Plaque Counts Research Articles

Male-specific RNA coliphages detected by plaque assay and RT-PCR in tropical river waters and animal fecal matter

Male-specific RNA coliphages (FRNA) have been recommended as indicators of fecal contamination and of the virological quality of water. In this study, 16 river water and 183 animal fecal samples were examined for the presence of FRNA coliphages by a plaque assay using Salmonella typhimurium WG49 and WG25 to differentiate between male-specific and somatic phages, a RNase spot test to differentiate between DNA and RNA phages and a reverse transcriptase-polymerase chain reaction (RT-PCR) for the specific identification of FRNA phages. The overall recovery rate for F-specific coliphages was 8.0%. (4.4% from animal fecal matter and 50% from river water samples). Plaque counts were generally low (< 6×102 pfu per g feces or ml water), with FRNA (6.5%) and Male-specific DNA coliphages (FDNA) (7.0%) phages occurring at almost equal frequencies. The RT-PCR was positive in all FRNA plaques and was able to identify FRNA phages in mixed populations of FRNA, FDNA and somatic phages.

Neuropathologic Correlates of Apathy in Alzheimer’s Disease

Apathy is the most commonly observed behavioral disturbance in Alzheimer’s disease (AD) and has been suggested to be frontally mediated. Neuritic plaque (NP) and neurofibrillary tangle (NFT) counts were performed for 8 brain regions in 29 subjects with definite AD. Neuropsychiatric Inventory (NPI) for autopsied subjects was obtained from questioning of caregivers of subjects included in the study. Chronic apathy and total NPI composite scores correlated with anterior cingulate NFT counts (r = 0.518, p = 0.01, and r = 0.438, p = 0.032). This analysis suggests that chronic apathy in AD correlates with a greater anterior cingulate NFT burden and that chronic behavioral changes are more reflective than acute changes of disease pathology.

Neuropathologic Correlates of Activities of Daily Living in Alzheimer Disease

Functional status, reflected by measures of activities of daily living (ADLs), deteriorates as Alzheimer disease (AD) progresses. Decline in activities of daily living may be mediated by executive and frontal lobe dysfunction. The objective of this study was to examine the relationship between activities of daily living and pathologic burden in Alzheimer disease. Twenty two subjects with definite Alzheimer disease were selected from the UCLA ADRC neuropathology database. A total activities of daily living score was derived from the Retrospective Collateral Dementia Interview-Revised (RCDI-R) questionnaire, which was administered to caregivers of autopsied subjects included in the study. Neuritic plaque (NP) and neurofibrillary tangle (NFT) counts were performed for 8 brain regions. There was a significant positive correlation between total activities of daily living score (higher scores indicate more disability) and mean neuritic plaques and neurofibrillary tangle counts (r = 0.671, P = 0.001, and r = 0.542, P = 0.009, resp), as well as CA1 and prosubiculum neuritic plaques and neurofibrillary tangle counts, right and left orbital frontal neuritic plaques counts, and occipital neuritic plaques count. Total activities of daily living score did not correlate with age at death, age at symptom onset, dementia duration, gender, or education. Deteriorating activities of daily living in Alzheimer Disease subjects correlate with greater overall pathologic burden and possibly selectively with involvement of the medial temporal, occipital, and orbital frontal regions.

Association of the endothelial nitric oxide synthase (ecNOS) gene polymorphism with carotid atherosclerosis in type 2 diabetes

This study was done to see whether 27-base pair repeats polymorphism in intron 4 of ecNOS gene is associated with carotid atherosclerosis in type 2 diabetic patients. The polymorphism was identified by polymerase chain reaction (PCR). Ultrasound parameters of carotid atherosclerosis were analyzed in relation to the genotype in 210 patients with type 2 diabetes. The ecNOS4a allele was detected in 34 (16.2%) of this study group. With the exception of the plaque count ( P = 0.069), all other parameters obtained by ultrasound examination of carotid arteries were significantly correlated with presence of ecNOS4a allele ( P < 0.05). As all the measured carotid parameters correlated well each other, we selected the total mean carotid IMT (intima-media thickness) value to be used for this analysis. In the multivariate analysis including several variables such as age, sex, hypertension, LDL cholesterol, waist–hip ratio, and fasting insulin, all determined to be significant by univariate analysis, ecNOS4a allele had a significant correlation with total mean IMT ( P < 0.001). In conclusion, the ecNOS4a allele is associated with carotid atherosclerosis in type 2 diabetic patients in Korea.

Alterations in zinc transporter protein-1 (ZnT-1) in the brain of subjects with mild cognitive impairment, early, and late-stage Alzheimer's disease.

Several studies show increased levels of zinc (Zn) in the Alzheimer's disease (AD) brain. More recently, alterations in synaptic Zn and Zn transporter proteins (ZnT) have been implicated in the accumulation of amyloid plaques in an animal model of AD. To determine if alterations in ZnT proteins are present in AD brain, we measured levels of ZnT-1, the protein responsible for export of Zn to the extracellular space in the amygdala (AMY), hippocampus/parahippocampal gyrus (HPG), superior and middle temporal gyrus (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of 19 AD and 14 age-matched control subjects. To determine if alterations of ZnT-1 occur early in the progression of AD, we analyzed protein levels in the HPG, SMTG and CER of 5 subjects with mild cognitive impairment (MCI), 5 subjects with early AD (EAD) and 4 appropriately age-matched controls. Western blot and dot-blot analysis showed statistically significant (p 0.05) elevations of ZnT-1 in AD AMY, HPG, and IPL and significantly depleted ZnT-1 in AD SMTG compared to age-matched control subjects. We also observed statistically significant elevations of ZnT-1 in the HPG of EAD subjects compared with controls. In contrast to late-stage AD subjects, ZnT-1 levels were significantly decreased in HPG of subjects with MCI and were significantly elevated in the SMTG of both MCI and EAD subjects compared with age-matched controls. Correlation analysis of ZnT-1 levels and senile plaque (SP) and neurofibrillary tangle (NFT) counts in the AMY and CA1 and subiculum of AD HPG showed a significant (p 0.05) positive correlation with SP counts and a trend towards a significant (p = 0.12) positive correlation with NFT counts in AMY. Overall, our results show alterations in one of the key proteins responsible for maintenance of Zn homeostasis early in the progression of AD suggesting that alterations in Zn balance could be involved in the pathogenesis of neuron degeneration and amyloid deposition in AD.

Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis

BackgroundRespiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in infants and young children. Although several experimental RSV vaccines are under investigation, immuno therapy is the only treatment currently available. In assessing the immunogenicity of various vaccine formulations, a plaque reduction neutralization assay for the evaluation of RSV neutralizing antibody has been widely used. The method produces reliable results, but it is tedious and labor intensive as it relies on manual counting by laboratory personnel. To facilitate evaluation of phase II and phase III vaccine clinical trials, a more rapid, reliable and efficient neutralization assay is needed.ResultsAn improved microneutralization assay for quantifying RSV neutralizing antibodies was developed using an ImmunoSpot® Series I Analyzer (Cellular Technology Ltd., Cleveland, OH) for automated plaque counting. The method is an improvement of the established classical microneutralization assay in which immunostained plaques on transparent tissue culture plates are counted manually under a dissecting microscope. Image analyzer technology allows for fully automated counting of plaques distributed throughout an entire well. Adjustments, such as the use of opaque tissue culture plates and the TMB substrate, True Blue™ (KPL, Gaithersburg, MD), were required to adapt the assay for optimal detection of plaques by the image analyzer. The suitability and the accuracy of the method for counting RSV plaques were determined by comparative testing of a reference serum and two control sera by manual and automated counting methods. The results showed that the two methods were highly correlated (R = 0.9580) and the titers generated by them were within two-fold.ConclusionOur results demonstrate that the semi-automated assay is rapid and reliable. It provides results within two fold to the classical plaque microneutralization assay and is readily applied to the evaluation of neutralizing antibody titers in sera obtained from epidemiology or vaccine clinical trials.

In VivoMagnetic Resonance Microimaging of Individual Amyloid Plaques in Alzheimer's Transgenic Mice

The ability to detect individual Alzheimer's amyloid plaques in vivo by magnetic resonance microimaging (MRI) should improve diagnosis and also accelerate discovery of effective therapeutic agents for Alzheimer's disease (AD). Here, we perform in vivo and ex vivo MRI on double transgenic AD mice as well as wild-type mice at varying ages and correlate these with thioflavin-S and iron staining histology. Quantitative counts of individual plaques on MRI increase with age and correlate with histologically determined plaque burden. Plaques 20 microm in diameter can be detected in AD mice as young as 3 months of age with ex vivo MRI. Plaques 35 microm in diameter can be detected by 9 months of age with in vivo MRI. In vivo MRI of individual Alzheimer's amyloid plaques provides a noninvasive estimate of plaque burden in transgenic AD mice that might be useful in assessing the efficacy of amyloid reduction therapies.

Lack of protective effect of estrogens on cerebral Aβ levels in intact female and male APP transgenic mice

AbstractIn previous experiments, treatment with either 17β‐estradiol or 17α‐estradiol decreased cerebral β‐amyloid peptide (Aβ) levels in ovariectomized female APPSWE transgenic mice, a mouse model for Alzheimer disease‐like pathology. However, no effect of ovariectomy itself on cerebral Aβ levels was observed. In the current study, intact female and male APPIND,SWE transgenic mice were treated chronically with oral 17β‐estradiol or 17α‐estradiol. These treatments did not reduce cerebral Aβ levels or amyloid plaque counts vs. vehicle treated mice. In fact, 17β‐estradiol treatment of the intact female mice led to a significant increase in cerebral Aβ levels. Drug Dev. Res. 66:136–141, 2006. © 2006 Wiley‐Liss, Inc.

Mixed plaques: Statistical evidence how plaque assays may underestimate virus concentrations

Even at very low concentrations human pathogenic viruses may result in infection and possibly subsequent disease. Ideally, viruses are quantified by use of cell culture assays to determine their infectivity. Plaque assays are common tools for enumeration of viruses in inocula and this process is straightforward when a plaque results from the offspring of a single infectious virus particle. In the course of a study on the usefulness of sewage monitoring for surveillance of polio-virus transmission, sewage samples containing a mixture of two live polio vaccine strains (type 1 and type 3) were analyzed. The total poliovirus concentration in plaque forming units (pfu) was estimated by means of a monolayer plaque assay on L20B cells. Subsequent typing of virus directly by neutralisation of virus from excised plaques revealed the occurrence of plaques containing both type 1 and type 3 virus. This means that there must be plaques that originate from more than one initial infectious virus particle. As a consequence, the estimated virus concentration is incorrect. We present statistical methods that utilize these mixed plaque counts to estimate the concentrations of either virus type in our sewage samples. We can also calculate a correction factor for the error in virus concentration, which would result from equating a pfu to a single infectious particle. Since many quantitative methods in microbiology are based on colony counts, we conclude that such counts should be interpreted with caution, especially when data are used in quantitative microbial risk assessment to estimate the public health impact.

Enhanced contrast of bacteriophage plaques in Salmonella with ferric ammonium citrate and sodium thiosulfate (FACST) and tetrazolium red (TZR)

Visualization of bacteriophage plaques may be enhanced by addition of ferric ammonium citrate and sodium thiosulfate (FACST) or 2,3,5-triphenyltetrazolium chloride (tetrazolium red, TZR) to the soft agar layer of a traditional bacteriophage plaque assay. Background color from these reagents improved contrast between clear plaques and turbid host lawns in trypticase soy agar (TSA) plates. Enhancement by FACST is based on reaction with hydrogen sulfide gas (H 2S) produced by some strains of bacteria and was tested here using H 2S+ and H 2S− strains of Salmonella enterica subsp. enterica with a bacteriophage ( Podoviridae) isolated from swine lagoon effluent. Only the H 2S+ strain produced dark brown-black color in FACST-amended agar. Both strains showed bright pinkish-red color in TZR-amended agar. Color intensity for both reagents decreased with decreasing concentrations of the reagents. Contrast in FACST-amended plates appeared greater than that with TZR, but diminished after 12 h, while contrast in TZR-amended plates remained constant. At the concentrations tested, neither reagent affected plaque counts in the H2S+ strain. The FACST should be useful in bacteriophage plaque assays with H 2S+ strains of Salmonella and other H 2S+ bacteria.

Memories of Biology, Mathematics and Ivan

AbstractThe plaque assay years at Pittsburgh that included efforts to optimize the detection for single immunoglobulin M (IgM) and IgG antibody‐producing cells are described. Sensitivity of the assay and precision of the plaque count was established, and a series of response curves were obtained that enabled to calculate the frequency of precursors and to identify the target of the antigen. If one ignored a number of anomalies of the curves and made the simple assumption that a single hit by antigen on a pre‐committed precursor yielded a clone of antibody‐forming cells, estimates of some 700 target cells (per mouse spleen) were obtained, figures not too far from the number that Ivan would later find.Years at the Paul Ehrlich Institute and visits to the Basel Institute for Immunology are described.

Early life linguistic ability, late life cognitive function, and neuropathology: findings from the Nun Study

The relationships between early life variables, cognitive function, and neuropathology were examined in participants in the Nun Study who were between the ages of 75 and 95. Our early life variable was idea density, which is a measure of linguistic ability, derived from autobiographies written at a mean age of 22 years. Six discrete categories of cognitive function, including mild cognitive impairments, were evaluated, using the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) battery of cognitive tests. Neuropathologic data included Braak staging, neurofibrillary tangle and senile plaque counts, brain weight, degree of cerebral atrophy, severity of atherosclerosis, and the presence of brain infarcts. Early-life idea density was significantly related to the categories of late-life cognitive function, including mild cognitive impairments: low idea density was associated with greater impairment. Low idea density also was significantly associated with lower brain weight, higher degree of cerebral atrophy, more severe neurofibrillary pathology, and the likelihood of meeting neuropathologic criteria for Alzheimer's disease.

Right prosubiculum amyloid plaque density correlates with anosognosia in Alzheimer’s disease

Background: Anosognosia is a common manifestation of Alzheimer’s disease. There is an association between impaired awareness and frontal-executive cognitive deficits. Anosognosia is also correlated with decreased metabolism in the right...

A simplified plaque assay for respiratory syncytial virus—direct visualization of plaques without immunostaining

Due to their generally small size, detection of respiratory syncytial virus (RSV) plaques commonly relies on immunostaining using either polyclonal antisera or a pool of monoclonal antibodies against the surface fusion glycoprotein. Disadvantages include the costs of the antibodies and substrate, and microscopic examination is often needed for counting of plaques. By optimizing many parameters, greatly improved plaque assays for RSV A2, Long and RSV B 18537 strains using Vero or HEp-2 cells have been developed. Although many groups use Vero cells for plaquing, after optimization plaques were much larger in HEp-2 cells. Both cell types needed to be confluent at infection and agarose was more suitable than carboxymethyl cellulose for the overlay. Overlay medium for HEp-2 was DMEM/F12, 0.3% agarose, and for Veros it was Liebovitz L15, 5% fetal calf serum, 0.3–0.5% agarose. After 5–7 days, monolayers were fixed with 1% formal saline overnight, agarose was removed and monolayers were stained with 0.05% neutral red in water. Plaques were readily visible by eye and plates could be dried and stored permanently. Parainfluenza virus type 3 also formed large plaques in HEp-2 cells under the conditions optimal for RSV.

A cluster analysis model for caries risk assessment

Cluster analysis was applied to determine, the natural grouping of individuals, among sixty 8–10-year-old children, and to identify the most significant set of markers for risk assessment. The risk clusters were obtained with initial clinical and bacteriological measurements including dmf + DMFS, active caries, mutans streptococci and lactobacilli counts in plaque or saliva on two media, and Snyder’s test results. The morbidity clusters were constructed with the final clinical indexes and incidence after 18 months (dependent variables). A risk cluster was identified that included the following significant initial variables; dmf + DMFS, active caries, counts of mutans streptococci from plaque on TSY20B and lactobacilli in saliva, and Snyder’s test results. This set of markers identified 86% of the children at high risk who developed high morbidity, as well as 94% of children in the low-risk cluster who developed low or no caries. The results of this investigation provide the basis to develop a system for caries risk assessment.

Characterization of foot-and-mouth disease serotype Asia1 viruses grown in the presence of polyclonal antisera in serology and nucleotide sequence analysis

Foot-and-mouth disease viruses (FMDV) have a high rate of mutation and spontaneous mutants can be readily. isolated in the laboratory. In this study, plaque purified FMDV Asial vaccine strains (IND 63/72 and IND 491/97) were passaged in-vitro in Baby Hamster Kidney-21 cell monolayers in the presence of sub-neutralizing levels of antiviral polyclonal sera (APS), raised in guinea pigs against the purified and inactivated whole virus particles of IND 63/72, IND 491/97 and IND 13/01. After serial passages under selective immune pressure, the viruses starts growing in the presence of undiluted sera and showed certain characteristics like an increased resistance to neutralization by APS and reduction in plaque counts on titration in plaque assay. Cross-neutralization of these viruses with above-mentioned APS revealed selection of three complete and one partial polyclonal antibody resistant (PAR) viruses based on the 'r' value in micro neutralization test. Alterations were detected at several amino acid residues in the structural protein-coding P1 region. Many of the residues inferred to be positively selected sites in other serotypes of this virus were also prone to substitution under immune selection pressure in Asia1 virus. The present work extends the finding that selection exerted by host antibody also plays a major role in the rapid evolution of FMDV Asia1, as observed in other serotypes.

Estrogen treatment improves spatial learning in APP + PS1 mice but does not affect beta amyloid accumulation and plaque formation

We investigated the effects of ovariectomy (OVX) and 17β-estradiol (0.18 mg per pellet) treatment on spatial learning and memory, hippocampal beta amyloid (Aβ) levels, and amyloid plaque counts in double transgenic mice (A/P) carrying mutated amyloid precursor protein (APPswe) and presenilin-1 (PS1-A246E). After OVX at 3 months of age, the mice received estrogen treatment for the last 3 months of their lifetime before they were killed at 6, 9, or 12 months of age. Estrogen treatment in A/P OVX mice increased the number of correct choices in a position discrimination task in the T-maze, and slightly improved their performance in a win-stay task (1/8 arms baited) in the radial arm maze (RAM). However, estrogen treatment did not reverse the Aβ-dependent cognitive deficits of A/P mice in the water maze (WM) spatial navigation task. Furthermore, ovariectomy or estrogen treatment in OVX and sham-operated A/P mice had no effect on hippocampal amyloid accumulation. These results show that the estrogen treatment in a transgenic mouse model of Alzheimer's disease (AD) improves performance in the same learning and memory tasks as in the normal C57BL/6J mice. However, the estrogen effects in these mice appeared to be unrelated to Aβ-induced cognitive deficits. Our results do not support the idea that estrogen treatment decreases the risk or alleviates the symptoms of Alzheimer's disease by inhibiting the accumulation of Aβ or formation of amyloid plaques.

Brain aging in acquired immunodeficiency syndrome: increased ubiquitin-protein conjugate is correlated with decreased synaptic protein but not amyloid plaque accumulation.

Two neuropathological changes that are linked with biological and pathological aging were examined in subjects with end-stage acquired immunodeficiency syndrome (AIDS). Autopsy brain specimens were examined from 25 people who died from complications of AIDS and 25 comparison subjects who were human immunodeficiency virus (HIV)-negative, matched for age, gender, ethnicity, and postmortem time interval. These adults were stratified into three age groups: elderly (62 to 75 years), intermediate (55 to 60 years), and young (21 to 42 years). Ubiquitin-stained dotlike deposits (Ub-dots) and diffuse extracellular plaques containing the beta-amyloid (Abeta) fragment of the amyloid precursor protein (Abeta plaque) were both increased significantly in the hippocampal formation of older subjects. In subjects with AIDS, Ub-dots were increased whereas Abeta plaque counts were not significantly different. Western blotting confirmed that high-molecular-weight ubiquitin-protein conjugates (HMW-Ub-conj) were increased in AIDS. The band intensity of one HMW-Ub-conj species with an approximate molecular mass of 145 kDa was correlated significantly with increased acute phase inflammatory protein (a-1-antichymotrypsin) and decreased synaptophysin and growth-associated protein-43 band intensities. These results raise the possibility that HIV-related brain inflammation disturbs neuronal protein turnover through the ubiquitin-proteasome apparatus, and might increase the prevalence of age-associated neurodegenerative diseases by decreasing synaptic protein turnover through the proteasome.

Effects of Feeding Lactobacillus spp. on the Level of Cell Glutathione Sulphydryl and Immunoglobulin M in ICR Mice

Effects of feeding seven strains of Lactobacillus spp. on the level of cell glutathione sulphydryl (GSH) in spleen, liver and erythrocyte of the ICR mice and on the level of immunoglobulin M in the spleen were determined. The level of cell glutathione sulphydryl in the spleen was dependent on the strain of Lactobacilli, it was significantly higher in the mice fed with L. casei CU 001, L. rhamnosus CU 02, L. acidophilus NCFM and L. casei YIT9018 (p>0.05). The level of cell glutathione sulphydryl in the liver increased in the mice fed with L. casei YIT9018, L. acidophilus NCFM, L. casei CU 001 (p>0.05), the level of glutathione sulphydryl of the erythrocyte showed significantly higher value than control mice when fed with L. acidophilus NCFM, L. casei YIT9018, L. casei CU 001 (p>0.05). The level of immunoglobulin M in the spleen of ICR mice expressed as the plaque count revealed significantly higher value than the control mice when fed with L. casei CU 001, L. acidophilus NCFM and L.casei YIT 9018.

Assays for B Lymphocyte Function

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation.