Biomarkers and microRNA
Investigation of the KIR4.1 potassium channel as a putative antigen in patients with multiple sclerosis: a comparative study.
Brickshawana A, Hinson SR, Romero MF, Lucchinetti CF, Guo Y, Buttmann M, McKeon A, Pittock SJ, Chang MH, Chen AP, Kryzer TJ, Fryer JP, Jenkins SM, Cabre P, Lennon VA.
BACKGROUND: Antibodies have been implicated in the pathogenicity of multiple sclerosis by findings of immunoglobulins in patients' CSF and often IgG and complement in lesions, and by a 2012 report that nearly half of patients' serum samples contain IgG specific for a glial potassium-channel, KIR4.1. We aimed to establish the frequency of KIR4.1-binding IgG in serum and CSF of patients with multiple sclerosis, and whether KIR4.1 immunoreactivity is retained or lost in demyelinating lesions.
METHODS: Using ELISA with a KIR4.1 peptide, we tested archival serum from 229 population-based and 57 clinic-based patients with multiple sclerosis, 99 healthy controls, and 109 disease controls, and CSF from 25 patients with multiple sclerosis and 22 disease controls. We tested all CSF and serum samples from 50 of the clinic-based patients with multiple sclerosis on cells expressing functional KIR4.1, using cell-based immunofluorescence and immunoprecipitation (solubilised recombinant human KIR4.1). We assessed KIR4.1 immunoreactivity in archival brain samples from 15 patients with histopathologically confirmed multiple sclerosis (22 plaques [eight early active, eight inactive, and six remyelinated], 13 periplaque regions and eight normal-appearing white-matter and grey-matter regions) and from three controls with non-neurological diseases.
FINDINGS: Three of 286 serum samples from patients with multiple sclerosis and two of 208 serum samples from controls showed KIR4.1 reactivity on ELISA; none of the CSF samples from patients or controls showed KIR4.1 reactivity. IgG in none of the 50 serum samples from clinic-based patients immunoprecipitated KIR4.1, but a commercial KIR4.1-specific control IgG did. By immunofluorescence, one of 50 serum samples from patients with multiple sclerosis yielded faint plasmalemmal staining on both KIR4.1-expressing and non-expressing cells; 16 bound faintly to intracellular components. In all cases, IgG binding was quenched by absorption with liver powder or lysates from non-transfected cells. Binding by the KIR4.1-specific control IgG was quenched only by lysates containing KIR4.1. IgG in none of the 25 CSF samples from patients with multiple sclerosis bound to KIR4.1-transfected cells. Glial KIR4.1 immunoreactivity was increased relative to expression in healthy control brain in all active demyelinating lesions, remyelinated lesions, and periplaque white matter regions.
INTERPRETATION: We did not detect KIR4.1-specific IgG in serum or CSF from patients with multiple sclerosis or KIR4.1 loss from glia in multiple sclerosis lesions. Serological testing for KIR4.1-specific IgG is unlikely to aid diagnosis of multiple sclerosis. The target antigen of multiple sclerosis remains elusive.
FUNDING: The National Institutes of Health, the National Multiple Sclerosis Society, and the Mayo Clinic Robert and Arlene Kogod Center on Aging.
Source: Lancet Neurol. 2014 Jul 4. pii: S1474-4422(14)70141-3. doi: 10.1016/S1474-4422(14)70141-3. [Epub ahead of print] & Pubmed PMID: 25008548 (15/07/14)
The use of cytokine signature patterns: separating drug naïve, interferon and natalizumab-treated multiple sclerosis patients.
O'Connell KE1, Mok T, Sweeney B, Ryan AM, Dev KK.
Multiple sclerosis (MS) is an inflammatory illness characterised by demyelination and axonal neurodegeneration. Here, we used serum samples from MS patients to demonstrate if "cytokine signature" patterns can separate different patient groups better than using single cytokines.
In this case, we used cytokine profiling to demonstrate if "cytokine signature" patterns can separate MS patients treated with interferon or natalizumab from drug naïve patients.
Serum levels of eight individual cytokines (TNFα, IFNγ, S100B, IL-1β, IL-6, IL-8, IL-17 and IL-23) in MS patients treated with interferons (n = 11) and natalizumab (n = 14) were measured and, in general, showed reduced levels compared to drug naïve MS patients (n = 12).
More evident changes were seen when analyzing "cytokine signatures" (i.e. summed value of all eight cytokines), which showed that patients treated with natalizumab and interferons showed significantly reduced cytokine signature levels than drug naïve MS patients.
Moreover, patients treated with natalizumab were separated from drug naïve patients by almost 100% fidelity and that patients treated with natalizumab also had reduced levels of pro-inflammatory cytokines compared to patients treated with interferon.
Overall, this study provides an example showing that the use of "cytokine signatures" may provide benefits over the analysis of single cytokines for the development of potential biomarkers.
Source: Autoimmunity. 2014 Jun 30:1-7. [Epub ahead of print] & Pubmed PMID: 24974887 (07/07/14)
For some, the disease multiple sclerosis (MS) attacks its victims slowly and progressively over a period of many years. For others, it strikes without warning in fits and starts. But all patients share one thing in common: the disease had long been present in their nervous systems, hiding under the radar from even the most sophisticated detection methods. But now, scientists at the Gladstone Institutes have devised a new molecular sensor that can detect MS at its earliest stages -- even before the onset of physical signs.
In a new study from the laboratory of Gladstone Investigator Katerina Akassoglou, PhD, scientists reveal in animal models that the heightened activity of a protein called thrombin in the brain could serve as an early indicator of MS. By developing a fluorescently labeled probe specifically designed to track thrombin, the team found that active thrombin could be detected at the earliest phases of MS -- and that this active thrombin correlates with disease severity. These findings, reported online in Annals of Neurology, could spur the development of a much-needed early-detection method for this devastating disease.
MS, which afflicts millions of people worldwide, develops when the body's immune system attacks the protective myelin sheath that surrounds nerve cells. This attack damages the nerve cells, leading to a host of symptoms that include numbness, fatigue, difficulty walking, paralysis and loss of vision. While some drugs can delay these symptoms, they do not treat the disease's underlying causes -- causes that researchers are only just beginning to understand.
Last year, Dr. Akassoglou and her team found that a key step in the progression of MS is the disruption of the blood brain barrier (BBB). This barrier physically separates the brain from the blood circulation and if it breaks down, a blood protein called fibrinogen seeps into the brain. When this happens, thrombin responds by converting fibrinogen into fibrin -- a protein that should normally not be present in the brain. As fibrin builds up in the brain, it triggers an immune response that leads to the degradation of the nerve cells' myelin sheath, over time contributing to the progression of MS.
"We already knew that the buildup of fibrin appears early in the development of MS -- both in animal models and in human patients, so we wondered whether thrombin activity could in turn serve as an early marker of disease." said Dr. Akassoglou, who directs the Gladstone Center for In Vivo Imaging Research (CIVIR). She is also a professor of neurology at the University of California, San Francisco, with which Gladstone is affiliated. "In fact, we were able to detect thrombin activity even in our animal models -- before they exhibited any of the disease's neurological signs."
In laboratory experiments on mice modified to mimic the signs of MS, the team employed an Activatable Cell-Penetrating Peptide (ACPP), a special type of molecular probe that delivers fluorescent agents to a region of interest. For this study, they developed a thrombin-specific ACPP that could track thrombin activity in mice as the disease progressed. They then carefully analyzed where -- and at what stage of disease -- thrombin activity was found.
"We detected heightened thrombin activity at specific disease 'hot-spots,' regions where neuronal damage developed over time," said Gladstone Staff Research Scientist Dimitrios Davalos, PhD, associate director of the CIVIR and one of the paper's lead authors. "And when we compared those results to those of a separate, healthy control group of mice, we saw that thrombin activity in the control group was wholly absent."
