyed. We discuss possible mechanisms for sex specific differences including gender differences in immune response and organ vulnerability, reproductive capacity including pregnancy, sex hormones, genetic predisposition, parental inheritance, and epigenetics. Evidence demonstrates that gender has a significant influence on the development of autoimmune disease. Thus, considerations of gender should be at the forefront of all studies that attempt to define mechanisms that underpin autoimmune disease.

Body mass index and dietary intervention: implications for prognosis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult onset, neurodegenerative disease that is characterized by the loss of upper (corticospinal) and lower motor neurons. ALS is a multifactorial disease whereby a combination of genetic and environmental factors may contribute to disease pathogenesis. While the majority of studies indicate that the underlying causes for ALS pathology may be due to multiple defects at the cellular level, factors that have recently been identified to be associated with survival could lead to the development of beneficial interventions. In ALS, a higher pre-morbid body mass index (BMI) and the maintenance of BMI and nutritional state is associated with improved outcome. This review will focus on the associations between body composition and adiposity relative to disease duration and risk, and will discuss current evidence that supports the benefits of improving energy balance, and the maintenance of body mass through nutritional intervention in ALS.

Growth hormone secretion is correlated with neuromuscular innervation rather than motor neuron number in early-symptomatic male amyotrophic lateral sclerosis mice.

GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1(G93A) mice throughout the course of ALS disease. Male wild-type and hSOD1(G93A) mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1(G93A) mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1(G93A) mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.

Reduced levels of interleukin 33 and increased levels of soluble ST2 in subjects with amyotrophic lateral sclerosis.

Interleukin 33 (IL-33) is a cytokine that functions as an alarmin and is released from damaged tissue. The receptor for IL-33 is ST2, which exists as membrane bound and soluble forms. Levels of IL-33 and soluble ST2 (sST2) are elevated in some inflammatory diseases. Amyotrophic lateral sclerosis (ALS) is a disease that is due to loss of motor neurones, with some neuro-inflammation at the site of pathology. This study was performed to measure levels of IL-33 and sST2 in ALS. Serum was obtained from subjects with ALS (n=42) and healthy controls (n=38). Levels of IL-33 and s ST2 were measured with ELISA. The level of Il-33 was significantly lower in ALS subjects than healthy controls, and the levels of sST2 were significantly higher. The lower levels of IL-33 could be due to degradation of IL-33 by caspases released from apoptotic cells. However the levels of IL-33 could also be lower due to effects of sST2 which acts as a receptor for IL-33. The levels of sST2 could reflect inflammation in ALS.

Impairments to the GH-IGF-I axis in hSOD1G93A mice give insight into possible mechanisms of GH dysregulation in patients with amyotrophic lateral sclerosis.

GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.

The relationship between Bayesian motor unit number estimation and histological measurements of motor neurons in wild-type and SOD1(G93A) mice.

OBJECTIVE: To assess the relationship between Bayesian MUNE and histological motor neuron counts in wild-type mice and in an animal model of ALS. METHODS: We performed Bayesian MUNE paired with histological counts of motor neurons in the lumbar spinal cord of wild-type mice and transgenic SOD1(G93A) mice that show progressive weakness over time. We evaluated the number of acetylcholine endplates that were innervated by a presynaptic nerve. RESULTS: In wild-type mice, the motor unit number in the gastrocnemius muscle estimated by Bayesian MUNE was approximately half the number of motor neurons in the region of the spinal cord that contains the cell bodies of the motor neurons supplying the hindlimb crural flexor muscles. In SOD1(G93A) mice, motor neuron numbers declined over time. This was associated with motor endplate denervation at the end-stage of disease. CONCLUSION: The number of motor neurons in the spinal cord of wild-type mice is proportional to the number of motor units estimated by Bayesian MUNE. In SOD1(G93A) mice, there is a lower number of estimated motor units compared to the number of spinal cord motor neurons at the end-stage of disease, and this is associated with disruption of the neuromuscular junction. SIGNIFICANCE: Our finding that the Bayesian MUNE method gives estimates of motor unit numbers that are proportional to the numbers of motor neurons in the spinal cord supports the clinical use of Bayesian MUNE in monitoring motor unit loss in ALS patients.

The Role of immune and inflammatory mechanisms in ALS.

