Towards early diagnosis of Alzheimer's disease: advances in immune-related blood biomarkers and computational approaches.

Alzheimer's disease has an increasing prevalence in the population world-wide, yet current diagnostic methods based on recommended biomarkers are only available in specialized clinics. Due to these circumstances, Alzheimer's disease is usually diagnosed late, which contrasts with the currently available treatment options that are only effective for patients at an early stage. Blood-based biomarkers could fill in the gap of easily accessible and low-cost methods for early diagnosis of the disease. In particular, immune-based blood-biomarkers might be a promising option, given the recently discovered cross-talk of immune cells of the central nervous system with those in the peripheral immune system. Here, we give a background on recent advances in research on brain-immune system cross-talk in Alzheimer's disease and review machine learning approaches, which can combine multiple biomarkers with further information (e.g. age, sex, APOE genotype) into predictive models supporting an earlier diagnosis. In addition, mechanistic modeling approaches, such as agent-based modeling open the possibility to model and analyze cell dynamics over time. This review aims to provide an overview of the current state of immune-system related blood-based biomarkers and their potential for the early diagnosis of Alzheimer's disease.

Immunomodulation by poly-YE reduces organophosphate-induced brain damage.

Accidental organophosphate poisoning resulting from environmental or occupational exposure, as well as the deliberate use of nerve agents on the battlefield or by terrorists, remain major threats for multi-casualty events, with no effective therapies yet available. Even transient exposure to organophosphorous compounds may lead to brain damage associated with microglial activation and to long-lasting neurological and psychological deficits. Regulation of the microglial response by adaptive immunity was previously shown to reduce the consequences of acute insult to the central nervous system (CNS). Here, we tested whether an immunization-based treatment that affects the properties of T regulatory cells (Tregs) can reduce brain damage following organophosphate intoxication, as a supplement to the standard antidotal protocol. Rats were intoxicated by acute exposure to the nerve agent soman, or the organophosphate pesticide, paraoxon, and after 24 h were treated with the immunomodulator, poly-YE. A single injection of poly-YE resulted in a significant increase in neuronal survival and tissue preservation. The beneficial effect of poly-YE treatment was associated with specific recruitment of CD4(+) T cells into the brain, reduced microglial activation, and an increase in the levels of brain derived neurotrophic factor (BDNF) in the piriform cortex. These results suggest therapeutic intervention with poly-YE as an immunomodulatory supplementary approach against consequences of organophosphate-induced brain damage.

Excess circulating alternatively activated myeloid (M2) cells accelerate ALS progression while inhibiting experimental autoimmune encephalomyelitis.

BACKGROUND: Circulating immune cells including autoreactive T cells and monocytes have been documented as key players in maintaining, protecting and repairing the central nervous system (CNS) in health and disease. Here, we hypothesized that neurodegenerative diseases might be associated, similarly to tumors, with increased levels of circulating peripheral myeloid derived suppressor cells (MDSCs), representing a subset of suppressor cells that often expand under pathological conditions and inhibit possible recruitment of helper T cells needed for fighting off the disease. METHODS AND FINDINGS: We tested this working hypothesis in amyotrophic lateral sclerosis (ALS) and its mouse model, which are characterized by a rapid progression once clinical symptoms are evident. Adaptive transfer of alternatively activated myeloid (M2) cells, which homed to the spleen and exhibited immune suppressive activity in G93A mutant superoxide dismutase-1 (mSOD1) mice at a stage before emergence of disease symptoms, resulted in earlier appearance of disease symptoms and shorter life expectancy. The same protocol mitigated the inflammation-induced disease model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), which requires circulating T cells for disease induction. Analysis of whole peripheral blood samples obtained from 28 patients suffering from sporadic ALS (sALS), revealed a two-fold increase in the percentage of circulating MDSCs (LIN(-/Low)HLA-DR(-)CD33(+)) compared to controls. CONCLUSIONS: Taken together, these results emphasize the distinct requirements for fighting the inflammatory neurodegenerative disease, multiple sclerosis, and the neurodegenerative disease, ALS, though both share a local inflammatory component. Moreover, the increased levels of circulating MDSCs in ALS patients indicates the operation of systemic mechanisms that might lead to an impairment of T cell reactivity needed to overcome the disease conditions within the CNS. This high level of suppressive immune cells might represent a risk factor and a novel target for therapeutic intervention in ALS at least at the early stage.