"Our results are proof of principle that a thrombin-specific molecular probe could be used as an early-detection method," added former Gladstone Postdoctoral Researcher Kim Baeten, PhD, the paper's other lead author.
The team's results offered significant support for the notion that thrombin activity is directly tied to the degradation of nerve cell's myelin sheath -- and the subsequent destruction of nerve cells -- that characterizes MS. But they also shed light on what has been a long-standing mystery: the underlying molecular processes that kick-start the progression of MS.
"In the future," said Dr. Akassoglou, "this thrombin-specific ACPP could be developed to one day allow for early patient diagnosis and therapeutic intervention -- including a way to effectively monitor how patients are responding to the latest treatments."
Dimitrios Davalos, Kim M. Baeten, Michael A. Whitney, Eric S. Mullins, Beth Friedman, Emilia S. Olson, Jae Kyu Ryu, Dimitri S. Smirnoff, Mark A. Petersen, Catherine Bedard, Jay L. Degen, Roger Y. Tsien, Katerina Akassoglou. Early detection of thrombin activity in neuroinflammatory disease. Annals of Neurology, 2013; DOI: 10.1002/ana.24078
Source: ScienceDaily Copyright 2013 by ScienceDaily, LLC (05/12/13)
A protein involved in blood clotting may be a new indicator to help detect multiple sclerosis (MS) lesions before symptoms arise. The presence of the clotting protein, thrombin, signals an early stage of the disease when the blood-brain barrier is breached and the brain’s immune response is set into motion. The research was presented at Neuroscience 2013, the annual meeting of the Society for Neuroscience and the world’s largest source of emerging news about brain science and health.
30,000 scientists are attending this meeting.
“Our research shows this indicator is a promising approach for detecting MS-like lesions early, even before major symptoms appear,” said senior author Katerina Akassoglou, Ph.D., of the Gladstone Institutes and the University of California, San Francisco. “Such sensitive indicators could act as red flags that signal neuroinflammatory changes in the brain not only in MS, but also in other diseases such as Alzheimer’s.”
MS is a debilitating disorder that can be intermittent or progressive, and causes numbness, fatigue, difficulty walking, paralysis, and loss of vision in 2 million people worldwide. MS arises when the body’s immune system attacks its own myelin sheaths, the protective coverings that surround neurons and allow signals to move from one cell to the next.
The researchers found that thrombin, usually a beneficial protein involved in blood clotting, builds up in the central nervous system as MS progresses. Thrombin enters in the brain together with fibrinogen, another clotting protein when the protective barrier between the blood and brain becomes leaky. Thrombin converts the fibrinogen to fibrin which activates brain’s immune cells that break down the protective myelin sheath that surrounds neurons in the central nervous system. Because thrombin levels increase as the disease progresses, the researchers conclude that it could be used as an early detector of the disease.
In their studies, the researchers used a mouse model and demonstrated that MS symptoms increased as thrombin levels rose. Early detection of MS could result in more successful treatment of the disease.
Research was supported with funds from the National Multiple Sclerosis Society, the Nancy Davis Foundation for Multiple Sclerosis, and the National Institutes of Health. Dr. Akassoglou outlined her findings in a press conference held on Sunday, November 10, and this summary was distributed with the press release. The scientific presentation of Dr. Akassoglou’s work will be delivered on Monday, Novermber 11.
Source: Bioquick News (11/11/13)
Biomarkers predict second MS attack(07/10/13)
In patients who experienced a first attack of multiple sclerosis (MS) symptoms, risk of a second attack was associated with a variety of baseline factors, researchers said here.
Measurements of brain lesions with MRI, oligoclonal bands in cerebrospinal fluid (CSF), and visual and sensorimotor evoked potentials were, collectively, indicators for patients at relatively high risk of a second attack within 12 months, reported Vittorio Martinelli, MD, of San Raffaele Hospital in Milan.
And, in a separate pan-European study, levels of the chitinase 3-like 1 (CHI3L1) CSF protein were a moderately strong predictor of second attacks in a similar patient population, said Ester Cantó, a PhD student a Vall d'Hebron University in Barcelona.
Using a cutoff value of 190 ng/mL, patients with higher CHI3L1 values showed a median time to develop clinically definite MS by 2005 McDonald criteria of 12.4 months (95% CI 11.8-13.0), compared with 39.5 months in patients with lower CHI3L1 values (95% CI 29.5-50.4), she said.
Both studies were reported at the European Committee for Treatment and Research in Multiple Sclerosis annual meeting, where attention has been particularly focused on the earliest stages of MS.
To qualify for a diagnosis, patients must have experienced two bouts of symptoms separated in time. A first attack is called clinically isolated syndrome (CIS).
In some patients, years may pass until they experience a second attack and are diagnosed with MS -- or never. Others may experience renewed symptoms in as little as 6 months.
Physicians now want to initiate disease-modifying therapies right away in patients with such aggressive pathology, but currently there are no standards for identifying them.
Martinelli noted that previous studies, using different criteria to stratify patients, have found very different rates of conversion from CIS to MS.
Those studies had established some risk factors for conversion: young age, presence of oligoclonal bands in CSF, past steroid treatment, and large MRI lesion burdens.
But narrowing them to establish a reliable predictive model would be useful, he suggested.
Improved Prediction with Conventional Tests
In a step toward that goal, he and colleagues at San Raffaele examined data collected prospectively from 227 CIS patients seen from 2000 through 2010. The data represented results of standard tests for patients with suspected CIS including MRI scans, CSF analysis for oligoclonal bands, and multimodal (visual and sensorimotor) evoked potential testing.
Mean follow-up was 6.8 years (standard deviation 2.8). Mean time from symptom onset to hospitalization and CIS diagnosis was 1.7 months (SD 1.4).
MRI findings at baseline indicated three-quarters of patients had monofocal lesions, with the remainder showing a multifocal distribution. Total T2 lesion counts were zero or one in 12% of patients, from two to nine in 59%, and 10 or more in 29%. Half the patients had gadolinium-enhancing lesions and 74% had oligoclonal bands in CSF.
Just over 30% converted to clinically definite MS within 2 years; through the entire follow-up, 53% converted.
Statistical analysis showed that three factors were most strongly associated with early conversion to MS:
Having more than two T2 lesions
A positive test for oligoclonal bands in CSF
Being in the top quartile of multimodal evoked potentials (indicating impaired peripheral nerve function)
Among patients lacking these three factors, some 15% converted to MS within 12 months, whereas 40% of those showing all three converted.
"Integration of MRI, CSF, and multimodal evoked potential data is essential for an accurate and personalized risk management in patients with CIS," Martinelli said.
CSF Protein Marker
The study reported by Cantó involved CSF samples and clinical data from 813 CIS patients and 559 controls recruited from 15 centers across Europe. Controls included 121 patients with inflammatory neurological diseases that were not CIS or MS.
Her team focused on CHI3L1 due to its appearance in an earlier proteomics discovery project aimed at finding CSF biomarkers that could distinguish early converters from CIS to MS. The current study was designed to validate those findings in a larger, well controlled sample from multiple centers.
In addition, Cantó said, the researchers wanted to understand more about the origins of CHI3L1 within the nervous system.
CIS patients in the study had provided CSF samples during their diagnostic workups. With mean follow-up of 5.4 years, a total of 60% eventually received an MS diagnosis according to 2005 McDonald criteria and 51.5% by the more restrictive Poser criteria.
Across the entire cohort of CIS patients (irrespective of conversion), mean CHI3L1 levels were higher than those in non-inflammatory neurological disease controls by 70 ng/mL, but lower than those in the controls with inflammatory neurological disease by 329 ng/mL (both P<0.0001).
Levels were higher by 40 ng/mL (95% CI 25-56) in patients who converted to MS during follow-up than in those who didn't (P<0.00001), Cantó said.
