Quantitative and qualitative analysis of stability for 16 serum immunoregulators over 50 freeze-thaw cycles.

OBJECTIVES: To evaluate the reliability of data from the assay of bio-archived specimens, a 50-freeze-thaw-cycle (FTC) degradation study of fresh sera was conducted to test the stability of 16 immunoregulators. METHODS: Twenty de-identified serum specimens were obtained from volunteers at United Health Services-Wilson Memorial Hospital. Specimens were stored at -20°C and underwent daily 1 h thawing and subsequent freezing for each FTC over 50 consecutive days. Immunoregulator concentrations were assessed via enzyme-linked immunosorbent assay (ELISA) in participant samples at 2 FTC (baseline), 25 FTC, and 50 FTC. Specific immunoregulators observed in the study were C-reactive protein (CRP), interleukin (IL)-1α, 4, 6, 8, 10, monocyte chemoattractant protein-1 (MCP-1, CCL2), monocyte chemoattractant protein-2 (MCP-2, CCL8), eotaxin-1, thymus-and-activation-regulated chemokine (TARC, CCL17), regulated on activation normal T-cell expressed and secreted (RANTES, CCL5), growth-regulated oncogene-alpha (GRO-α, CXCL1), small inducible cytokine A1 (I-309, CCL1), interferon-gamma (IFN-γ), interferon-gamma inducible protein-10 (IP-10, CXCL10), and tumor necrosis factor-alpha (TNF-α). RESULTS: Quantitative stability of serum immunoregulators: Serum CRP, IL-8, IL-10, IFN-γ, IP-10, and eotaxin-1 levels appear to be statistically equivalent from baseline to 50 FTC (p ≤ .05). Retention of patterns in serum immunoregulators: patterns across FTC were retained for TARC (age) and CRP, IFN-γ, and MCP-2 (sex). CONCLUSIONS: While the effect of multiple FTC on serum immunoregulator levels may not replicate prolonged freezer storage, the results of this study provide valuable information on the robustness of immunoregulators for research using bio-archived sera.

Impact of white-tailed deer on the spread of Borrelia burgdorferi.

There is a public perception that the white-tailed deer Odocoileus virginianus (Artiodactyla: Cervidae) is the main reservoir supporting the maintenance and spread of the causative agent of Lyme disease, Borrelia burgdorferi. This study examines the pathogen prevalence rate of Borrelia in adult Ixodes scapularis (Ixodida: Ixodidae), the black-legged tick, collected from white-tailed deer and compares it with pathogen prevalence rates in adult ticks gathered by dragging vegetation in two contiguous counties west of the Hudson Valley in upstate New York. In both Broome and Chenango Counties, attached and unattached ticks harvested from white-tailed deer had significantly lower prevalences of B. burgdorferi than those collected from vegetation. No attached ticks on deer (n = 148) in either county, and only 2.4 and 7.3% of unattached ticks (n = 389) in Broome and Chenango Counties, respectively, were harbouring the pathogen. This contrasts with the finding that 40.8% of ticks in Broome County and 46.8% of ticks in Chenango County collected from vegetation harboured the pathogen. These data suggest that a mechanism in white-tailed deer may aid in clearing the pathogen from attached deer ticks, although white-tailed deer do contribute to the spatial distribution of deer tick populations and also serve as deadend host breeding sites for ticks.

Six-year follow-up of a point-source exposure to CWD contaminated venison in an Upstate New York community: risk behaviours and health outcomes 2005-2011.

