Methamphetamine Increases Brain Viral Load and Activates Natural Killer Cells in Simian Immunodeficiency Virus-Infected Monkeys

Methamphetamine Increases Brain Viral Load and Activates Natural Killer Cells in Simian Immunodeficiency Virus-Infected Monkeys

The common marmoset is a new world primate belonging to the Callitrichidae family weighing between 350 and 400g. The virus infects the brain (encephalitis) and the surrounding membrane (meningitis). Ninety percent of all reptiles carry and shed salmonella in their feces. Risks from monkey bites include serious wound infections, herpes B virus, and rabies. Monkey business. The incubation period in humans varies from 12 hours to 5 days, depending on the dose received, and can be longer following exposure to low doses or removal of therapeutics. When she began developing symptoms, her concerns were downplayed and Emory doctors failed to recognize the likely life-threatening disease she had contracted in spite of the history of her case.

The B virus in monkeys does not sicken the monkeys but when passed to humans leads to deadly infections of the brain. Infection with B virus is extremely rare in humans. Beside McGeorge, another former laboratory employee also is infected with the virus. Snakes are the most common “pet” reptiles and have the potential to inflict serious injury through a bite or constriction. At necropsy, performed after terminal anesthesia, animals were intracardially perfused with sterile PBS containing 1 U/ml heparin. For the purposes of this review, the focus is on the use of marmosets in high biocontainment, highlighting how they reflect human disease. Cells from centrifuged EDTA-treated blood (buffy coat) or 70-μm nylon-mesh sieved cells from spleen and lymph nodes were subjected to Histopaque (Sigma-Aldrich, St.

Louis, MO) gradient for isolation of the mononuclear fraction. For the organs, tissues were removed at necropsy after intravascular perfusion. While herpes B is relatively harmless among monkeys, among humans it can cause vomiting, headache, loss of motor control, and, eventually, infection of the central nervous symptom. anthracis (Ames strain) in a head-only exposure chamber within a class III biosafety cabinet to determine the median lethal dose (LD50). Beth was working as a research assistant at Yerkes Regional Primate Research Center at Emory University in Atlanta in the fall of 1997 where she was engaged in behavioral research on hormonal influences in Rhesus Macaques — or the Rhesus Monkeys — and was in the process of applying to continue her pursuit of graduate studies and research in the field of biological/psychological sciences. “It’s a terrible disease,” said Koehler. Isolated cells were stained with labeled antibodies in PBS containing 2% fetal calf serum and 0.01% NaN3.

For cell surface phenotyping, the antibodies used were anti-CD14-PE (clone M5E2, BD Pharmingen, San Diego, CA), anti-CD16-FITC (clone 3G8, BD Pharmingen), anti-monkey CD3-biotin (clone FN-18, Invitrogen Biosource, Carlsbad, CA) followed by streptavidin-PerCP or -APC (BD Pharmingen), anti-human CD8-PE, -FITC, or -PeCy5 (clone DK25, Dako, Carpinteria, CA), or isotype controls (BD Pharmingen). In addition, monkeys have been known to transmit the Ebola virus, Monkey Pox, and other deadly illnesses. For functional (CD107a/degranulation or cytokine) assessment samples were treated with protein transport blockers. As the disease progresses, nausea, vomiting, diarrhea and abdominal pain are often observed. Cells were washed and cell surface-stained with anti-CD3 and anti-CD8 as above. They were then surface-stained with anti-CD107a (BD Pharmingen) or treated with Cytofix/Cyoperm solution (BD Pharmingen), washed in Perm/Wash Buffer (BD Pharmingen), and incubated with anti-interferon-γ (BD Pharmingen) or anti-tumor necrosis factor-α (BD Pharmingen) in Perm/Wash buffer for 30 minutes. Cells were kept in 3% paraformaldehyde and acquired in a FACSCalibur instrument using CellQuest software.

Analysis was performed using FlowJo 6.2.1 software. All animals were culture-positive for B. Beth tried calling the Office of Infectious Diseases and was told she needed a referral before she could be seen. Types of macaque monkeys known to carry herpes B are Tibetan macaques, lion-tailed macaques and crab-eating macaques. Group comparisons were performed using the tests described in the text and figure legends. The difference between the means was considered significant at P < 0.05. Tests were performed using Excel (Microsoft Corporation, Redmond, WA) and Prism software (GraphPad Software Inc., San Diego, CA) for Macintosh. Studies on the pathogenesis of SIV infection are often complicated by the inclusion of animals with an aberrant, rapidly progressive disease course that does not reflect human HIV infection, and such animals lack the development of the initial immune response to the infection. In addition to liver tissues, a marked reduction in the intensity of HLA-DR staining was also observed in lymph nodes in Lassa virus-infected marmosets. Three animals did not show control of viral load and were sacrificed because of early development of simian AIDS. The other six animals developed an acute viremia with viral load reaching an average of 7.0 (log10) at 2 weeks postinoculation (p.i.), which then dropped and began at a steady-state level at 8 weeks p.i. . Plasma viral load and blood immune cell numbers in PBS- and Meth-treated animals. Mediastinal hemorrhage and/or edema were considered to be present when the thymus or pericardial sac was affected; this occurred in four monkeys (29%). At 19 weeks p.i. The case was unusual in that infection is relatively rare -- there have been only 40 cases between 1933 and 1994 -- and most infections result from bites or scratches. Importantly, we mimicked the amounts of Meth used by chronic abusers with our drug administration schedule. Human chronic Meth abusers, who generally increase dose with use, reach up to 1 g/day. In a recent high-resolution magnetic resonance imaging study demonstrating Meth-induced defects in human brains, the subjects averaged 3.4 g of Meth/week.21 Assuming an average of 70 kg body weight and 50% purity of the Meth (the average according to US Department of Justice statistics), this would be 24 to 25 mg/kg/week. Because a typical abuser takes Meth 1 to 3 times a day on >20 days a month,22 we used a modification of our escalating dose regimen, giving drug 5 days a week, twice a day, and further increasing the dose to reach a final dose of 2.5 mg/kg twice daily, after a 5 week ramp-up, for a total of 25 mg/kg/week, approximating the amounts used by chronic Meth abusers. The family Filoviridae predominantly consists of two genera – Ebolavirus (EBOV) and Marburgvirus (MARV).
Methamphetamine Increases Brain Viral Load and Activates Natural Killer Cells in Simian Immunodeficiency Virus-Infected Monkeys

The other three animals received PBS injections on the same schedule. All animals were sacrificed at 42 weeks p.i. Meth treatment did not alter the plasma viral load over the course of the experiment . Although cerebrospinal fluid was sampled less frequently, no difference was found in the viral load in the cerebrospinal fluid (data not shown). Small mucosal hemorrhages were characteristic of all hemorrhages in the intestinal tract. Histopathological examination of the brain did not reveal SIV-specific lesions. Immunohistochemical examination of the brain for lymphocytes and macrophages showed occasional perivascular lymphocytes and macrophages , with no differences between the groups.

Although there was no appreciable microglial activation, astrocyte activation was present and was more pronounced in Meth-treated animals than in PBS-treated controls . Photomicrographs of CNS immunohistochemical analyses. Left (A, C, E, and F): PBS-treated animals. Right (B, D, G, and H): Meth-treated animals. Marmosets experienced anorexia coinciding with the onset of fever. Immune cells were isolated from the brain and assessed by fluorescence-activated cell sorter analysis, revealing that the proportion of CD4+, CD8+, and NK cells in the brain did not differ between groups (CD4 T cells: 1.65 ± 0.75% in PBS group and 1.28 ± 0.47% in Meth group; CD8 T cells: 3.57 ± 1.02% in PBS group and 2.10 ± 0.85% in Meth group; NK cells: 2.40 ± 1.62% in PBS group and 2.33 ± 0.3% in Meth group). Meth treatment did not alter the overall percentage of CD11b+ cells (microglia and macrophages) in the brain; in both groups these cells comprised more than 85% of immune cells isolated from the brain.

Interestingly, significantly more virus was found in the brains of the Meth-treated animals than in the brains of the control animals . Viral load in the brain. Minimal to mild suppurative inflammation was present in at least one lymph node in 50% of animals, usually in the intrathoracic lymph nodes or the mandibular lymph node. **P < 0.01 in two-way analysis of variance. ** ... Although their proportions did not differ, to examine potential activation/activity of the brain-infiltrating cells as well as lymphocytes, we assessed their release of granule contents through analysis of cell surface expression of CD107a (lysosomal-associated membrane protein 1), a marker of secretory lysosomes present on the cell surface, after degranulation, representing the functional ability to secrete cytokines and/or cytotoxic molecules.23 In addition, we assessed the percentage of cells producing cytokines, on a single-cell basis by intracellular staining. Increased amounts of CD107a were present on the cell surface of CD8+ T cells and NK cells in the Meth-treated group in the brain, blood, and lymphoid organs; increased CD107a was also present in the CD4+ T cells in the brain , indicating a higher degree of active cell degranulation. Intracellular cytokine staining indicated that in the Meth-treated animals, NK cells were markedly affected, exhibiting higher levels of tumor necrosis factor-α and interferon-γ production in the brain and other sites . Phenotypic changes were also observed in the brain macrophages, which were identified by the presence of CD14, and further analyzed for the activation marker CD16. The coagulopathy observed in humans at times exists in the absence of rash: only 50% of patients infected with EBOV develop a maculopapular rash[47]. Brain cell suspension scatter plots of one representative control animal and one Meth-treated animal show the increase of CD14+CD16+ macrophages within the CD11b+ gate. On average there were almost double the number of CD14+CD16+ macrophages in the brains of Meth-treated animals compared with the number in the PBS group . Interestingly, although the majority of these CD14+CD16+ cells in the brain expressed high levels of CCR5 in both SIV-infected groups regardless of the treatment, in the Meth-treated group the CD14+CD16low macrophages had a higher percentage of CCR5-highly expressing cells . Representative CCR5 expression histograms illustrate the high levels of CCR5 on CD14+CD16+ cells (left panel) in both groups and the up-regulation of CCR5 within the CD14+ CD16low compartment (right panel) in Meth-treated animals (red lines) compared with PBS-treated animals (blue lines) . 2; microscopic view, Fig. Subpopulations of macrophages in the brains of PBS- or Meth-treated SIV-infected monkeys. A: Total CD11b, and percentage of CD14+CD16+ and CD14+CD16low within CD11b+-gated cells in PBS-treated (white bar) or Meth-treated ... Here we report changes in the virus-host interaction in SIV-infected macaques as a result of Meth administration. Although no change in plasma viral load was found after Meth administration, Meth led to a significantly increased viral load in the brain and increased numbers of activated brain macrophages. Interestingly, effects on peripheral as well as brain lymphoid cells were found, with markedly increased NK cell activation. We performed this study because of the overlap in individuals who use Meth and who are HIV-infected and because of the potential interacting effects of these agents on the immune system as well as the brain. A decrease in leukocytes was observed in NA EEEV-infected marmosets within 24-48 hr of infection, followed by marked leukocytosis prior to death or euthanasia. Increased virus was present in the brain of Meth-treated animals as well as an increase in CD14+CD16+ inflammatory macrophages, known targets for SIV/HIV infection in the brain.24,25 These cells express high levels of CCR5, the SIV coreceptor; in addition there is also increased expression of CCR5 on CD14+CD16low macrophages in Meth-treated animals. This result suggests that the increase in brain viral load is correlated to an enrichment of target cells. At this stage of infection, we did not find significant inflammatory foci, either in the presence or absence of Meth treatment. The increased macrophages in Meth-treated animals are probably diffuse within the brain, although histopathological studies are limited to a small proportion of the brain volume. In the brain, necrotizing vasculitis was observed in 2 of the 14 animals and may have contributed to some of the other lesions present, including hemorrhages and leukomalacia. The expression of CD107a showed a significant increase in CD8 and NK cells in Meth-treated animals in comparison with PBS controls. The increased expression of CD107a correlated with an increased capacity to secrete tumor necrosis factor-α and interferon-γ in NK cells but not in the CD8 compartment; however we did not examine other important molecules produced by CD8 T cells in SIV/HIV infection, such as perforin and granzymes.19 These markers suggest that in Meth-treated animals there is more NK cell activation and possibly CD8+ T-cell activation than in PBS-treated animals. Studies in HIV and SIV infection reveal that NK cells do not seem to play a major role in viral control.30,31 Given the nature of NK cells and the broad range of activation, we speculate that Meth may be causing a relatively nonspecific activation of NK cells, but further work is required to assess the true basis of this effect. Neuropathologically the only difference noted between the groups was increased astrocyte activation in the monkeys administered Meth. Reactive astrocytes represent a fairly nonspecific finding and are seen in response to SIV/HIV infection as well as to other neuropathological insults. Meth use may lead to increased astrocyte activation21; in addition, the increased brain virus and activated NK cells may also have contributed to this finding in the Meth-treated animals. Older people were more often associated with increased SARS pathogenicity and death resulting from acute respiratory distress syndrome[59,60]. Meth raises concentrations of dopamine, which can directly affect immune cells.32,33 Regarding NK cells, both stimulatory and inhibitory effects of Meth have been found in mice and monkeys.34,35,36 However, the markedly different Meth pharmacokinetics in rodents as opposed to primates (approximately a 10-fold shorter half-life in rodents) complicates interpretation of rodent studies.37 Furthermore, these are largely acute studies, and chronic administration studies such as ours have not been performed to examine the effects of Meth on immune parameters. In addition to the activation of NK cells, the introduction of a Meth administration regimen during stable SIV infection led to an increase in activated macrophages and virus in the brain, which are probably connected events and both linked to the potential development of CNS disease. Meth may act on immune cells both directly and indirectly, affecting different functional levels in distinct ways. Given the prevalence of Meth use in HIV-infected and HIV at-risk populations, Meth represents a significant comorbid factor in HIV-induced disease, particularly in the brain.

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