CHI3L1 levels remained an independent risk factor (P<0.00001) for early conversion when analyzed in a model that also included age at CIS onset, presence of oligoclonal bands, and so-called Barkhof criteria based on MRI findings.
In addition to predicting early conversion to MS, CHI3L1 levels above 190 ng/mL also predicted more rapid disability progression, Cantó said.
Mean time to expanded disability status scale (EDSS) scores of 3.0 was 156 months in those with high values compared with 215 months in those with low values (P=1×10-9).
A histopathology study, using cells from CSF obtained from five CIS patients and five controls with non-inflammatory neurological disease, showed that CHI3L1 expression was localized in MS-type lesions and reactive astrocytes and other activated immune cells.
Cantó said these latter results suggested that elevations in the marker "reflect the degree of astrocyte activation secondary to inflammation."
Both studies had no commercial funding. Quidel provided assays for CHI3L1 but otherwise played no role in the study by Cantó and colleagues.
Martinelli reported relationships with Biogen Dompe, Merck Serono, Bayer Schering, Teva, and Sanofi. Other investigators in his study had relationships with these firms and Genmab.
Cantó reported no conflicts of interest. Other investigators in her study reported relationships with Bayer Schering, Merck Serono, Biogen Idec, Teva, Sanofi, Novartis, Roche, Elan, Genmab, UCB, Wyeth, and others.
Primary source: European Committee for Treatment and Research in Multiple Sclerosis
Source reference: Martinelli V, et al. "Predicting early conversion to multiple sclerosis in patients with clinically isolated syndromes: the importance of an integrated modeling of risk factors" ECTRIMS 2013; Abstract 155.
Additional source: European Committee for Treatment and Research in Multiple Sclerosis
Source reference:Canto E, et al. "Validation of cerebrospinal fluid chitinase 3-like 1 as a prognostic biomarker of conversion to multiple sclerosis and disease severity in patients with clinically isolated syndromes" ECTRIMS 2013; Abstract 156.
Source: MedPage Today © 2013 MedPage Today, LLC (07/10/13)
A research team lead by Violaine Harris, Ph.D., at the Tisch MS Research Center of New York, has just published findings on a new method of measuring disease activity in patients with multiple sclerosis (MS) (Harris, et al, Cerebrospinal fluid fetuin-A is a biomarker of active multiple sclerosis, Multiple Sclerosis Journal, Epub: 2/25/2013 doi: 10.1177/1352458513477923 ahead of print).
This important biomarker discovery is based on spinal fluid measurement of Fetuin-A levels obtained over the course of several years of clinical and pathological studies of MS patients as well as experimental models of the disease. Dr. Harris's findings are likely to change the process for making treatment decisions in MS patients.
Current MS treatment is designed to stop disease activity in the brain and spinal cord with the goal of arresting disease progression and disability. According to Dr. Saud A. Sadiq, the senior author on the study, "these findings will provide a measurable method of monitoring the effectiveness of treatment much like determining blood sugar levels are assayed for diabetic patients. Many patients with MS on treatment report 'worsening' despite stable MRI findings. Addition of Fetuin-A measurement will help better evaluate disease activity in such patients."
The Tisch MS Research Team continues to study the underlying mechanisms of elevation of spinal fluid Fetuin-A to determine its exact role in multiple sclerosis.
ABOUT TISCH MS RESEARCH CENTER OF NEW YORK For over twenty years, Dr. Saud A. Sadiq has believed that combining excellence in clinical care with innovative research targeted at finding the cure for multiple sclerosis would set an exemplary standard in the treatment of people with MS. Today, the Tisch MS Research Center of New York embodies this new model of healthcare, in which your doctor is also your researcher. Dr. Sadiq helps those with MS by conducting cutting-edge, patient-based research to ensure unparalleled care. The close relationship of the non-profit research center and its affiliated clinical practice (International Multiple Sclerosis Management Practice) enables the testing of new MS treatments and accelerates the pace at which research discoveries move from lab bench to bedside. The Tisch MS Research Center of New York aims to identify the disease trigger, optimize treatments for patients and repair the damage caused by multiple sclerosis.
Source: Tisch MS Research Center of New York & PR Newswire Copyright (C) 2013 PR Newswire (30/07/13)
MicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls.
Methods: Heparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells.
MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).
Results: We identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls.
By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS.
The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA.
Conclusion: These findings indicate that microRNA may be important regulatory molecules in T-cells in MS.
Author: Margareta JernÃ¥sClas MalmestrÃ¶mMarkus AxelssonIntawat NookaewHans WadenvikJan LyckeBob Olsson
Credits/Source: BMC Immunology 2013, 14:32
Source: 7thSpace Interactive © 2013 7thSpace Interactive (30/07/13)
A blood test could one day provide a fast, inexpensive way for multiple sclerosis patients to assess the effectiveness of their medications and track disease flares.
While the test is still years away from market, the new biotechnology may prove invaluable for people with multiple sclerosis (MS), a degenerative disease that affects the brain, spinal cord and optic nerve, causing disability, chronic fatigue and pain, among other symptoms. Currently, doctors and patients rely on MRIs, which are expensive and time-consuming, to evaluate the progress of MS and the effects of treatment.
“You can't just walk in and say, ‘Hey I’m here.’ You have to schedule [the MRI], and somebody has to read it,” said Carolyne Reed, 62, a real estate agent in Tuscon, Arizona who was diagnosed with MS in her late forties.
“I'd rather know sooner rather than later,” Reed added, acknowledging that MRI results take time. “The not knowing, the constant feeling of being in limbo is very, very difficult."
Researchers at the University of California, San Francisco, and the Stanford University School of Medicine have identified a blood biomarker called TOB1 that they believe points to MS disease activity, according to a study published in the June edition of The Journal of Experimental Medicine.
TOB1 is only one MS biomarker out of many possibilities. When the immune system begins to attack the brain or the central nervous system, it creates inflammation, which can be picked up in a blood test. Biomarkers can also point to debris left in the blood after an MS relapse. The more damage inside the body, the stronger the presence of a biomarker on the test.
An Easier Way to Monitor MS?
A biomarker, in the simplest terms, is something that can be precisely measured within the body, said Sergio Baranzini, PhD, a molecular geneticist at the University of California, San Francisco. Biomarkers like cerebrospinal fluid, blood, urine, saliva, stool and tears play a role in diagnosing and tracking a host of illnesses, from strep throat to HIV and cancer.
"All these bodily fluids contain information about a disease process," Dr. Baranzini said. "The trick is to correlate the production of this substance and the disease process that we’re trying to track."
And that's what makes research so challenging a shortage of qualified doctors. A blood test would be simple to schedule at any laboratory and provide faster results.
“A couple weeks is a while to struggle with something and wonder if it's going to be this way forever,” Reed added.
To Be Effective, a Blood Test Must Outperform MRI
In order to make a difference for people with MS, any new blood test must outperform the MRI. If a blood test is less expensive and more convenient, but no more accurate than an MRI, there’s no reason to transfer over, said Thomas Stewart, a physician assistant at Rocky Mountain Multiple Sclerosis Center.
“You got this blood marker showing inflammation? So what?” Stewart said. “That doesn’t really get us very far. We’ve already got some markers for disease activity, you’ve got MRI. You have to be more sensitive than that.”
"The blood biomarkers would be a big advance, [but] it’s a little bit of a pipe dream right now," Dr. Bermel said, agreeing with Stewart's assessment. "Right now we don’t have MS divided into sub-types enough to create a blood test like that."
If it lives up to it’s potential, an MS blood test would reduce some of the financial burden, and the wait time, of treating multiple sclerosis. And though it's not on the market yet, Barazini said the technology should be available within a decade.
“I think it would give you more control over a lot of aspects of your life,” Reed said. “It is very difficult trying to live every day not knowing if the sword is going to fall on your head. This [test] can take some of the unknown out of it.”
Source: Everyday Health Copyright © 2013 Everyday Health Media, LLC (17/07/13)