Polymerase chain reaction-based national surveillance programme to determine the distribution and prevalence of Taylorella equigenitalis in South African horses.

REASONS FOR PERFORMING STUDY: The response to the first outbreak of contagious equine metritis in South Africa included pioneering a web-based platform to coordinate key aspects of a national, real-time polymerase chain reaction (qPCR)-based stallion screening programme to determine the distribution and prevalence of Taylorella equigenitalis in stallions and exposed mares. OBJECTIVES: To define the hypothesised pre-existing status of T. equigenitalis in the South African equine population and progression of the epidemiological investigation via the implementation of a molecular diagnostic-based surveillance programme. STUDY DESIGN: Retrospective case series. METHODS: Screening for T. equigenitalis was via a qPCR assay on genital swabs obtained from predilection sites in stallions and mares with subsequent confirmation using bacterial culture according to prescribed methods. RESULTS: The initial outbreak investigation identified 4 horses including the index stallion and mare. Traceback of in-contact horses identified 26 horses, including a subpopulation focus at the South African Lipizzaner Centre where 24/33 resident stallions tested positive for T. equigenitalis on qPCR. The national screening programme identified an additional 9 stallions. A total of 39 horses (36 stallions and 3 mares) tested positive for T. equigenitalis by qPCR and T. equigenitalis was isolated from 23 of these stallions and 2 of these mares. In addition to the index property, an artificial breeding centre where the index case was first identified, an additional 12 properties with infected horses were identified in 3/9 provinces. Horses on 11 of these 12 properties were directly linked to the index property. Two incidents of T. equigenitalis transmission associated with artificial insemination were recorded. CONCLUSIONS: T. equigenitalis was present in a subpopulation focus within the South African horse population prior to the outbreak identification in April 2011. Horizontal fomite-associated spread was the most probable route of transmission between stallions. The targeted surveillance of stallions and exposed mares using a qPCR-based screening programme expedited investigation of the distribution and prevalence of T. equigenitalis infection in South African horses. The application of qPCR provided a sensitive and practical screening test for identification of T. equigenitalis-positive animals as part of an emergency response to the first identified cases of T. equigenitalis infection in South African horses.

Inhibitory control across the life span.

Findings from previous research suggest that inhibitory control improves during early childhood and declines during late adulthood. Very few researchers, however, have examined life-span changes in this ability in single studies. Within this life-span context, we investigated 1 type of inhibitory control--the ability to inhibit aprepotent response and generate an incompatible response--in individuals ranging from 6 to 82 years of age. Examination of raw reaction time data revealed a significantly larger inhibitory control effect for children and older adults than for young adults. Using proportional and z score transformations, we demonstrated that a processing speed explanation is sufficient to account for the differences in performance between children and young adults; this explanation, however, did not adequately explain the discrepancy between young and older adults. Taken together, these findings suggest that, above and beyond differences in processing speed, inhibitory control was less efficient in older adults. Our findings are consistent with the assertion that inhibitory control develops quite early and declines at the later end of the developmental spectrum.

Context processing in older adults: evidence for a theory relating cognitive control to neurobiology in healthy aging.

A theory of cognitive aging is presented in which healthy older adults are hypothesized to suffer from disturbances in the processing of context that impair cognitive control function across multiple domains, including attention, inhibition, and working memory. These cognitive disturbances are postulated to be directly related to age-related decline in the function of the dopamine (DA) system in the prefrontal cortex (PFC). A connectionist computational model is described that implements specific mechanisms for the role of DA and PFC in context processing. The behavioral predictions of the model were tested in a large sample of older (N = 81) and young (N = 175) adults performing variants of a simple cognitive control task that placed differential demands on context processing. Older adults exhibited both performance decrements and, counterintuitively, performance improvements that are in close agreement with model predictions.

Exploring the relationship between age, executive abilities, and psychomotor speed.

Age-related declines in executive abilities have been widely reported and are thought to result from neuropathological changes in the prefrontal cortex. Some investigators have suggested that age-related changes in cognition may be the result of slowed information processing speed rather than declines in specific cognitive abilities. We examined the relationships among age, executive abilities, and psychomotor speed in 40 older adults and 46 young adults. Both verbal and nonverbal tasks were administered that measured 2 aspects of executive ability: set formation and set shifting. Executive and psychomotor speed tasks were paired based on similarities in basic task demands. Our results revealed that poorer executive performance was associated with increasing age. Further, although psychomotor speed attenuated the relationship, age accounted for a unique and significant proportion of variance in executive performance after controlling for psychomotor speed. These results suggest that age has an effect on prefrontally mediated executive abilities that cannot be explained solely in terms of psychomotor slowing.

Identification of a novel receptor kinase that phosphorylates a phospholipase C-linked muscarinic receptor.

Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied beta-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by beta-adrenergic receptor kinase ( beta-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for beta-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C-linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser345-Leu463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30-60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparin-Sepharose. The purified protein kinase represented an approximately 18,600-fold purification and was a single polypeptide with a molecular weight of approximately 40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, beta-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.

Molecular determinants of the V3 loop of human immunodeficiency virus type 1 glycoprotein gp120 responsible for controlling cell tropism.

We and others have identified the major determinant of cell tropism in human immunodeficiency virus type 1 (HIV-1) as the V3 loop of glycoprotein gp120. We have conducted a detailed study of two molecularly cloned isolates of HIV-1, HIVJR-CSF and HIVNL4-3, that differ in their tropism for immortalized CD4+ cell lines, by constructing a series of site-directed mutations within the V3 loop of HIVJR-CSF based on the sequence of HIVNL4-3. The phenotypes of these mutants fall into two classes, those which are viable and those which are not. A spontaneous mutant with significantly altered growth properties was also recovered and found to have an additional single amino acid change in the V3 loop sequence. The carboxy-terminal beta-strand part of the V3 loop is the major determinant of cell tropism. However, the results presented here indicate that the functional role of the V3 loop sequences can only be interpreted properly in the context of the original gp120 backbone from which they were derived. These findings show that over-simplistic interpretation of sequence data derived from unknown mixtures of HIV variants in infected persons may be highly misleading.

Phosphorylation of a phosphoinositidase C-linked muscarinic receptor by a novel kinase distinct from beta-adrenergic receptor kinase.

Muscarinic receptor kinase activity previously described in intact CHO cells transfected with human m3-muscarinic receptor cDNA (CHO-m3 cells) [Tobin, A.B and Nahorski, S.R (1993) J. Biol. Chem. 268, 9817-9823] was found to be associated, at least in part, with a crude membrane fraction of CHO-m3 cell lysates. Phosphorylation of the m3-muscarinic receptor was agonist dependent, reaching a maximum after 10 min exposure to carbachol (1 mM) and was completely blocked by atropine (10 microM). m3-Muscarinic receptor phosphorylation was insensitive to Zn2+ (0.1 mM) and heparin (1 microgram/ml), concentrations that inhibit endogenous beta-adrenergic receptor kinase activity present in CHO-m3 cells strongly suggesting that the m3-muscarinic receptor kinase is distinct from beta-adrenergic receptor kinase. A role for protein kinase C can also be eliminated on the basis that the potent protein kinase C inhibitor, Ro-318220 (1 microM), had no effect on agonist-mediated m3-muscarinic receptor phosphorylation. Further, the inability of calcium (300 microM), cAMP (0.2 mM) and cGMP (0.2 mM) to elevate the basal phosphorylation state of m3-muscarinic receptors eliminates a role for protein kinases regulated by these second messengers. Finally, agonist mediated phosphorylation appears to be independent of G-protein activation as both GDP-beta-S (500 microM) and GTP-gamma-S (100 microM) did not influence m3-muscarinic receptor phosphorylation.

V3 sequences of paired HIV-1 isolates from blood and cerebrospinal fluid cluster according to host and show variation related to the clinical stage of disease.

The spread of HIV-1 to the nervous system occurs early during infection in a large number of asymptomatic virus carriers. In order to address the question whether the brain is targeted by a group of HIV-1 variants with increased neurotropic properties we have obtained 20 paired isolates from the blood and cerebrospinal fluid (CSF) of patients with varying severity of HIV-1 infection. We determined the nucleotide sequence of variable region 3 (V3) of the gp 120 envelope protein and studied the growth capacity of the virus isolates in primary monocyte/macrophage cultures. Blood and CSF V3 sequences could be grouped according to the host, whereas particular amino acid sequences related to tissue specificity were not identified. Moreover, the majority of HIV-1 isolates, independently of the tissue source, replicated in macrophage cultures. The isolates derived from the brain and blood compartments thus appeared genetically similar and could not be grouped according to their replicative capacities. The genetic distance between paired CSF and blood sequences was more pronounced in the group of patients with AIDS than in asymptomatic virus carriers. These observations indicate that the viruses present in the blood and CSF in the early stage of infection are genetically similar. Different mechanisms of adaptation or immunomodulation of the virus in CSF and blood may account for the increased intra-patient variability observed in the AIDS group.

Human immunodeficiency virus type 1 is present in the cerebrospinal fluid of a majority of infected individuals.

Cerebrospinal fluid (CSF) specimens from 63 patients with different severities of human immunodeficiency virus (HIV-1) infection, including asymptomatic virus carriers, were examined for the presence of HIV-1 by using polymerase chain reaction (PCR) and virus isolation. Polyadenylated RNA, presumably associated with virus particles, was extracted and reverse transcribed, and the pol region was amplified in a nested PCR. Virus could be detected in 90% of the CSF specimens examined by PCR, and data on isolation of virus from CSF were in agreement with these figures. In fact, when several CSF specimens from the same individual were studied, HIV-1 could be isolated from 80% of the patients. The presence of the viral RNA in CSF was independent of the clinical stage of infection and of neurological symptoms. These results show that the spread of HIV-1 to the brain represents an early event during infection and occurs in the majority of asymptomatic individuals.

Lack of autologous neutralizing antibodies in the cerebrospinal fluid of HIV-1 infected individuals.

The cerebrospinal fluids (CSF) and sera from HIV-1-infected individuals at different clinical stages were monitored for neutralizing activity against CSF-derived HIV-1 isolates. None of the CSF samples and only one of seven serum samples could neutralize the autologous CSF isolate. CSF samples collected one to two years later from the same patients also lacked autologous neutralizing antibodies against these isolates. However, some CSF samples were able to neutralize heterologous CSF isolates albeit in low titers. HIV antibody positive control sera could readily neutralize all of the CSF isolates demonstrating that these isolates were not resistant to neutralization per se. IgG antibodies against the HIV-1 envelope protein and, specifically, against the V3 loop of HIV-1 gp120 (MN) were present in some CSF samples, although the samples lacked neutralizing activity. In summary, this study demonstrates a lack of autologous neutralizing antibodies in CSF samples when assayed against CSF-derived HIV-1 isolates.

Brain-derived cells can be infected with HIV isolates derived from both blood and brain.

HIV type 1 and 2 isolates derived from brain and blood of infected individuals were used to infect astrocytic cells of tumor origin. Infection was monitored by polymerase chain reaction. The majority of the isolates infected the glioma cells, independently of the source of isolation. Added to the fact that the majority of primary HIV isolates infect cells of the monocyte/macrophage lineage, these results indicate that primary blood and brain HIV strains have similar target cells. The production of virus from infected astrocytes was detected only upon infection with two macrophage-adapted strains. Also in this case, the number of infected cells was very low and only one in 5000 cells carried the proviral HIV genome.

Simian immunodeficiency virus (SIVsm) isolation from blood and brain of experimentally infected macaques.

The possibility of using simian immunodeficiency virus (SIV)-infected macaques to study pathogenic events linked to HIV infection of the brain prompted us to investigate some of the virological features in SIV-infected macaques. Nine cynomolgus macaques were inoculated with SIVsm and killed at different times. We successfully isolated virus from the blood of all the animals and from the brains of eight. These results point to the early and regular spread of this lentivirus to the brain. Neutralizing activity was studied in the serum and cerebrospinal fluid specimens obtained from these macaques against a selected group of isolates. Cerebrospinal fluid did not show any neutralizing activity. Our findings integrate the observations from HIV-1 infection in man and indicate that SIV infection of macaques is a useful model for studying pathogenic events of brain infection.

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid.

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.

Development and validation of in vitro induction assays for toxic halogenated aromatic mixtures: a review.

Halogenated aromatic industrial compounds, typified by the polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) have been identified as residues in almost every component of the global ecosystem. Risk assessment of the complex mixtures of halogenated aromatics found in environmental samples is complicated by analytical problems and the lack of toxicological information on individual compounds and mixtures. Research in our laboratory has focused on the development and vadidation of the in vitro aryl hydrocarbon hydroxylase (AHH) induction assay in rat hepatoma H-4-II E cells in culture for quantitating individual toxic halogenated aryl hydrocarbons and their mixtures. For several PCB, PCDD, PCDF congeners, their mixed bromo/chloro analogs and reconstituted mixtures there was an excellent linear correlation between their -log ED50 values for AHH induction in rat hepatoma cells and their -log ED50 values for in vivo hepatic microsomal AHH induction, inhibition of body weight gain and thymic atrophy in the rat. It has also been shown for selected compounds that there was a good correlation between their in vitro AHH induction potencies and their effects in guinea pigs (AHH induction, inhibition of body weight gain) and mice (immunotoxicity). This assay system has been utilized to quantitative the "2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents" present in extracts from diverse sources including fly ash from a municipal incinerator and pyrolyzed brominated flame retardants which contain a complex mixture of halogenated dibenzo-p-dioxins and dibenzofurans.

Quantitative structure-activity relationships: analysis of interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2-substituted analogues with rat, mouse, guinea pig, and hamster cytosolic receptor.

The competitive receptor binding affinities of thirteen 2-substituted 3,7,8-trichlorodibenzo-p-dioxins to hepatic cytosol from rat, mouse, guinea pig, and hamster were determined with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin as the radioligand. Multiple parameter linear regression analysis of the binding data from the four species gave the following equations: pEC50 (rat) = 7.196 + 0.600 pi - 0.255 delta Es - 1.683 HB pEC50 (mouse) = 6.365 + 1.641 pi + 1.206 sigma 0 pEC50 (hamster) = 7.416 + 1.026 pi + 0.509 delta Es + 0.748 sigma 0 pEC50 (guinea pig) = 6.892 + 1.035 pi where pi, delta Es, HB, sigma 0, and Vw are physicochemical parameters for substituent lipophilicity, steric effect, hydrogen bonding capacity, electronegativity, and van der Waals volume (relative to H), respectively. These equations demonstrate that there are important species differences in the receptor protein binding site interactions with the substituted analogues. These data, coupled with the known species differences in the molecular properties of the receptor proteins, are evidence for a heterologous nature of the receptor between mammalian species. Multiple parameter linear regression analysis of the relative aryl hydrocarbon hydroxylase (AHH) induction potencies of these analogues in rat hepatoma H-4-II E-cells in culture gave the following equation. The correlation pEC50 (AHH induction) = 3.208 + 0.950 pEC50 (rat binding) - 0.955 delta B5 between receptor binding and AHH induction was dependent on a steric parameter (delta B5, STERIMOL) and the results suggest that an additional substituent-dependent process (e.g., an activation step) may be required after initial ligand-receptor binding for the ultimate expression of the receptor-mediated response (i.e., AHH induction).

The effects of organochlorine pesticides as inducers of testosterone and benzo[a]pyrene hydroxylases.

p,p'-DDE, phenobarbital, dieldrin heptachlor, chlordane and toxaphene induced rat liver microsomes exhibited increased formation of the 4,5-dihydrodiol, 3,6-quinone, 9- and 3-hydroxymetabolites of benzo[a]pyrene and the latter three compounds also induced an increase in the rate of formation of the 9,10-dihydrodiol metabolite. Lindane was inactive as an inducer of benzo[a]pyrene hydroxylase. With the exception of lindane, all the organochlorine pesticides and PB induced testosterone 16 alpha- and 16 beta-hydroxylases; in contrast lindane induced testosterone 6 alpha-, 7 alpha- and 6 beta-hydroxylases and PB also induced testosterone 15 beta-hydroxylase and androstenedione formation. Using a battery of monooxygenase enzyme assays it was evident that there were significant differences between PB and several organochlorine pesticides as inducers of rat hepatic cytochrome P-450-dependent monooxygenases.

The cytosolic receptor binding affinities and AHH induction potencies of 29 polynuclear aromatic hydrocarbons.

The dose-response rat hepatic cytosolic receptor-binding avidities, aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) induction potencies in rat hepatoma H-4-II E cells in culture were determined for 29 polynuclear aromatic hydrocarbons. It was apparent that the magnitude of the EC50 values for these in vitro responses were strongly dependent on structure. Dibenz[a,h]anthracene (1.6 X 10(-8) M), 7-methylbenz[a]anthracene (1.6 X 10(-8) M), 3-methylcholanthrene (2.8 X 10(-8) M) and picene (4.5 X 10(-8) m) exhibited the highest affinity for the receptor protein and these compounds were only 5-fold less active the 2,3,7,8-tetrachlorodibenzo-p-dioxin (1 X 10(-8) M). All of the compounds which were active in the receptor-binding and monooxygenase enzyme-induction assays possessed one common structural feature, namely the presence of a phenanthrene structure fused with at least 1 benzo ring. The results also demonstrated that there was not any apparent correlation between the receptor-binding avidities and in vitro monooxygenase enzyme-induction potencies for the most active polynuclear aromatic hydrocarbons.

Polychlorinated dibenzo-p-dioxins: quantitative in vitro and in vivo structure-activity relationships.

There were marked effects of structure on the activities of 14 polychlorinated dibenzo-p-dioxins (PCDDs) as competitive ligands for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor and as inducers of aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (EROD) in rat hepatoma H-4-II E cells in culture. 2,3,7,8-TCDD was the most active compound in both assays and several PCDD congeners which were fully substituted in the lateral 2, 3, 7 and 8 positions but also contained additional chlorosubstituents in non-lateral 1, 4, 6 and 9 positions were less active. It was also evident that there was a decrease in in vitro binding and induction activities with decreasing lateral chlorine substitution. Although comparable structure-activity relationships (SARs) for the PCDDs were observed for the induction and receptor binding assays, there was not a linear or rank order correlation between the 2 sets of data. Several in vivo biologic and toxic activities of 2,3,7-trichloro-, 2,3,7,8- and 1,3,7,8-tetrachloro-, 1,2,4,7,8- and 1,2,3,7,8-pentachloro- and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin were determined in a dose-response fashion in immature male Wistar rats. The ED50 values for hepatic microsomal AHH and EROD induction, body weight loss and thymic atrophy were obtained. There was an excellent linear correlation between the -log EC50 values for AHH or EROD induction in cell culture and the -log ED50 values for enzyme induction, body weight loss and thymic atrophy in the rat. The in vitro enzyme induction data could be used to quantitatively estimate the toxicity of the PCDD congeners in the rat: this latter correlation has previously been observed for a series of polychlorinated dibenzofurans.

Polychlorinated dibenzofurans as 2,3,7,8-TCDD antagonists: in vitro inhibition of monooxygenase enzyme induction.

2,4,6,8- and 1,3,6,8-tetrachlorodibenzofuran (TCDF) competitively displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from the rat cytosolic receptor protein and their EC50 values were 1.5 X 10(-6) and 1.25 X 10(-7) M, respectively. In contrast to their relatively high binding avidities these TCDF isomers were poor inducers of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase in rat hepatoma H-4-II E cells in culture (EC50 greater than 10(-5) M). Coadministration of different concentrations of 2,4,6,8- and 1,3,6,8-TCDF (10(-5), 10(-6) and 10(-7) M) with 2 X 10(-10) M, 2,3,7,8-TCDD (a dose which elicits 80% of the maximal induction response) resulted in significant decreases in the expected (additive) induction of benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase by the mixture. Thus the partial agonists, 1,3,6,8- and 2,4,6,8-TCDF, antagonize the receptor-mediated enzyme induction activity of 2,3,7,8-TCDD presumably via competitive displacement of 2,3,7,8-TCDD from the receptor protein. In contrast, coadministration of 2,3,7,8-TCDF and 2,3,7,8-TCDD gave additive enzyme induction responses. The identification of the 2,3,7,8-TCDD antagonists represents a new class of halogenated aryl hydrocarbons.

Modulation of rat hepatic microsomal testosterone hydroxylases by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related toxic isostereomers.

The effects of several toxic halogenated aryl hydrocarbons including 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, 2,3,4,7,8-pentachlorodibenzofuran, 3,3',4,4'-tetrabromobiphenyl, 3,3',4,4'5-pentachlorobiphenyl, and 3,3',4,4'-tetrachloroazobenzene on hepatic microsomal testosterone hydroxylases were determined in the immature male rat. All of these compounds induced hepatic testosterone 7 alpha-hydroxylase and inhibited testosterone 6 beta-, 16 alpha-, 16 beta-, and 2 alpha-hydroxylases and androstenedione formation. It was observed that there was a good correlation between the increase in testosterone 7 alpha-hydroxylase by the halogenated hydrocarbons and their ability to cause body weight loss in the exposed rats. Comparable linear correlations were observed between toxicity and the decreased activities of other testosterone hydroxylases. The role of altered testosterone metabolism in the toxicity of halogenated aryl hydrocarbons is unknown.