Saliva Diagnosis as a Disease Predictor.

BACKGROUND: Saliva, the most readily available body fluid, is the product of genes which are in constant activity throughout life. Measurement of saliva can predict the onset of some diseases years before their accumulation in vulnerable tissues causes clinical signs to appear. The purpose of this study was is to demonstrate current applications of saliva analysis and to predict and prevent disease progression. METHODS: We measured levels of Abeta42, C-reactive proteins (CRPs), and tumornecrosis factors (TNFs) in saliva from both healthy and fatal diseased cases such as cancer, Alzheimer's disease (AD), and coronary heart disease by ELISA-mediated techniques. We also immunostained human tissue sections with antibodies specific to these proteins to demonstrate the data are comparable. RESULTS: We found all the proteins expressed constantly in saliva from healthy controls but increased in diseased cases. This was accompanied by data from immunohistochemistry. It was also found that these proteins wereexpressed in high amounts in some healthy controls, which reflects high risk for the onset of diseases such as AD and heart diseases. CONCLUSIONS: It is concluded that measuring changes in essential gene products in saliva can predict onset of fatal diseases and open the door to effective protection measures, thus preventing premature death.

Application of multisegment injection on quantification of creatinine and standard addition analysis of urinary 5-hydroxyindoleacetic acid simultaneously with creatinine normalization.

In this paper, the development of a simple dilute-and-shoot method for quantifying urinary creatinine by CE-ESI-MS was described. The creatinine analysis time was about 7 min/sample by conventional single injection (SI) method and can be significantly reduced to less than 2 min/sample with multi-segment injection (MSI). In addition, the standard addition analysis of 5-hydroxyindole-3-acetic acid (5-HIAA) and creatinine normalization was performed within one run by the MSI technique, and the total analysis time was 14-min faster compared to the SI method for analyzing the same set of samples. The uses of isotopic and non-isotopic internal standards (ISs) were compared. Creatinine-(methyl-13 C) and 5-hydroxyindole-4,6,7-D3 -3-acetic-D2 acid (5-HIAA-D5 ) used as isotopic ISs can provide both accurate and precise results. In contrast, 1,5,5-trimethylhydantoin (1,5,5-TH) used as the non-isotopic IS for creatinine may cause a bias of over 13% in SI method and even worse when the MSI technique was used. Another compound, 2-methyl-3-indoleacetic acid (2-MIAA), was determined not suitable for MSI analysis of 5-HIAA due to endogenous interferences despite its acceptable performance in conventional methods of analysis.

Conquering Alzheimer's Disease by Self Treatment.

The means are now at hand to conquer Alzheimer's disease (AD). The method is to identify those at risk for the disease before clinical signs develop. That is followed by implementing measures that can effectively prevent disease development. Since biotechnology markers have shown that AD commences at least a decade before cognitive deficits set in, there is an extended window of opportunity to successfully prevent disease development. Methods of identifying those at risk include positron electron microscopy for AD senile plaques, blood or saliva analysis for elevation of the amyloid-ß protein fragment terminating at position 42, and cerebrospinal fluid analysis showing a decrease in content of this protein. Of the modalities available, saliva is by far the simplest and least invasive. Once identified, those at risk can prevent disease development through self treatment by consumption of non-steroidal anti-inflammatory drugs, adhering to a Mediterranean diet, and consuming antioxidants such as quercitin which is contained in coffee.

Alzheimer's Disease Can Be Spared by Nonsteroidal Anti-Inflammatory Drugs.

Alzheimer's disease (AD) is characterized by deposits of amyloid-ß protein (Aß) in brain which become foci of inflammation. Neurons are destroyed by this inflammatory process, leading to the cognitive deficits which define AD clinical onset. Epidemiological studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) can ameliorate this destructive process if they are started well before clinical signs develop. Biomarker studies indicate that the disease process starts at least a decade before cognitive deficits appear. This pre-clinical onset explains the NSAID effect. It also opens a window of opportunity for preventive treatment that can be met with a simple diagnostic test. Salivary levels of Aß42 may fulfill that need. They can be measured by a simple ELISA test we have developed using commercially available reagents. By this ELISA test, normal controls, who are not at risk for AD, have levels of Aß42 close to 20 pg/ml. AD cases, as well as high level controls, secrete levels in the range of 40-85 pg/ml. Widespread application of this test to detect high level controls, followed by NSAID consumption, could substantially reduce the prevalence of AD.

Biological Imaging with Medium-Sensitive Bichromatic Flexible Fluorescent Dyes.