OBJECTIVES: It is currently unknown whether chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, is transmissible to humans. Reported on here are the behavioural risk factors and health conditions associated with a six-year follow-up of a known point-source exposure to a CWD infected deer in an Upstate New York community. STUDY DESIGN: Longitudinal. METHODS: The Oneida County Chronic Wasting Disease Surveillance Project was launched in 2005 in response to a point-source exposure to a CWD infected deer at a March 2005 Sportsmen's feast in Upstate New York. Eighty-one exposed individuals participated in the 2005 baseline data collection, and were sent follow-up questionnaires following each deer hunting season between 2005 and 2011. RESULTS: Over a six year period, participants reported a reduction in overall venison consumption. Participants reported no significant changes in health conditions, although several conditions (vision loss, heart disease, type 2 diabetes, weight changes, hypertension, and arthritis), were significantly associated with age. CONCLUSIONS: To this day, this incident remains the only known large-scale point-source exposure to a CWD infected deer. Prion diseases can incubate for multiple decades before the manifestation of clinical symptoms; thus, continued surveillance of this exposed study population represents a unique opportunity to assess the risk of CWD transmission to humans. This project is uniquely situated to provide the first epidemiological evidence of CWD transmission to humans, should it occur.

Intraneuronal aluminum accumulation in amyotrophic lateral sclerosis and Parkinsonism-dementia of Guam.

Scanning electron microscopy with energy-dispersive x-ray spectrometry was used to analyze the elemental content of neurofibrillary tangle (NFT)-bearing and NFT-free neurons within the Sommer's sector (H1 region) of the hippocampus in Guamanian Chamorros with amyotrophic lateral sclerosis and parkinsonism-dementia and in neurologically normal controls. Preliminary data indicate prominent accumulation of aluminum within the nuclear region and perikaryal cytoplasm of NFT-bearing hippocampal neurons, regardless of the underlying neurological diagnosis. These findings further extend the association between intraneuronal aluminum and NFT formation and support the hypothesis that environmental factors are related to the neurodegenerative changes seen in the Chamorro population.

Role of DNA dynamics in Alzheimer's disease.

DNA is a dynamic molecule, the conformation of which plays a major role in biological function. The non-B-form of DNA conformations are reported in the patho-physiology of diseases like Fragile X-syndrome, Huntington's chorea, Alzheimer's and others. Recently, our laboratory discovered the presence of Z-DNA in the hippocampal region of severely affected Alzheimer's disease (AD) brain samples. Alternate purine-pyrimidine bases, potential sequences adopting Z-DNA, are present in the promoter regions of AD specific genes like amyloid precursor protein (APP), Presenilin and ApoE. Thus, Z-DNA might be involved in the expression of these pathologically important genes. In the present review, we have focused on the possible mechanisms/hypothetical models of Z-DNA transition and its implications in AD. We propose that Z-DNA is formed in the promoter region of the APP, and Presenilin genes and Z-DNA may absorb the negative supercoils at that region. This decreases the supercoil density, altering the domain's native supercoiling state and facilitates the binding of effectors, which positively regulate gene expression of AD-related genes like APP and Presenilin. Further, it is presumed that Z-DNA may be involved in the down regulation of genes involved in Abeta clearance, anti-oxidant and defense mechanisms in AD. The proposed working model is novel and reveals possible triggering factors or precursors, which regulate the modulation of the supercoiling level of DNA involving putative Z-DNA forming sequences and regulatory proteins binding to DNA in AD.

TAU as a susceptibility gene for amyotropic lateral sclerosis-parkinsonism dementia complex of Guam.

BACKGROUND: A Guam variant of amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia complex (PDC-G) are found in the Chamorro people of Guam. Both disorders have overlapping neuropathologic findings, with neurofibrillary tangles in spinal cord and brain. The cause of ALS-G-PDC-G is unknown, although inheritance and environment appear important. Because neurofibrillary tangles containing tau protein are present in ALS-G-PDC-G, and because mutations in the tau gene (TAU) cause autosomal dominant frontotemporal dementia, TAU was examined as a candidate gene for ALS-G-PDC-G. METHODS: TAU was evaluated by DNA sequence analysis in subjects with ALS-G-PDC-G, by linkage analysis of TAU polymorphisms in an extended pedigree from the village of Umatac, and by evaluation of linkage disequilibrium with polymorphic markers flanking and within TAU. RESULTS: Linkage disequilibrium between ALS-G-PDC-G and the TAU polymorphism CA3662 was observed. For this 2-allele system, PDC and ALS cases were significantly less likely than Guamanian controls to have the 1 allele (4.9% and 2% vs 11.5%, respectively; Fisher exact P =.007). DNA sequence analysis of TAU coding regions did not demonstrate a mutation responsible for ALS-G-PDC-G. Analysis of TAU genotypes in an extended pedigree of subjects from Umatac showed obligate recombinants between TAU and ALS-G-PDC-G. Linkage analysis of the Umatac pedigree indicates that TAU is not the major gene for ALS-G-PDC-G. CONCLUSIONS: The genetic association between ALS-G-PDC-G implicates TAU in the genetic susceptibility to ALS-G-PDC-G. TAU may be a modifying gene increasing risk for ALS-G-PDC-G in the presence of another, as yet, unidentified gene.

Disappearance of high-incidence amyotrophic lateral sclerosis and parkinsonism-dementia on Guam.

The high incidence rates of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) occurring among the Chamorros of Guam have declined to rates only slightly higher than those observed in the continental United States. This decline has occurred principally among males, especially those born after 1920 and living in areas where calcium and magnesium levels are low in soil and water. The male-to-female ratio among affected patients now approaches unity, compared with ratios of 2 to 1 for ALS and 3 to 1 for PD three decades ago. These changes are consistent with the hypothesis that the previously high incidence resulted from defects in mineral metabolism and secondary hyperparathyroidism, provoked by nutritional deficiencies of calcium and magnesium, with resultant deposition of calcium and aluminum in neurons.

Brain destruction alone does not elevate brain aluminum.

Graphite furnace atomic-absorption spectroscopy was used to measure aluminum concentrations in brain samples from 33 patients dying from a variety of neurologic diseases. Four samples from patients dying of nonneurologic causes also were studied. Nine samples (one from each of nine patients) of Creutzfeldt-Jakob disease brain contained normal amounts of aluminum. Aluminum was increased in 9 of 18 brain specimens with seven different pathologic processes. This included three of seven Alzheimer disease, two of three Huntington disease, two of two Parkinson disease, one of one progressive supranuclear palsy, one of one acoustic neuroma, one of two cerebrovascular disease, and one of two Guamanian amyotrophic lateral sclerosis (ALS). Aluminum was normal in the remaining samples (four normal, two ALS, one multiple sclerosis, one Pick disease, and two Guamanian parkinsonism-dementia). The significance of high aluminum values is not clear, but the normal values from the Creutzfeldt-Jakob cases imply that neuronal destruction per se need not lead to accumulation of aluminum in the brain.

Amyotrophic lateral sclerosis and parkinsonism-dementia on Guam: a 30-year evaluation of clinical and neuropathologic trends.

We reviewed the records of 279 Guamanian Chamorro patients with amyotrophic lateral sclerosis (ALS) and 293 patients with parkinsonism-dementia (PD), who had onset of symptoms between 1950 and 1979, to determine if there were changes in the clinical and neuropathologic features that might clarify the declining incidence rates in the past decade. There were no major temporal changes in the frequencies of physical findings or histopathologic features, but in the past three decades, an increase in age at onset was observed for both ALS and PD. There was also a shorter duration of illness in ALS and a longer duration in PD. Good correlation was found between the clinical and pathologic findings for both ALS and PD throughout this period.

ALS and PDC of Guam: forty-year follow-up.

BACKGROUND: It was noticed in the mid-1950s that the incidence of ALS and parkinsonism--dementia complex (PDC) were much higher on Guam than anywhere else in the world. In 1958, a registry of patients and controls was established to ascertain the familial and genetic aspects of these diseases. Patients and individually matched controls and their relatives were registered from 1958 to 1963. The registry was updated and analyzed in 1998 through 1999. OBJECTIVE: To ascertain whether first-degree relatives of patients had a higher risk for developing ALS or PDC than relatives of controls. METHODS: During the period of 1958 to 1963, 126 new patients and 126 individually matched controls and their respective first-degree relatives and spouses were evaluated neurologically and registered. Forty years later, the number of new cases among the patient and control relatives were compared to an expected number of new cases based on the age- and sex-specific incidence of ALS and PDC in the population at large. RESULTS: From 1958 to 1999, there were 102 new ALS or PDC cases among relatives of patients and 33 among relatives of controls. These values were compared with the derived expected values. There were more observed than expected new cases among patients' relatives, and less observed cases than expected among the controls' relatives. CONCLUSIONS: Relatives of patients with ALS or PDC have significantly higher risks for developing the disease than the Guamanian population, whereas relatives of controls have significantly lower risks.

Potential role of an additive genetic component in the cause of amyotrophic lateral sclerosis and parkinsonism-dementia in the western Pacific.

Amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) are neurological degenerative disorders that occur in three high incidence foci in the western Pacific: among the Chamorros of Guam and the Commonwealth of the Northern Marianas Islands, among Japanese on the Kii peninsula of Honshu Island, and among the Auyu and Jakai peoples of southern West New Guinea. Previous studies have implicated both genetic susceptibility and environmental risk factors in the causation and familial clustering of these disorders. The data analyzed consist of 2,026 individuals in nuclear families ascertained on Guam through two mechanisms: (1) nuclear families were included in the study if one or both parents in the family were affected with ALS or PD or both; and (2) a group of "controls" was selected by obtaining nuclear families where neither parent was affected and both had lived through the age of risk. Clinically, ALS and PD are two distinct disorders. However, preliminary analyses indicated that combining all three diagnoses into one affected diagnosis for genetic analyses (thereby assuming any genetic effect on susceptibility to the two disorders was due to the same genetic mechanism) was reasonable. An age, sex and birth cohort-specific liability was defined and segregation analysis was performed under both logistic and normal models for this liability at the time of disease onset. Under either model, purely environmental, Mendelian dominant and Mendelian recessive hypotheses could be rejected, but a two-allele additive major locus hypothesis could not be rejected.

Blood groups, immunoglobulin allotypes and dermatoglyphic features of patients with amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

Blood group frequencies, immunoglobulin allotypes, and dermatoglyphic patterns were determined on patients with amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD), two chronic, degenerative, neurologic disorders of unknown cause found commonly among the Chamorros of the Mariana Islands, in an attempt to identify a specific genetic or phenetic marker associated with either disorder. With the exception of the Kidd system, no significant differences were found in blood group frequencies nor in immunoglobulin allotypes between ALS patients, PD patients, and unaffected controls. The dermatoglyphic analysis demonstrated that ALS patients had higher frequencies of palmar patterns and accessory triradii in the IV interdigital area, and PD patients had significantly higher frequencies of complete simian creases and of palmar patterns in the thenar/I interdigital area than unaffected controls. The frequencies of the remaining dermatoglyphic traits showed no significant differences. We conclude that none of the marker systems tested show a particular pattern of association in patients and controls or a genetic predisposition to either disorder, and that early identification of at-risk individuals remains elusive.

Search for a red cell enzyme or serum protein marker in amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

Frequency distributions were determined for 24 red cell enzyme and four serum protein systems, in an attempt to identify a genetic marker associated with either amyotrophic lateral sclerosis (ALS) or parkinsonism-dementia (PD), two progressive and fatal neurological disorders of unknown cause found with unusually high incidence among the Chamorros of Guam and the Northern Mariana Islands. No striking associations were identified between either disorder and any of the gene markers tested. Thus, no genetic cause is known for either disease; local environmental factors are most likely involved in pathogenesis.

The Cu/Zn superoxide dismutase gene in ALS and parkinsonism-dementia of Guam.

Guam is one of three endemic foci whose indigenous (Chamorro) people have an unusually high incidence of fatal neurodegenerative disorders, amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia (PD). Recently, mutations in the Cu/Zn superoxide dismutase (SOD-1) gene have been identified in some familial cases of ALS. To investigate if mutations in the SOD-1 gene are also involved in the pathogenesis of ALS and PD of Guam, we analyzed the SOD-1 gene in Chamorros. No mutations were found in Chamorros with ALS or PD, indicating that mutations in the SOD-1 gene do not underlie the high-incidence neurodegenerative disorders of Guam.

The distribution and prevalence of group A arbovirus neutralizing antibodies among human populations in Southeast Asia and the Pacific islands.

Plaque reduction neutralization tests, using five group A arboviruses (chikungunya, Ross River, Getah, Bebaru and Sindbis), were done on sera from human populations in 44 Southeast Asia and Pacific island localities. Specificity of the plaque neutralization test was determined by examining convalescent sera from patients with known alphavirus infections. Chikungunya-specific neutralizing antibodies were demonstrated in sera of persons living in South Vietnam, Northern Malaysia, Indonesia (Kalimantan and Sulawesi), as well as Luzon, Marinduque, Cebu and Mindanao islands in the Philippines. Evidence of Ross River virus infection was found among populations living in West New Guinea and Papua New Guinea mainland, the Bismark Archipelago, Rossel Island and the Solomon Islands. There appeared to be no geographic overlap in the distribution of chikungunya and Ross River viruses, with the separation in their distribution corresponding with Weber's line in the Pacific. Sindbis neutralizing antibodies were found in 7 of 21 populations sampled, but in general the prevalence of infection was low. Four sera, from Vietnam, Malaysia and Mindanao gave monospecific reactions with Getah virus. No evidence of specific Bebaru virus infection was detected. The epidemiology of these five alphaviruses in Southeast Asia and the Pacific islands is discussed.

Human T-lymphotropic virus type I infection in the Solomon Islands.

To ascertain the prevalence of human T-lymphotropic virus type I (HTLV-I) infection and the occurrence of diseases caused by HTLV-I in the Solomon Islands, we tested 1141 sera from 851 patients (317 females and 534 males), who were hospitalized at the Central Hospital in Honiara between February 1984 and November 1988, for antibodies to HTLV-I using an enzyme-linked immunosorbent assay (ELISA). Sera from 69 of 81 ELISA-positive patients and from 56 ELISA-negative patients were then tested by Western analysis. As verified by strict Western immunoblot criteria, the overall HTLV-I seroprevalence was 2.2% (19/851). Age- and gender-specific prevalence data indicated an age-related acquisition of infection with no sexual predominance. No diagnosis category was over-represented among the seropositive patients. HTLV-I-specific antibodies were found in serum and cerebrospinal fluid samples from one of six patients with spastic paraparesis. As in other Melanesian populations, the majority of ELISA-positive sera could not be confirmed by Western analysis. Reactivity to three or more gag-encoded proteins was found in 85% (45/53) of ELISA-positive, Western blot-indeterminate sera, and 30% (16/53) reacted to p19 and an env gene product but lacked reactivity to p24. Whether or not the high frequency of indeterminate HTLV-I Western immunoblots in the Solomon Islands is indicative of incomplete specific reactivity to HTLV-I or the existence of antigenically related retroviruses is being investigated.

Slow dendritic transport of dissociated mouse hippocampal neurons exposed to aluminum.

We determined the influence of aluminum on dendritic transport, using an in vitro system of dissociated mouse hippocampal neurons. Newly synthesized RNA from dissociated mouse hippocampal neurons was more slowly transported into dendrites in the presence of aluminum chloride when compared to those without the addition of aluminum chloride to the culture medium. Suppression of dendritic transport of newly synthesized RNA may be responsible for the dendritic degeneration observed in aluminum neurotoxicity, eventually leading to neuronal degeneration.

Thiamin mono- and pyrophosphatase activities from brain homogenate of Guamanian amyotrophic lateral sclerosis and parkinsonism-dementia patients.

Thiamin-pyrophosphatase (TPPase) and thiamin-monophosphatase (TMPase) were determined using a spectrophotometric method at various pH values (5.5, 7.5, and 9.0) in brain tissue obtained at autopsy from amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) patients from Guam and from Guamanian patients who died from other diseases (controls). TPPase separation by thin-layer polyacrylamide gel isoelectric focusing (IEF) was also performed using both gray and white matter. TPPase content, chemically determined at pH 9.0, was found to be significantly reduced in the frontal cortex of ALS and PD patients compared to controls. TMPase content, on the contrary, was unchanged. IEF analysis showed 9 clear-cut bands with TPPase activity in the pH range 5.4-7.2 and a broad band at pH 4.7-5.2. The enzymatic activity was higher in gray than in white matter. In one patient the pattern was clearly different, with two additional bands observed at pH 7.1 and 6.7, and thought to be due to genetic microheterogeneity.

Pacific paradigms of environmentally-induced neurological disorders: clinical, epidemiological and molecular perspectives.

During the past quarter century biomedical scientists have begun to recognize the unique opportunities for studying disease etiology and mechanisms of pathogenesis in non-Western anthropological populations with focal, endemic diseases. Such natural experiments as they are called, are important paradigms for solving etiological and epidemiological problems of widespread medical significance, with an ultimate goal towards treatment and prevention. The systematic search for etiological factors and mechanisms of pathogenesis of neurodegenerative disorders is perhaps nowhere better exemplified than in the western Pacific. During the past three decades, the opportunistic and multidisciplinary study of hyperendemic foci of amyotrophic lateral sclerosis and parkinsonism-dementia which occur in different cultures, in different ecological zones and among genetically divergent populations have served as natural models that have had a major impact on our thinking and enhanced our understanding of these and other neurodegenerative disorders such as Alzheimer disease and the process of early neuronal aging. Our cross-disciplinary approach to these intriguing neurobiological problems and the accumulated epidemiological, genetic, cellular and molecular evidence strongly implicates environmental factors in their causation, specifically the role of aluminum and its interaction with calcium in neuronal degeneration. As a direct consequence of our studies in these Pacific populations, we have undertaken the long-term development of experimental models of neuronal degeneration, in an attempt to understand the cellular and molecular mechanisms by which these toxicants affect the central nervous system. Our experimental studies have resulted in the establishment of an aluminum-induced chronic myelopathy in rabbits and the development of neurofilamentous lesions after low-dose aluminum administration in cell culture. These studies clearly demonstrate the philosophy that chronic rather than acute experimental models of toxicity are necessary in order to enhance our understanding of human neurodegenerative disorders with long-latency and slow progression. Finally, the ultimate significance of these Pacific paradigms may well depend on our ability to comprehensively evaluate and synthesize the growing body of relevant scientific data from other human disorders and from widely divergent academic fields, as well as our ability to recognize emerging new models in nature.

Potentiation in the neurotoxic induction of experimental chronic neurodegenerative disorders: N-butyl benzenesulfonamide and aluminum chloride.

Repeated monthly intracisternal inoculations of N-butyl benzenesulfonamide induced a chronic, slowly progressive myelopathy in young adult New Zealand white rabbits that was manifested by hyperreflexia, spasticity, hypertonia, gait impairment and altered tonic immobility responses. The neuropathological features consisted of scattered neuroaxonal spheroids, fusiform distention of the intramedullary portions of the spinal cord ventral roots and, as defined by microtubule-associated protein-2 (MAP 2) immunoreactivity, an initial distention and subsequent loss of dendritic processes in neurons of the nucleus motoris lateralis with the perikaryon of these cells remaining intact. A similar chronic progressive myelopathy was induced by repeated low dose intracisternal inoculations of aluminum chloride in New Zealand white rabbits. However, the neuropathological changes were more extensive and consisted of dendritic, axonal and perikaryal inclusions of phosphorylated and nonphosphorylated neurofilament localized to spinal motor neurons in the nucleus motoris medialis, substantia grisea intermedia and select brainstem nuclei with only minimal involvement of the nucleus motoris lateralis. The co-administration of these two neurotoxins over the course of 8 months induced striking behavioral changes as well as a fulminant myelopathy. This was accompanied by a loss of neuronal perikarya in the nucleus motoris accompanied by a loss of neuronal perikarya in the nucleus motoris lateralis and topographically extensive neocortical neurofilamentous degeneration. These features suggest that potentiation occurs when the two toxins are co-administered, a view supported by an estimation of the co-neurotoxicity coefficient (CNC greater than 1). Our results have implications for understanding human neurodegenerative disorders in which potentiation of insults may occur, producing a clinical and neuropathological disease state not expected from either agent alone.