Amyotrophic lateral sclerosis (ALS) is a severe progressive neurodegenerative disease. The cause is unknown, but genetic abnormalities have been identified in subjects with familial ALS and also in subjects with sporadic ALS. Environmental factors such as occupational exposure have been shown to be risk factors for the development of ALS. Patients differ in their clinical features and differ in the clinical course of disease. Immune abnormalities have been found in the central nervous system by pathological studies and also in the blood and CSF of subjects with ALS. Inflammation and immune abnormalities are also found in animals with a model of ALS due to mutations in the SOD1 gene. Previously it has been considered that immune abnormalities might contribute to the pathogenesis of disease. However more recently it has become apparent that an immune response can occur as a response to damage to the nervous system and this can be protective.

Levels of phosphorylated axonal neurofilament subunit H (pNfH) are increased in acute ischemic stroke.

For the study of stroke outcomes, there is the need for measurements of severity of stroke damage. Phosphorylated neurofilament heavy protein (pNfH) levels are elevated in axonal injury. We have measured levels of pNfH in stroke and correlated these levels with measures of stroke severity. Blood samples were collected from 54 ischaemic stroke patients at day 1, week 1 (days 7-10) and weeks 3-6, and an ELISA was used to measure pNfH levels in each patient at each time-point. Serum pNfH levels were significantly elevated in stroke patients compared to healthy controls. The levels were low at day 1, higher at day 7 and reached a peak at week 3, the latest day that we assessed. Significant associations were found between the pNfH levels at week 3 and early and stroke severity, size and outcome. Blood pNfH levels that reflect the severity of ischaemic stroke, are correlated with outcome and rise during the weeks after stroke. This may be a useful measure of tissue damage in stroke.

T cells from patients with Guillain-Barré syndrome produce interferon-gamma in response to stimulation with the ganglioside GM1.

Guillain-Barré syndrome (GBS) is an acquired demyelinating neuropathy, characterized by infiltration of peripheral nerves with macrophages and T cells. There have been reports of antibodies to glycolipids in GBS. We have previously found T cell reactivity to glycolipids in patients with the demyelinating form of GBS. This study was performed to characterize the cytokines produced by these T cells. Peripheral blood lymphocytes from patients with GBS, chronic inflammatory demyelinating polyradiculoneuropathy, healthy control patients and other neuropathies were incubated with the ganglioside GM1 and transferred to enzyme-linked immunospot plates. The average number per well of spot-forming cells (SFC) in the absence of antigen was counted. The average spontaneous SFC number was subtracted from the average SFC number in the presence of GM1, to produce a corrected SFC. There was significantly increased production of interferon-gamma but not interleukin-5 in response to stimulation with the ganglioside GM1. This could indicate that SFC have a role in pathogenesis of disease.

Sexual dimorphism in autoimmune disease.

We briefly survey the concept of autoimmunity and nominate the range of autoimmune diseases that include multisystemic and organ-specific disorders, and cite prevalences of autoimmune diseases in males and females, in humans and in experimental animals. Most human autoimmune diseases such as systemic lupus erythematosus (SLE), multiple sclerosis (MS) and autoimmune thyroid disease, have an increased incidence and prevalence in females, but a few others such as autoimmune diabetes, the Guillain Barré syndrome (GBS) and psoriasis are increased in males. Animal models of autoimmunity show an equivalent sexual dimorphism. The possible reasons for the differing incidence and prevalence of autoimmune diseases in females and males engage our attention. Environmental exposures may differ for females and males. There are innate differences in the function of the female and male immune systems, and there is some evidence for differences between females and males in the ability of a target organ for autoimmunity to withstand damage. In seeking reasons for these differences, we review the role of sex hormones in immunity and include results of trials of hormone therapy in autoimmune diseases. The association of autoimmunity and pregnancy, a female-specific condition, is discussed, and the claimed effects of lymphoid cell microchimerism on provocation of autoimmunity are reviewed. Genetic predisposition is an important factor in autoimmune disease and we particularly focus on genes on the X and Y chromosomes, the role of X chromosome inactivation, and the interaction of the sex of the patient with other genetic factors. The possible role of epigenetic mechanisms, including environmental influences, is then surveyed. We assert that sex is a vital variable that must be considered in all immunological studies, as it should be at all levels of biological research.