A novel immune-based therapy for stroke induces neuroprotection and supports neurogenesis.

The ability of the central nervous system to cope with stressful conditions was shown to be dependent on proper T-cell-mediated immune response. Because the therapeutic window for neuroprotection after acute insults such as stroke is relatively narrow, we searched for a procedure that would allow the relevant T cells to be recruited rapidly. Permanent middle cerebral artery occlusion was induced in adult rats. To facilitate a rapid poststroke T cell activity, rats were treated with poly-YE using different regimens. Control and poly-YE-treated rats were assessed for functional recovery using neurological severity score and Morris water maze. Neuroprotection, neurogenesis, growth factor expression, and microglial phenotype were assessed using histological and immunofluorescence methods. Administration of poly-YE as late as 24 hours after middle cerebral artery occlusion yielded a beneficial effect manifested by better neurological performance, reduced neuronal loss, attenuation of behavioral deficits, and increased hippocampal and cortical neurogenesis. This compound affected the subacute phase by modulating microglial response and by increasing local production of insulin-like growth factor-I, known to be a key player in neuronal survival and neurogenesis. The relative wide therapeutic window, coupled with its efficacy in attenuating further degeneration and enhancing restoration, makes poly-YE a promising immune-based candidate for stroke therapy.

Immune-based therapy for spinal cord repair: autologous macrophages and beyond.

Spinal cord injury is a devastating condition of the central nervous system (CNS), often resulting in severe loss of tissue, functional impairment, and only limited repair. Studies over the last few years have shown that response to the insult and spontaneous attempts at repair are multiphasic processes, with varying and sometimes conflicting requirements. This knowledge has led to novel strategies of therapeutic intervention. Our view is that a pivotal role in repair, maintenance, healing, and cell renewal in the CNS, as in other tissues, is played by the immune system. The mode and timing of intervention must be carefully selected, however, as the capacity of the CNS to tolerate local repair mechanisms is limited. Studies have shown that the spontaneously evoked early innate response to CNS injury is characterized by invasion of neutrophils and is unfavorable for cell survival. This is followed by a response of the resident innate immune cells (microglia), which however cannot supply all the needs of the damaged tissue; moreover, once evoked, and for as long as the damage persists, the microglial response remains beyond the capacity of the CNS to tolerate it. Immune-based clinical intervention is most effective in improving functional and morphological recovery when delayed for a certain period. Effective intervention might be in the form of (1) local injection of "alternatively activated" macrophages, (2) systemic injection of dendritic cells specific to CNS antigens, or (3) T-cell-based vaccination. The treatment of choice depends on the severity of the insult, the site of injury, the therapeutic window, and safety considerations.

Clinical experience using incubated autologous macrophages as a treatment for complete spinal cord injury: phase I study results.

OBJECT: A Phase I, open-label nonrandomized study was conducted to assess the safety and tolerability of incubated autologous macrophages administered to patients with acute complete spinal cord injury (SCI). METHODS: This therapy was first tested in rat models of spinal cord transection and contusion, in which it was shown to promote motor recovery. The procedure developed for clinical use consists of isolating monocytes from patient blood and incubating them ex vivo with autologous dermis. The resulting incubated autologous macrophages were injected into the patient's spinal cord immediately caudal to the lesion within 14 days of injury. Patients underwent preoperative and follow-up neurological assessment (American Spinal Injury Association [ASIA] standards), electrophysiological monitoring (motor evoked and/or somatosensory evoked potentials), magnetic resonance imaging, and safety monitoring. Before macrophage administration, complete neurological functional loss (ASIA Grade A) was confirmed in all patients. Of the eight patients in the study, three recovered clinically significant neurological motor and sensory function (ASIA Grade C status). During the study period, some adverse events were encountered, the most serious of which involved two cases of pulmonary embolism and one case of osteomyelitis that were treated and resolved without further complication. These and other adverse events appear to be similar to those encountered in other spinal cord-injured patients and are not considered a consequence of the experimental therapy. CONCLUSIONS: It is concluded that incubated autologous macrophage cell therapy is well tolerated in patients with acute SCI. Further clinical evaluation is warranted.

Features of skin-coincubated macrophages that promote recovery from spinal cord injury.

Uncontrolled inflammation is considered to exacerbate the neuronal loss that follows spinal cord trauma. However, controlled inflammation response appears to be beneficial. Skin-coincubated macrophages injected into contused spinal cord of rats resulted in improved motor recovery and reduced spinal cyst formation. The macrophages express elevated levels of cell-surface molecules CD80, CD86, CD54 and MHC-II, markers characteristic of antigen presenting cells (APCs). Additionally, skin-coincubation elevates secretion of interleukin-1 beta (IL-1 beta) and Brain-Derived Neurotrophic Factor (BDNF), and reduces secretion of tumor necrosis factor alpha (TNF-alpha). We propose that macrophages activated by skin-coincubation bolster neuroprotective immune activity in the spinal cord, making the environment less cytotoxic and less hostile to axonal regeneration.

Vaccination with dendritic cells pulsed with peptides of myelin basic protein promotes functional recovery from spinal cord injury.

Injury-induced self-destructive processes cause significant functional loss after incomplete spinal cord injury (SCI). Cellular elements of both the innate (macrophage) and the adaptive (T-cell) immune response can, if properly activated and controlled, promote post-traumatic regrowth and protection after SCI. Dendritic cells (DCs) trigger activation of effector and regulatory T-cells, providing a link between the functions of the innate and the adaptive immune systems. They also initiate and control the body's response to pathogenic agents and regulate immune responses to both foreign and self-antigens. Here we show that post-injury injection of bone marrow-derived DCs pulsed with encephalitogenic or nonencephalitogenic peptides derived from myelin basic protein, when administered (either systemically or locally by injection into the lesion site) up to 12 d after the injury, led to significant and pronounced recovery from severe incomplete SCI. No significant protection was seen in DC recipients deprived of mature T-cells. Flow cytometry, RT-PCR, and proliferation assays indicated that the DCs prepared and used here were mature and immunogenic. Taken together, the results suggest that the DC-mediated neuroprotection was achieved via the induction of a systemic T-cell-dependent immune response. Better preservation of neural tissue and diminished formation of cysts and scar tissue accompanied the improved functional recovery in DC-treated rats. The use of antigen-specific DCs may represent an effective way to obtain, via transient induction of an autoimmune response, the maximal benefit of immune-mediated repair and maintenance as well as protection against self-destructive compounds.

Beneficial effect of orally administered myelin basic protein in EAE-susceptible Lewis rats in a model of acute CNS degeneration.

Axonal injury in the central nervous system (CNS) results in the degeneration of directly damaged fibers and also in the secondary degeneration of fibers that escaped the primary insult. Studies have shown that a protective T cell-mediated autoimmunity directed against myelin-related self-antigens is a physiological response to CNS insult, spontaneously elicited in strains that are constitutionally resistant to experimental autoimmune encephalomyelitis (EAE) but not in EAE-susceptible strains. The protective response following axonal injury can be induced in susceptible rats and boosted in resistant rats by passive or active immunization with myelin-related antigens. Here we show that oral administration of low-dose myelin basic protein (MBP) over a 5-day period is beneficial for post-traumatic survival of neurons in Lewis (EAE-susceptible) rats. Protection was accompanied by increased expression of the costimulatory molecule B7.2 in the traumatized nerves, similar to that seen after passive transfer of MBP-specific T cells. These results support the contention that properly controlled autoimmunity is the body's defense mechanism against non-infective insults. Oral immunization with MBP can be viewed as a way to control the autoimmunity capable of fighting off the consequences of CNS injury in EAE-susceptible strains.

Therapeutic vaccination for closed head injury.

Closed head injury often has a devastating outcome, partly because the insult, like other injuries to the central nervous system (CNS), triggers self-destructive processes. During studies of the response to other CNS insults, it was unexpectedly discovered that the immune system, if well controlled, provides protection against self-destructive activities. Here we show that in mice with closed head injury, the immune system plays a key role in the spontaneous recovery. Strain-related differences were observed in the ability to harness a T cell-dependent protective mechanism against the effects of the injury. We further show that the trauma-induced deficit could be reduced, both functionally and anatomically, by post-traumatic vaccination with Cop-1, a synthetic copolymer used to treat patients with multiple sclerosis and found (using a different treatment protocol) to effectively counteract the loss of neurons caused by axonal injury or glutamate-induced toxicity. We suggest that a compound such as Cop-1 can be safely developed as a therapeutic vaccine to boost the body's immune repair mechanisms, thereby providing multifactorial protection against the consequences of brain trauma.

Neuroprotective effect of memantine in different retinal injury models in rats.

PURPOSE: To evaluate the neuroprotective effect of memantine, an NMDA receptor channel blocker, in two retinal ganglion cell (RGC) injury models in rats. METHODS: Neuroprotective effect of memantine was tested in partial optic nerve injury and chronic ocular hypertensive models. In the optic nerve injury model, memantine (0.1 - 30 mg/kg) was injected intraperitoneally immediately after injury. Two weeks later, optic nerve function was determined by measuring compound action potential and surviving RGC was determined by retrograde labeling with dextran tetramethyl rhodamine. Chronic ocular hypertension was attained by laser photocoagulation of episcleral and limbal veins. Memantine (5 or 10 mg/kg) was administered continuously each day with an osmotic pump, either immediately after or 10 days after first laser photocoagulation, for 3 weeks, after which RGC survival was determined. RESULTS: Two weeks after partial optic nerve injury, there was approximately 80% reduction in RGC number. Memantine (5 mg/kg) caused a twofold increase in compound action potential amplitude and a 1.7-fold increase in survival of RGCs, respectively. In the chronic ocular hypertension model there was 37% decrease in RGCs after 3 weeks of elevated intraocular pressure. Memantine (10 mg/kg daily) reduced ganglion cell loss to 12% when applied immediately after first laser photocoagulation, and prevented any further loss when applied 10 days after first laser photocoagulation. CONCLUSION: The protective effect of memantine suggests that excessive stimulation of NMDA receptors by glutamate is involved in causing cell damage in these RGC injury models.

Myelin specific Th1 cells are necessary for post-traumatic protective autoimmunity.

Myelin-specific encephalitogenic T cells, when passively transferred into rats or mice, cause an experimental autoimmune disease. Previous studies by our group have shown that (a) the same cells also significantly reduce post-traumatic degeneration in these animals after injury to the central nervous system, (b) this beneficial autoimmunity is a physiological response, and (c) animals differ in their ability to resist injurious conditions, and the ability to resist post-traumatic degeneration correlates with resistance to the development of an autoimmune disease. Here we show that optic nerve neurons in both resistant and susceptible rat strains can be protected from secondary degeneration after crush injury by immunization with myelin basic protein emulsified in complete or incomplete Freund's adjuvant. We provide evidence that potentially destructive autoimmunity (causing autoimmune disease) and beneficial autoimmunity (causing improved neuronal survival) both result from activity of the same myelin-specific, proinflammatory Th1 cells. We further show that following passive transfer of such Th1 cells, the expression of their beneficial potential depends on the activity of an additional T cell (CD4(+)) population. By identifying the additional cellular component of autoimmune neuroprotection, we may be able to take meaningful steps toward achieving neuroprotection without risk of accompanying autoimmune disease.

Immune-related mechanisms participating in resistance and susceptibility to glutamate toxicity.

Glutamate is an essential neurotransmitter in the CNS. However, at abnormally high concentrations it becomes cytotoxic. Recent studies in our laboratory showed that glutamate evokes T cell-mediated protective mechanisms. The aim of the present study was to examine the nature of the glutamate receptors and signalling pathways that participate in immune protection against glutamate toxicity. We show, using the mouse visual system, that glutamate-induced toxicity is strain dependent, not only with respect to the amount of neuronal loss it causes, but also in the pathways it activates. In strains that are genetically endowed with the ability to manifest a T cell-dependent neuroprotective response to glutamate insult, neuronal losses due to glutamate toxicity were relatively small, and treatment with NMDA-receptor antagonist worsened the outcome of exposure to glutamate. In contrast, in mice devoid of T cell-dependent endogenous protection, NMDA receptor antagonist reduced the glutamate-induced neuronal loss. In all strains, blockage of the AMPA/KA receptor was beneficial. Pharmacological (with alpha2-adrenoceptor agonist) or molecular intervention (using either mice overexpressing Bcl-2, or DAP-kinase knockout mice) protected retinal ganglion cells from glutamate toxicity but not from the toxicity of NMDA. The results suggest that glutamate-induced neuronal toxicity involves multiple glutamate receptors, the types and relative contributions of which, vary among strains. We suggest that a multifactorial protection, based on an immune mechanism independent of the specific pathway through which glutamate exerts its toxicity, is likely to be a safer, more comprehensive, and hence more effective strategy for neuroprotection. It might suggest that, because of individual differences, the pharmacological use of NMDA-antagonist for neuroprotective purposes might have an adverse effect, even if the affinity is low.

Resistance of retinal ganglion cells to an increase in intraocular pressure is immune-dependent.

PURPOSE: Glaucoma is widely accepted as a neurodegenerative disease in which retinal ganglion cell (RGC) loss is initiated by a primary insult to the optic nerve head, caused, for example, by increased intraocular pressure (IOP). In some cases, the surviving RGCs, despite adequate IOP control, may continue to degenerate as a result of their heightened susceptibility to self-destructive processes evoked by the initial damage. In animal models of mechanical or biochemical injury to the optic nerve or retina, a T-cell-mediated immune response evoked by the insult helps to reduce this ongoing loss. The current study was conducted to find out whether the ability to resist the IOP-induced loss of RGCs in a rat model is affected by the immune system. METHODS: The ocular veins and limbal plexus of rats of two strains differing in their resistance to experimental autoimmune encephalomyelitis (EAE) and in their ability to manifest a beneficial autoimmune response were laser irradiated twice to induce an increase in IOP. The pressure was measured weekly, and RGC losses were assessed 3 and 6 weeks after the first irradiation. To verify the existence of a relationship between the immune system and RGC survival, we assessed neuronal survival in Sprague-Dawley (SPD) rats devoid of mature T cells as well as after transferring splenocytes from Fisher rats, an EAE-resistant rat strain capable of manifesting T-cell-mediated neuroprotection, to rats of a major histocompatibility complex (MHC)-matched EAE-susceptible strain (Lewis), in which the ability to manifest such protective immunity is limited. RESULTS: Both 3 and 6 weeks after the increase in IOP was initiated, the number of surviving RGCs in SPD rats, a strain in which a beneficial autoimmune response can be evoked spontaneously, was significantly higher than in Lewis rats. Moreover, in SPD rats that were thymectomized at birth, the number of surviving RGCs after an increase in IOP as adults was significantly diminished. Passive transfer of splenocytes from Fisher rats to Lewis rats significantly reduced the IOP-induced loss of RGCs in the latter. CONCLUSIONS: In rats of different strains, a similar increase in IOP results in differing amounts of RGC loss. This disparity was found to correlate with immune potency. These findings may explain why patients with glaucoma experience different degrees of visual loss after pressure reduction, even when the severity of the disease at the time of diagnosis is similar. The results have far-reaching prognostic and therapeutic implications.