TDP-43 proteinopathy in aging: Associations with risk-associated gene variants and with brain parenchymal thyroid hormone levels.

TDP-43 proteinopathy is very prevalent among the elderly (affecting at least 25% of individuals over 85 years of age) and is associated with substantial cognitive impairment. Risk factors implicated in age-related TDP-43 proteinopathy include commonly inherited gene variants, comorbid Alzheimer's disease pathology, and thyroid hormone dysfunction. To test parameters that are associated with aging-related TDP-43 pathology, we performed exploratory analyses of pathologic, genetic, and biochemical data derived from research volunteers in the University of Kentucky Alzheimer's Disease Center autopsy cohort (n = 136 subjects). Digital pathologic methods were used to discriminate and quantify both neuritic and intracytoplasmic TDP-43 pathology in the hippocampal formation. Overall, 46.4% of the cases were positive for TDP-43 intracellular inclusions, which is consistent with results in other prior community-based cohorts. The pathologies were correlated with hippocampal sclerosis of aging (HS-Aging) linked genotypes. We also assayed brain parenchymal thyroid hormone (triiodothyronine [T3] and thyroxine [T4]) levels. In cases with SLCO1A2/IAPP or ABCC9 risk associated genotypes, the T3/T4 ratio tended to be reduced (p = .051 using 2-tailed statistical test), and in cases with low T3/T4 ratios (bottom quintile), there was a higher likelihood of HS-Aging pathology (p = .025 using 2-tailed statistical test). This is intriguing because the SLCO1A2/IAPP and ABCC9 risk associated genotypes have been associated with altered expression of the astrocytic thyroid hormone receptor (protein product of the nearby gene SLCO1C1). These data indicate that dysregulation of thyroid hormone signaling may play a role in age-related TDP-43 proteinopathy.

Genomics and CSF analyses implicate thyroid hormone in hippocampal sclerosis of aging.

We report evidence of a novel pathogenetic mechanism in which thyroid hormone dysregulation contributes to dementia in elderly persons. Two single nucleotide polymorphisms (SNPs) on chromosome 12p12 were the initial foci of our study: rs704180 and rs73069071. These SNPs were identified by separate research groups as risk alleles for non-Alzheimer's neurodegeneration. We found that the rs73069071 risk genotype was associated with hippocampal sclerosis (HS) pathology among people with the rs704180 risk genotype (National Alzheimer's Coordinating Center/Alzheimer's Disease Genetic Consortium data; n = 2113, including 241 autopsy-confirmed HS cases). Furthermore, both rs704180 and rs73069071 risk genotypes were associated with widespread brain atrophy visualized by MRI (Alzheimer's Disease Neuroimaging Initiative data; n = 1239). In human brain samples from the Braineac database, both rs704180 and rs73069071 risk genotypes were associated with variation in expression of ABCC9, a gene which encodes a metabolic sensor protein in astrocytes. The rs73069071 risk genotype was also associated with altered expression of a nearby astrocyte-expressed gene, SLCO1C1. Analyses of human brain gene expression databases indicated that the chromosome 12p12 locus may regulate particular astrocyte-expressed genes induced by the active form of thyroid hormone, triiodothyronine (T3). This is informative biologically, because the SLCO1C1 protein transports thyroid hormone into astrocytes from blood. Guided by the genomic data, we tested the hypothesis that altered thyroid hormone levels could be detected in cerebrospinal fluid (CSF) obtained from persons with HS pathology. Total T3 levels in CSF were elevated in HS cases (p < 0.04 in two separately analyzed groups), but not in Alzheimer's disease cases, relative to controls. No change was detected in the serum levels of thyroid hormone (T3 or T4) in a subsample of HS cases prior to death. We conclude that brain thyroid hormone perturbation is a potential pathogenetic factor in HS that may also provide the basis for a novel CSF-based clinical biomarker.

The Antiphagocytic Activity of SeM of Streptococcus equi Requires Capsule.

Resistance to phagocytosis is a crucial virulence property of Streptococcus equi (Streptococcus equi subsp. equi; Se), the cause of equine strangles. The contribution and interdependence of capsule and SeM to killing in equine blood and neutrophils were investigated in naturally occurring strains of Se. Strains CF32, SF463 were capsule and SeM positive, strains Lex90, Lex93 were capsule negative and SeM positive and strains Se19, Se1-8 were capsule positive and SeM deficient. Phagocytosis and killing of Se19, Se1-8, Lex90 and Lex93 in equine blood and by neutrophils suspended in serum were significantly (P ≤ 0.02) greater compared to CF32 and SF463. The results indicate capsule and SeM are both required for resistance to phagocytosis and killing and that the anti-phagocytic property of SeM is greatly reduced in the absence of capsule.

Seroprevalence of Leptospira borgpetersenii serovar javanica infection among dairy cattle, rats and humans in the Cauvery river valley of southern India.

Leptospirosis is a major problem of dairy farms in Tamilnadu, India, resulting in abortions, stillbirths and infertility. Serologic and genetic analyses of samples from cattle, humans and rodents were performed in order to estimate infection prevalence and identify leptospiral species. Five hundred and fifteen sera and 76 urine samples were collected from dairy cattle on 25 farms including a farm that practiced rat control. Sera and kidney samples were also collected from field rats (Rattus norvegicus) in the vicinity of these farms. In addition, sera were collected from farm workers. Serum antibody was measured by the microscopic agglutination test. Leptospires isolated from blood, kidney, and urine were characterized as to serovar. Genomospecies were predicted using random amplified polymorphic DNA (RAPD) profiling. SecY gene sequencing was performed as a tool for tracing of source. Seroprevalence of 87.%, 51.% and 76.5% for cattle, rats and humans, respectively, was observed on endemic farms. Prevalences on a non-endemic farm were lower. Antibodies to Autumnalis, Javanica, Icterohaemorrhagiae and Pomona predominated in both cattle and rats. Thirteen isolates from rat kidneys were identified as serogroup Javanica, serovar Javanica. RAPD comparisons and secY gene sequencing identified these isolates as Leptospira borgpetersenii. These results altogether indicated that L. borgpetersenii was the dominant species in these areas with serovar Javanica apparently derived from rats which provided an important source of infection in cattle resulting a high incidence of infertility, abortion and.still-birth in the Cauvery river valley, Tiruchirappalli, Tamilnadu.

Alzheimer Disease Pathology-Associated Polymorphism in a Complex Variable Number of Tandem Repeat Region Within the MUC6 Gene, Near the AP2A2 Gene.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis.

Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection.

Comparison of specificities of serum antibody responses of horses to clinical infections caused by Streptococcus equi or zooepidemicus.

PROBLEM ADDRESSED: Streptococcus zooepidemicus (Sz) and its clonal derivative Streptococcus equi (Se) share greater than 96% DNA identity and elicit immune responses to many shared proteins. Identification of proteins uniquely targeted by the immune response to each infection would have diagnostic value. OBJECTIVE: The aim of the study was to compare serum antibody responses of horses infected by Se or Sz. METHODS AND APPROACH: Antibody levels were measured to panels of recombinant proteins of Sz and Se in sera of horses and ponies before and after experimental and naturally occurring invasive infections by these organisms. Antibody responses to an Se extract vaccine were also measured. Sera diluted 1:200 were assayed in triplicate using optimum concentrations of 9 and 14 immunoreactive proteins of Se and Sz, respectively. Bound IgG was detected using HRP-Protein G conjugate. RESULTS: Antibodies specific for SeM-N2, IdeE2, Se42.0 and Se75.3 (SEQ2190) were elicited by Se but not by Sz infection. Commercial Se extract vaccine did not elicit responses to IdeE2 or Se75.3. Sz infections resulted in significant (p<0.01) responses to Sz115, SzM, ScpC, SzP, MAP and streptokinase an indication these proteins are expressed during opportunistic invasions of the respiratory tract. FSR and HylC specific responses were unique to infections by Sz. CONCLUSIONS: The data indicate antibodies to IdeE2, Se75.3 and SeM-N2 may be used to distinguish infection by Se from that caused by the closely related Sz. Se infection, but not vaccination with Se extract elicits antibody to IdeE2 and Se75.3.

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.

A unique genotype of Leptospira interrogans serovar Pomona type kennewicki is associated with equine abortion.

Although serologic data indicate horses in N. America are exposed to a variety of leptospiral serovars, abortion is almost always associated with Leptospira interrogans serovar Pomona type kennewicki. A variety of wildlife including raccoons, white tailed deer, striped skunks, opossums, and red and grey foxes have been shown to host serovar Pomona and have therefore been suspect as sources of infection for pregnant mares. The aim of the present study was to examine genetic diversity in serovar Pomona type kennewicki in wildlife and in aborting mares. Our approach utilized PCR that targeted tandem repeats at the VNTR - 4 locus and a 1235 bp 5'-sequence of the lk73.5 (sph2) and adjacent upstream sequence unique to serovar Pomona. All isolates/specimens of equine origin in 1992 and 2008 yielded amplicons of 1235 and 595 bp, whereas 14 isolates/specimens from wildlife yielding a 1235 bp amplicon characteristic of serovar Pomona produced amplicons of 1300, 550 bp (3), 1300 bp (10), or 595 bp (6) with the VNTR - 4 primer set. Wildlife therefore hosted at least three different genetic variants of type kennewicki including the genetic variant that predominated in aborting mares. The data are consistent with other studies indicating specific genetic variants of type kennewicki show a strong tendency to be associated with a specific host. Levels of antibody in wildlife sera reactive with rLk73.5, rLig130 and sonicate of type kennewicki were poorly correlated with PCR data, although rLk73.5 was superior to rLig130 in detection of antibody responses. PCR is therefore a more reliable tool for studies of wildlife reservoirs of Leptospira sp. than serologic surveillance that targets host induced proteins or LPS-rich sonicate.

Immunisation of the equine uterus against Streptococcus equi subspecies zooepidemicus using an intranasal attenuated Salmonella vector.

Attenuated Salmonella enterica serovar Typhimurium MGN707, expressing the SzP protective protein of the MB9 serovar of Streptococcus equi subspecies zooepidemicus (SzP-MB9) was tested for its safety and efficacy as a nebulised intranasal vaccine against streptococcal uterine infections in mares. In a preliminary study, vaccinated mares (n=5) displayed serum, nasal and uterine responses (P<0.05) to S. Typhimurium lipopolysaccharide (St-LPS). Subsequently, vaccinated mares (expressor group, n=7), but not mares vaccinated with the vector only (control group, n=7), displayed significant increases in SzP-MB9 antibodies in serum, nasal and uterine washes (P<0.05). Assuming the uteri of all nine mares were free of streptococci prior to challenge with 6.3 x 10(9) colony forming units of S. e. zooepidemicus MB9, significantly fewer S. e. zooepidemicus were cultured from the uterine flushings of expressor-vaccinated mares (n=4) compared to control-vaccinated mares (n=5) (P<0.001). The only adverse reaction to vaccination was nasal haemorrhage in one mare.