A family of environment-sensitive shape-shifting molecules have been developed as flexible fluorescent (FlexFluor) dyes for biological imaging applications. These compounds feature a flexible bithiophene-based fluorophore that gives rise to different emission colors in lipophilic or hydrophilic environments, as well as side groups that can be synthetically modified with ease. FlexFluor dyes are the first fluorescent dyes in which emission color can be used to indicate lipid/water environments. The behavior of these dyes in different solvents was studied, and used to simultaneously highlight lipid and water contents in adipose and brain tissues using optical fluorescence microscopy.

Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-ß and Inhibitor of Fibril Assembly.

BACKGROUND: Identifying physiologically relevant binding partners of amyloid-ß (Aß) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. OBJECTIVE: We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aß42 and can modulate Aß fibril formation. METHODS: Specific interactions between LL-37 and Aß (with Aß in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aß42 alone, and LL-37/Aß complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). RESULTS: SPRi shows binding specificity between LL-37 and Aß, while TEM shows that LL-37 inhibits Aß42 fibril formation, particularly Aß's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aß42 from adopting its typical ß-type secondary structure. Microglia-mediated toxicities of LL-37 and Aß42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. CONCLUSION: Based on this body of evidence, we propose that LL-37 and Aß42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

A review of human diseases caused or exacerbated by aberrant complement activation.

Complement is the backbone of our innate immune system. It is of ancient evolutionary origin, being traced back to horseshoe crabs 350 million years ago. It consists today of more than 25 proteins which must work together like clockwork to distinguish friend from foe. Self-attack by the complement system can occur whenever it fails to do so. This failure has been reported to occur in an estimated 22 human diseases. A significant number of these are chronic degenerative neurological disorders. In some, there is overwhelming evidence that complement self-attack causes the disease. In many others, it is considered only to contribute to the overall pathology. Finding effective therapeutic agents should be a high priority for medical research. To date, the monoclonal antibody eculizumab is the only approved agent. Molecules under development include other monoclonal antibodies directed at C5, C3, and properdin, various aptamers to C3, and small molecules that are orally available.

A Method for Diagnosing Alzheimer's Disease Based on Salivary Amyloid-ß Protein 42 Levels.

We have developed a non-invasive method of diagnosing Alzheimer's disease (AD), which can also predict the risk of its future onset. It is based on measuring salivary levels of amyloid-ß protein terminating at position 42 (Aß42). Brain deposits of this peptide are characteristic of AD. Biomarker studies indicate that such brain deposits commence a decade or more prior to clinical onset of the disease. We report here that Aß42 is produced in all peripheral organs tested, thus establishing the generality of its production. We used this information to develop simple and sensitive tests to determine salivary Aß42 levels. The levels were first stabilized by adding thioflavin S as an anti-aggregation agent and sodium azide as an anti-bacterial agent. We then quantitated the Aß42 in a series of samples with ELISA type tests. Control cases showed almost identical levels of salivary Aß42 regardless of sex or age. All AD cases secreted levels of Aß42 more than double those of controls. Individuals at elevated risk of developing AD secreted levels comparable to the AD cases. The results establish that salivary Aß42 levels can be used to diagnose AD as well as to predict the risk of its future onset.

Inflammation, Antiinflammatory Agents, and Alzheimer's Disease: The Last 22 Years.

Two basic discoveries spurred research into inflammation as a driving force in the pathogenesis of Alzheimer's disease (AD). The first was the identification of activated microglia in association with the lesions. The second was the discovery that rheumatoid arthritics, who regularly consume anti-inflammatory agents, were relatively spared from the disease. These findings led to an exploration of the inflammatory pathways that were involved in AD pathogenesis. A pivotal advance was the discovery that amyloid-ß protein (Aß) activated the complement system. This focused attention on anti-inflammatories as blockers of complement activation. More than 15 epidemiological studies have since showed a sparing effect of non-steroidal anti-inflammatory drugs (NSAIDs) in AD. A consistent finding has been that the longer the NSAIDs were used prior to clinical diagnosis, the greater the sparing effect. The reason has since emerged from studies of biomarkers such as amyloid-ß (Aß) levels in the cerebrospinal fluid and Aß deposits in brain. They have established that the onset of AD commences at least a decade before cognitive decline permits clinical diagnosis. Such biomarker studies have revealed that a huge window of opportunity exists when application of NSAIDs, other anti-inflammatory agents, or complement activation blockers, could arrest further progress of AD, thus eliminating its manifestation. It can be anticipated that this principle will apply to many other chronic neurodegenerative diseases. Neuroinflammation, discovered in AD more than 30 years ago, has now become a major field of brain research today. Inhibiting it may be the key to successful treatment of many chronic neurological disorders.

Quercetin, not caffeine, is a major neuroprotective component in coffee.

Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson's disease and Alzheimer's disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-γ or interferon-γ released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-α and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFκB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson's disease and Alzheimer's disease.

Epigallocatechin Gallate (EGCG) Inhibits Alpha-Synuclein Aggregation: A Potential Agent for Parkinson's Disease.

Protein aggregation is a prominent feature of many neurodegenerative disorders including Parkinson's disease (PD). Aggregation of alpha-synuclein (SNCA) may underlie the pathology of PD. They are the main components of Lewy bodies and dystrophic neurites that are the intraneuronal inclusions characteristic of the disease. We have demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibited SNCA aggregation, which made it a candidate for therapeutic intervention in PD. Three methods were used: SNCA fibril formation inhibition by EGCG in incubates; inhibition of the SNCA fluorophore A-Syn-HiLyte488 binding to plated SNCA in microwells; and inhibition of the A-Syn-HiLyte488 probe binding to aggregated SNCA in postmortem PD tissue. Recombinant human SNCA was incubated under conditions that result in fibril formation. The aggregation was blocked by 100 nM EGCG in a concentration-dependent manner, as shown by an absence of thioflavin T binding. In the microplate assay system, the ED50 of EGCG inhibition of A-Syn-HiLyte488 binding to coated SNCA was 250 nM. In the PD tissue based assay, SNCA aggregates were recognized by incubation with 7 nM of A-Syn-HiLyte488. This binding was blocked by EGCG in a concentration dependent manner. The SNCA amino acid sites, which potentially interacted with EGCG, were detected on peptide membranes. It was implicated that EGCG binds to SNCA by instable hydrophobic interactions. In this study, we suggested that EGCG could be a potent remodeling agent of SNCA aggregates and a potential disease modifying drug for the treatment of PD and other α-synucleinopathies.

Common mechanisms involved in Alzheimer's disease and type 2 diabetes: a key role of chronic bacterial infection and inflammation.

Strong epidemiologic evidence and common molecular mechanisms support an association between Alzheimer's disease (AD) and type 2-diabetes. Local inflammation and amyloidosis occur in both diseases and are associated with periodontitis and various infectious agents. This article reviews the evidence for the presence of local inflammation and bacteria in type 2 diabetes and discusses host pathogen interactions in chronic inflammatory disorders. Chlamydophyla pneumoniae, Helicobacter pylori and spirochetes are demonstrated in association with dementia and brain lesions in AD and islet lesions in type 2 diabetes. The presence of pathogens in host tissues activates immune responses through Toll-like receptor signaling pathways. Evasion of pathogens from complement-mediated attack results in persistent infection, inflammation and amyloidosis. Amyloid beta and the pancreatic amyloid called amylin bind to lipid bilayers and produce Ca(2+) influx and bacteriolysis. Similarly to AD, accumulation of amylin deposits in type 2 diabetes may result from an innate immune response to chronic bacterial infections, which are known to be associated with amyloidosis. Further research based on an infectious origin of both AD and type 2 diabetes may lead to novel treatment strategies.

Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases.

BACKGROUND: Sodium thiosulfate (STS) is an industrial chemical which has also been approved for the treatment of certain rare medical conditions. These include cyanide poisoning and calciphylaxis in hemodialysis patients with end-stage kidney disease. Here, we investigated the anti-inflammatory activity of STS in our glial-mediated neuroinflammatory model. METHODS: Firstly, we measured glutathione (GSH) and hydrogen sulfide (H2S, SH(-)) levels in glial cells after treatment with sodium hydrosulfide (NaSH) or STS. We also measured released levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) from them. We used two cell viability assays, MTT and lactate dehydrogenase (LDH) release assays, to investigate glial-mediated neurotoxicity and anti-inflammatory effects of NaSH or STS. We also employed Western blot to examine activation of intracellular inflammatory pathways. RESULTS: We found that STS increases H2S and GSH expression in human microglia and astrocytes. When human microglia and astrocytes are activated by lipopolysaccharide (LPS)/interferon-γ (IFNγ) or IFNγ, they release materials that are toxic to differentiated SH-SY5Y cells. When the glial cells were treated with NaSH or STS, there was a significant enhancement of neuroprotection. The effect was concentration-dependent and incubation time-dependent. Such treatment reduced the release of TNFα and IL-6 and also attenuated activation of P38 MAPK and NFκB proteins. The compounds tested were not harmful when applied directly to all the cell types. CONCLUSIONS: Although NaSH was somewhat more powerful than STS in these in vitro assays, STS has already been approved as an orally available treatment. STS may therefore be a candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component.

Lack of support for bexarotene as a treatment for Alzheimer's disease.

Bexarotene has been reported to reduce brain amyloid-ß (Aß) levels and to improve cognitive function in transgenic mouse models of Alzheimer's disease (AD). Four groups failed to fully replicate the primary results but the original authors claimed overall support for the general conclusions. Because of its potential clinical importance, the current work studied the effects of bexarotene using two animal species and highly relevant paradigms. Rats were tested for the ability of bexarotene to prevent changes induced by an Aß challenge in the form intracerebroventricular (i.c.v) administration of 7PA2 conditioned medium (7PA2 CM) which contains high levels of Aß species. Bexarotene had no effect on the long-term potentiation of evoked extracellular field excitatory postsynaptic potentials induced by i.c.v. 7PA2 CM. It also had no effect following subcutaneous administration of 2, 5, 10 and 15 mg/kg on behavioral/cognitive impairment using an alternating-lever cyclic-ratio schedule of operant responding in the rat. The effects of bexarotene were further tested using the APPSwFILon, PSEN1*M146L*L286V transgenic mouse model of AD, starting at the time Aß deposits first begin to develop. Mice were sacrificed after 48 days of exposure to 100 mg bexarotene per day. No significant difference between test and control mice was found using a water-maze test, and no significant difference in the number of Aß deposits in cerebral cortex, using two different antibodies, was apparent. These results question the potential efficacy of bexarotene for AD treatment, even if instigated in the preclinical period prior to the onset of cognitive deficits reported for human AD. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

Inhibition of aberrant complement activation by a dimer of acetylsalicylic acid.

We here report synthesis for the first time of the acetyl salicylic acid dimer 5,5'-methylenebis(2-acetoxybenzoic acid) (DAS). DAS inhibits aberrant complement activation by selectively blocking factor D of the alternative complement pathway and C9 of the membrane attack complex. We have previously identified aurin tricarboxylic and its oligomers as promising agents in this regard. DAS is much more potent, inhibiting erythrocyte hemolysis by complement-activated serum with an IC50 in the 100-170 nanomolar range. There are numerous conditions where self-damage from the complement system has been implicated in the pathology, including such chronic degenerative diseases of aging as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and age-related macular degeneration. Consequently, there is a high priority for the discovery and development of agents that can successfully treat such conditions. DAS holds considerable promise for being such an agent.

Age-related increases in amyloid beta and membrane attack complex: evidence of inflammasome activation in the rodent eye.

BACKGROUND: The membrane attack complex (MAC) is a key player in the pathogenesis of age-related macular degeneration (AMD) and is a putative activator of the NLRP3 inflammasome. Amyloid beta (Aß), a component of drusen deposits, has also been implicated in inflammasome activation by our work and those of others. However, the interactions of MAC and Aß are still poorly understood, especially their roles in aging and retinal degenerative pathologies. Since inflammasome activation may represent a key cellular pathway underlying age-related chronic inflammation in the eye, the purpose of this study is to identify the effects associated with MAC and inflammasome activation in the retinal pigment epithelium (RPE)/choroid and to evaluate the therapeutic merits of MAC suppression. METHODS: Adult Long-Evans rats were divided into treatment and control groups. Treatment groups received oral aurin tricarboxylic acid complex (ATAC), a MAC inhibitor, in drinking-water, and control groups received drinking-water alone (No ATAC). Groups were sacrificed at 7.5 or 11.5 months, after approximately 40 days of ATAC treatment. To study age-related changes of Aß and MAC in RPE/choroid, naive animals were sacrificed at 2.5, 7.5, and 11.5 months. Eye tissues underwent immunohistochemistry and western blot analysis for MAC, Aß, NF-κB activation, as well as cleaved caspase-1 and IL-18. Vitreal samples were collected and assessed by multiplex assays for secreted levels of IL-18 and IL-1ß. Statistical analyses were performed, and significance level was set at p ≤ 0.05. RESULTS: In vivo studies demonstrated an age-dependent increase in MAC, Aß, and NF-κB activation in the RPE/choroid. Systemic ATAC resulted in a prominent reduction in MAC formation and a concomitant reduction in inflammasome activation measured by cleaved caspase-1 and secreted levels of IL-18 and IL-1ß, but not in NF-κB activation. In vitro studies demonstrated Aß-induced MAC formation on RPE cells. CONCLUSIONS: Age-dependent increases in Aß and MAC are present in the rodent outer retina. Our results suggest that suppressing MAC formation and subsequent inflammasome activation in the RPE/choroid may reduce chronic low-grade inflammation associated with IL-18 and IL-1ß in the outer retina.

The Neuroprotective Effects of Ratanasampil on Oxidative Stress-Mediated Neuronal Damage in Human Neuronal SH-SY5Y Cells.

We previously found that Ratanasampil (RNSP), a traditional Tibetan medicine, improves the cognitive function of mild-to-moderate AD patients living at high altitude, as well as learning and memory in an AD mouse model (Tg2576); however, mechanism underlying the effects of RNSP is unknown. In the present study, we investigated the effects and molecular mechanisms of RNSP on oxidative stress-induced neuronal toxicity using human neuroblastoma SH-SY5Y cells. Pretreatment with RNSP significantly ameliorated the hydrogen peroxide- (H2O2-) induced cytotoxicity of SH-SY5Y cells in a dose-dependent manner (up to 60 µg/mL). Furthermore, RNSP significantly reduced the H2O2-induced upregulation of 8-oxo-2'-deoxyguanosine (8-oxo-dG, the oxidative DNA damage marker) but significantly reversed the expression of repressor element-1 silencing transcription factor (REST) from H2O2 associated (100 µM) downregulation. Moreover, RNSP significantly attenuated the H2O2-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 (ERK 1/2) in SH-SY5Y cells. These observations strongly suggest that RNSP may protect the oxidative stress-induced neuronal damage that occurs through the properties of various antioxidants and inhibit the activation of MAPKs. We thus provide the principle molecular mechanisms of the effects of RNSP and indicate its role in the prevention and clinical management of AD.

Interferon-γ-induced neurotoxicity of human astrocytes.

Activated astrocytes, which can also be referred to as reactive astrocytes or astrogliosis, have been identified in affected regions of common neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis. Activated astrocytes may be beneficial, promoting neuronal survival due to their production of growth factors and neurotrophins. Activated astrocytes can also be detrimental to neighboring neurons in neuroinflammatory processes. Astrocytes exposed to certain inflammatory stimulants in vitro have been shown to release potentially neurotoxic molecules, including inflammatory cytokines, glutamate, nitric oxide and reactive oxygen species. It has recently been shown that adult human astrocytes stimulated with interferon-γ, a common inflammatory cytokine evidently present in neuropathological brains, exert potent neurotoxicity in vitro. This interferon- γ-induced astrocytic neurotoxicity is mediated by the activation of the Janus kinase-signal transducer and activator of transcription (STAT) 3 pathway in the astrocytes, and involves intracellular phosphorylation of STAT3 at tyrosine-705 residue. Therefore, control of STAT3 activation in human astrocytes may be a promising new therapeutic strategy for a broad spectrum of neurodegenerative and neuroinflammatory disorders where activated astrocytes may contribute to the pathology.

Human antimicrobial peptide LL-37 induces glial-mediated neuroinflammation.

LL-37 is the sole cathelicidin-derived antimicrobial peptide found in humans. It becomes active upon C-terminal cleavage of its inactive precursor hCAP18. In addition to antimicrobial action, it also functions as an innate immune system stimulant in many tissues of the body. Here we report that hCAP18 and LL-37 are expressed in all organs of the human body that were studied with the highest basic levels being expressed in the GI tract and the brain. Its expression and functional role in the central nerve system (CNS) has not previously been reported. We found increased expression of LL-37 in IFNγ-stimulated human astrocytes and their surrogate U373 cells, as well as in LPS/IFNγ-stimulated human microglia and their surrogate monocyte-derived THP-1 cells. We found that treatment of microglia, astrocytes, THP-1 cells and U373 cells with LL-37 induced secretion of the inflammatory cytokines IL-1ß and IL-6; the chemokines IL-8 and CCL-2, and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of LL-37 stimulation involves activation of intracellular proinflammatory pathways involving phospho-P38 MAP kinase and phospho-NFκB proteins. We blocked the inflammatory stimulant action of LL-37 by removing it with an anti-LL-37 antibody. The inflammatory effect was also prevented by treatment with inhibitors of PKC, PI3K and MEK-1/2 as well as with the intracellular Ca(2+)-chelator, BAPTA-AM. This indicates involvement of these intracellular pathways. Our data suggest that LL-37, in addition to its established roles, may play a role in the chronic neuroinflammation which is observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease.