Proton Magnetic Resonance Spectroscopy Reveals Neuroprotection by Oral Minocycline in a Nonhuman Primate Model of Accelerated NeuroAIDS

Background: Despite the advent of highly active anti-retroviral therapy (HAART), HIV-associated neurocognitive disorders continue to be a significant problem. In efforts to understand and alleviate neurocognitive deficits associated with HIV, we used an accelerated simian immunodeficiency virus (SIV) macaque model of NeuroAIDS to test whether minocycline is neuroprotective against lentiviral-induced neuronal injury. Methodology/Principal Findings: Eleven rhesus macaques were infected with SIV, depleted of CD8+ lymphocytes, and studied until eight weeks post inoculation (wpi). Seven animals received daily minocycline orally beginning at 4 wpi. Neuronal integrity was monitored in vivo by proton magnetic resonance spectroscopy and post-mortem by immunohistochemistry for synaptophysin (SYN), microtubule-associated protein 2 (MAP2), and neuronal counts. Astrogliosis and microglial activation were quantified by measuring glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (IBA-1), respectively. SIV infection followed by CD8+ cell depletion induced a progressive decline in neuronal integrity evidenced by declining N-acetylaspartate/creatine (NAA/Cr), which was arrested with minocycline treatment. The recovery of this ratio was due to increases in NAA, indicating neuronal recovery, and decreases in Cr, likely reflecting downregulation of glial cell activation. SYN, MAP2, and neuronal counts were found to be higher in minocycline-treated animals compared to untreated animals while GFAP and IBA-1 expression were decreased compared to controls. CSF and plasma viral loads were lower in MN-treated animals. Conclusions/Significance: In conclusion, oral minocycline alleviates neuronal damage induced by the AIDS virus

Are Porphyromonas gingivalis Outer Membrane Vesicles Microbullets for Sporadic Alzheimer’s Disease Manifestation?

Our research into Alzheimer’s disease (AD) focuses on the oral cavity and the brain, from which key evaluations of prospective and retrospective population based data have shown that chronic periodontal disease existing for ten-years or over doubles the risk for the sporadic form of AD. Furthermore, Porphyromonas gingivalis mono-infections in established periodontal lesions, or introducing its lipopolysachharide (LPS), as demonstrated in vivo studies, show hallmark pathology inclusive of extracellular amyloid plaques and phospho-tau bound neurofibrillary tangles with AD-like phenotype. Other studies have shown that if periodontitis remains untreated in human AD patients, cognitive decline ensues. This is a bi-directional relationship meaning that the converse is also true; treating periodontal disease in AD patients improves memory. Bacterial cultures and established oral biofilms generate vast numbers of microvesicles and P. gingivalis outer membrane vesicles encase key virulence factors (LPS, gingipains, capsule, fimbriae) as though they are complete destructive “microbullets” when shed in the host. This provides P. gingivalis additional arsenal to manipulate its entry into disparate organs, hijack phagocytosis, destroy tissues, and affect complement related genes whilst transducing the onset of proinflammatory signalling cascades. The resulting inflammatory mediators may be the cause of disease defining lesions and cognitive decline typical of clinical AD

Response of diminazene-resistant and diminazene-susceptible Trypanosoma congolense to trreatment with diminazene when occuring as a mixed infection in goats.

The research described in this thesis was carried out to investigate the phenotypic basis of resistance to diminazene (Berenil R) in Trypanosoma congolense. Earlier work has shown that, in both goats and mice, the majority of T. congolense trypanosomes which reappear following treatment with diminazene aceturate are sensitive to the dosage that was used. Such a phenomenon could be due to the ability of sensitive trypanosomes to survive treatment when mixed ,with resistant trypanosomes. Thus, the work described here was carried out to establish whether a diminazene-sensitive clone of T. congolense could survive treatment with diminazene aceturate at a dose of 7.0 mg/kg b.w. when mixed with a diminazene-resistant clone in goats. Since the study used two Savannahtype clones of T. congolense, the work necessitated the development of a polymerase chain reaction (peR) technique that could differentiate the two clones of T. congolense . .The 2 clones of T. congolense that were used were T. congolense IL 1180 . (a derivative of STIB 212 which is sensitive in goats to intramuscular [i/m] treatment with diminazene aceturate at a dose of 7.0 mg/kg b.w.) and T. congolense IL 3274 (a derivative of IL 2865, isolated from a cow in Burkina Faso, which is resistant in goats to i/m treatment with diminazene aceturate at a dose of 7.0 mg/kg b.w.). In order to distinguish the 2 clones, a peR technique was developed which utili sed a DNA sequence that is present in IL 1180, but not in IL 3274. A pair of 20 nt primers were developed on the basis of DNA sequence information for the ends of the cloned DNA sequence. The primers were then shown to amplify a 900 bp sequence from the plasmid in which the gene was cloned (P1616/5), and from genomic DNA of IL 1180. However, a similar product was not produced with IL 3274 genomic DNA. In further work, the 900 bp product amplified by PCR from p1616/5 was purified and labelled with 32p, in order to generate a probe specific for the trypanosome-specific PCR product. Using the above reagents, the first study endeavoured to establish the minimum amount of IL 1180 DNA that could be detected when mixed with 25 ng of IL 3274 DNA. When using ethidium bromide-staining of PCR products in an agarose gel, the minimum level of detection was 100 pg of IL 1180 DNA However, when such a gel was blotted and the filter hybridized with the [32p]_ labelled 900 bp product, the sensitivity was increased by 100-fold (i.e., to 1 pg). In an experiment to determine whether the diminazene-sensitive clone (IL 1180) could survive treatment with diminazene aceturate when mixed with a resistant clone (IL 3274), 24 goats were randomised into 3 groups of 5 goats each (Groups A, Band C) and 3 groups of 3 animals each (Groups D, E and F). Groups A and D were infected with IL 1180; Groups Band E were infected with IL 3274; and Groups C and F were infected with both clones simultaneously. All animals were infected intravenously, via the jugular vein, and animals that were treated were administered diminazene aceturate i/m at a dose of 7.0 mglkg b.w. Animals in Groups A and B were treated after all the goats in the respective groups had been detected parasitaemic. In contrast, goats in Group -C were not treated until all the animals in both Groups A and B had been detected parasitaemic, thereby ensuring that both trypanosome clones in these animals had fully developed by the time of treatment. Groups D, E and F served as nontreatment controls and also facilitated a comparison of the pathogenicity of the 2 clones individually and when mixed. Following treatment, all goats in all groups were monitored 3 times a week for 84 days for their levels of anaemia and parasitaemia. During the entire experiment, trypanosome stabilates were collected as follows from all animals; stabilates of goat blood, once a week; parasitaemic mouse blood as a result of inoculation with goat blood, once every 2 weeks; buffy-coat preparations of parasitaemic goat blood, twice a week. All 5 goats infected with IL 1180 and treated with diminazene aceturate (Group A) did not develop a relapse infection for the entire 84 days following treatment. This was in contrast to goats infected with IL 3274 (Group B) and those infected with both clones (Group C) in which 5 out of 5 and 4 out of 5 relapses occurred, respectively. All the non-treatment control goats infected with IL 3274 developed a severe anaemia (packed Cell Volume (PCV]< 12%). along with a high level of parasitaemia, and were therefore removed from the experiment since this level of anaemia was deemed fatal. In contrast, all nontreatment goats infected with IL 1180 maintained their PCV between 15% and 20% throughout the entire experiment. Finally, of the 3 non-treatment control goats infected with both clones, one developed a severe anaemia and was removed from the experiment; the remaining 2 maintained their PCV above 12% during the entire experimental period. Thus, on the basis of ability to maintain PCV, it can be concluded that the drug-resistant clone was more pathogenic than the drug-sensitive clone. In order to determine that IL 1180 was present in goats with mixed infections at the time of treatment (Group C), goat blood stabilates collected 3 days before treatment were expanded in irradiated mice. Goat buffy-coat preparations taken on the day of treatment, were also examined. However, while trypanosomes in goat blood were expanded in mice, trypanosomes in buffy-coats were examined directly. Goat blood stabilates of relapse trypanosome populations occurring in the same animals on days 18, 32, 46 and 60 following treatment were also examined; each stabilate was expanded in irradiated mice. The DNA from all the aforementioned samples were screened for the presence of IL 1180 using the PCR-technique described above, and the [32P]-labelled 900 bp probe. For each reaction, 25 ng of genomic DNA was used as the template. The results demonstrated that IL 1180 was present in all mixed infections prior to treatment, but was absent in all relapse populations following treatment at the level of detection of the radiolabelled probe (i.e., 1 pg/25 ng total DNA). The data therefore indicated that a diminazene-sensitive trypanosome population is unable to survive treatment with diminazene when mixed with a diminazeneresistant population

Evaluation of response to treatment of mixed trypanosome infection in goats using the polymerase chain reaction

Studies on infections with drug-resistant trypanosomes indicate that the majority of parasites in a population are sensitive to the drug dose used to select the population (Mamman et al., 1993). This study was initiated to examine whether a drug-resistant trypanosome population could influence the survival of a drug-sensitive population in mixed infections in goats. To identify both populations during the course of a mixed infection, a system for distinguishing between them was required. Arbitrary primer Polymerase Chain Reaction (PCR) was attempted, but was unable to distinguish the two populations if one was 10 percent or less of the total number of parasites. Using a DNA sequence that is only present in the diminazene-sensitive trypanosome, T. congolense IL 1180, a pair of 20 bp primers were designed, which, in a PCR, amplified a 900 bp sequence from IL 1180 but not IL 3274. The sensitivity of the PCR technique for detecting Il 1180 genomic DNA, when mixed with 25 ng total genomic DNA of IL 3274 was 100 pg by gel electrophoresis and ethidium bromide staining of the PCR products. Using the 900 bp PCR product as a 32 P-labelled probe in southern blots, the sensitivity was increased 100-fold. Three groups of 5 goats each were infected intravenously with either IL 1180 (group A), IL 3274 (group B) or both clones simultaneously (group C), and treated with diminazene aceturate at a does of 7.0 mg/kg b.wt following Development of parasitaemia. Animals in group C were treated after all animals in groups A and B had become parasitaemic. There other groups (groups D, E and F), consisting of 3 goats each, were similarly infected and kept as untreated controls. All group A animal sgot cured, while all in B and 4 in C relapsed. Trypanosomes were harvested from all animals every 2 weeks, beginning 3 days before treatment and up to 60 days post-treatment. Trypanosome DNA was analysed for the presence of IL 1180 DNA was absent in any post-treatment sample. It was therefore concluded that IL 1180 is unable to survive treatment with diminazene aceturate when mixed with IL 3274

Response of diminazene-resistance and diminazene-susceptible Trypanosoma congolense to treatment with diminazene when occuring as a mixed infection in goats

A study was carried out to determine whether a drug-resistant trypanosome population could influence the survival of a drug-sensitive population in mixed infections in goats. To identify both populations during the course of a mixed infection, a system for distinguishing them was developed; using a nucleotide sequence of cDNA that was derived from Trypanosoma congolense ILNat 3.3 (IL 1616), a pair of 20-mer primers was designed which, in a PCR, amplified a 900-bp sequence from the diminazene-senstive trypanosome, T. congolense IL 1180, but not the diminazene-resistant trypanosome, T. congolense IL 3247. The PCR technique detected 100 pg of IL 1180 DNA when mixed with 25 ng of total genomic DNA of IL 3274 as determined by gel electrophoresis and ethidium bromide-staining of the PCR products. Using the 900-bp PCR product as a 32 P-labelled probe on Southern blots, the sensitivity was increased 100-fold. Three groups of five goats each were infected with IL 1180 (group A.), IL 3274 (group B) or both clones simultaneously (group C), and treated with diminazene aceturate at a dose of 7.0 mg/kg body weight following detection of trypanosomes. Three other groups of three goats each were similarly infected and kept as untreated controls. All group A animals were cured, while all in group B and four animals in group C relapsed. Trypanosomes were harvested from all animals at regular intervals up to 60 days post treatment. Using the PCR techniques, IL 1180 DNA could not be detected in any post-treatment trypanosome DNA sample. It therefore appeared, on the basis of the sensitivity of the DNA detection systems used, that IL 1180 is unable to survive treatment with diminazene aceturate when mixed with IL 3274 in goats

IFN-gamma-induced IDO and WRS expression in microglia is differentially regulated by IL-4

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, has been implicated in the pathogenesis of various neurological disorders. IDO expression is induced by IFN-gamma and leads to neurotoxicity by generating quinolinic acid. Additionally, it inhibits the immune response through both tryptophan depletion and generating other tryptophan catabolites. IL-4 and IL-13 have been shown to control IDO expression by antagonizing the effects of IFN-gamma in different cell types. Here, we investigated the effects of these cytokines on IDO expression in microglia. Interestingly, we observed that both IL-4 and IL-13 greatly enhanced IFN-gamma-induced IDO expression. However, tryptophanyl-tRNA synthetase (WRS), which is coinduced with IDO by IFN-gamma, is downregulated by IL-4 and IL-13. The effect of IL-4 and IL-13 was independent of STAT-6. Modulation of IDO but not WRS was eliminated by inhibition of protein phosphatase 2A (PP2A) activity. The phosphatidylinositol 3-kinase (PI3K) pathway further differentiated the regulation of these two enzymes, as inhibiting the PI3K pathway eliminated IFN-gamma induction of IDO, whereas such inhibition greatly enhanced WRS expression. These findings show discordance between modulations of expression of two distinct enzymes utilizing tryptophan as a common substrate, and raise the possibility of their involvement in regulating immune responses in various neurological disorders

Regulation of Indoleamine 2,3-Dioxygenase Expression in Simian Immunodeficiency Virus-Infected Monkey Brains

The human immunodeficiency virus type 1-associated cognitive-motor disorder, including the AIDS dementia complex, is characterized by brain functional abnormalities that are associated with injury initiated by viral infection of the brain. Indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme in tryptophan catabolism in extrahepatic tissues, can lead to neurotoxicity through the generation of quinolinic acid and immunosuppression and can alter brain chemistry via depletion of tryptophan. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model of AIDS, we demonstrate that cells of the macrophage lineage are the main source for expression of IDO in the SIV-infected monkey brain. Animals with SIV encephalitis have the highest levels of IDO mRNA, and the level of IDO correlates with gamma interferon (IFN-γ) and viral load levels. In vitro studies on mouse microglia reveal that IFN-γ is the primary inducer of IDO expression. These findings demonstrate the link between IDO expression, IFN-γ levels, and brain pathology signs observed in neuro-AIDS