Guy A. Cabral

Acanthamoeba spp. as Agents of Disease in Humans

SUMMARY Acanthamoeba spp. are free-living amebae that inhabit a variety of air, soil, and water environments. However, these amebae can also act as opportunistic as well as nonopportunistic pathogens. They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis. Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba. The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role. The amebas life cycle includes an active feeding trophozoite stage and a dormant cyst stage. Trophozoites feed on bacteria, yeast, and algae. However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential. Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis. Treatment of Acanthamoeba infections has met with mixed results. However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients. Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized. Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease.

Differential expression of the CB2 cannabinoid receptor by rodent macrophages and macrophage-like cells in relation to cell activation

An in vitro model of multi-step activation, in which cells of macrophage lineage are driven sequentially through inflammatory, primed, and fully activated states, was employed to assess for cannabinoid receptor expression. Murine and rat peritoneal macrophages, murine RAW264.7 and P388D, macrophage-like cells, and neonatal rat brain cortex microglia expressed the cannabinoid receptor type 2 (CB2) differentially in relation to cell activation. The CB2 was undetectable in resident peritoneal macrophages, present at high levels in thioglycolate-elicited inflammatory and interferon gamma (IFNgamma)-primed peritoneal macrophages, and detected at significantly diminished levels in bacterial lipopolysaccharide (LPS)-activated peritoneal macrophages. A comparable pattern of differential expression of the CB2 was noted for murine macrophage-like cells and neonatal rat brain cortex microglia. The cannabinoid receptor type 1 (CB1) was not detected in peritoneal macrophages or murine macrophage-like cells regardless of cell activation state but was present in neonatal rat microglia at low levels. These results indicate that levels of the CB2 in cells of macrophage lineage undergo major modulatory changes in relation to cell activation. Furthermore, since inflammatory and primed macrophages express the highest levels of CB2, the functional activities of macrophages when in these respective states of activation may be the most sensitive to the action of cannabinoids.

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

The cannabinoid receptor family consists of two inhibitory G‐protein‐coupled receptors, CB1 and CB2. CB1 is distributed primarily in neural tissue, whereas CB2 is distributed predominately in immune cells. The distribution of cannabinoid receptors in neural tissue has been demonstrated by using ligand binding autoradiography with CP55,940, a high‐affinity cannabinoid receptor ligand, and in situ hybridization. However, the localization of CB1 within individual cells in the brain remains to be defined. In the present study, domain‐specific polyclonal antibody to amino acids 83–98 of CB1 was used to define the expression of the neural cannabinoid receptor at the histochemical level. The use of CB1‐specific antiserum is advantageous in view of recent reports that CB2 also is expressed in the brain and binds CP55,940. Thus, utilization of anti‐CB1 antiserum would allow for the specific detection of CB1 protein expression. The regional staining pattern for CB1 in rat brain was consistent with that reported for CB1 using ligand binding autoradiography and in situ hybridization. Intense immunoreactivity was present in the hippocampal formation, the basal ganglia, and the molecular layer of the cerebellum. Moderate immunohistochemical staining was observed in the olfactory bulb, piriform cortex, cerebral cortex, and the granular layer of the cerebellum. In addition, immunoreactive staining was concentrated on afferent projections and dendritic processes of neuronal cells and was present within cell bodies and on cell surfaces. These data indicate that the anti‐CB1 antibody is a sensitive probe for the unequivocal histological discrimination of CB1 protein expression. J. Neurosci. Res. 51:391–402, 1998. © 1998 Wiley‐Liss, Inc.

CB2 receptors in the brain: role in central immune function

Recently, it has been recognized that the cannabinoid receptor CB2 may play a functionally relevant role in the central nervous system (CNS). This role is mediated primarily through microglia, a resident population of cells in the CNS that is morphologically, phenotypically, and functionally related to macrophages. These cells also express the cannabinoid receptor CB1. The CB1 receptor (CB1R) is constitutively expressed at low levels while the CB2 receptor (CB2R) is expressed at higher levels and is modulated in relation to cell activation state. The relatively high levels of the CB2R correspond with microglia being in ‘responsive’ and ‘primed’ states, suggesting the existence of a ‘window’ of functional relevance during which activation of the CB2R modulates microglial activities. Signature activities of ‘responsive’ and ‘primed’ microglia are chemotaxis and antigen processing, respectively. The endocannabinoid 2‐arachidonylglycerol has been reported to stimulate a chemotactic response from these cells through the CB2R. In contrast, we have shown in vivo and in vitro that the exogenous cannabinoids delta‐9‐tetrahydrocannabinol and CP55940 inhibit the chemotactic response of microglia to Acanthamoeba culbertsoni, an opportunistic pathogen that is the causative agent of Granulomatous Amoebic Encephalitis, through activation of the CB2R. It is postulated that these exogenous cannabinoids superimpose an inhibitory effect on pro‐chemotactic endocannabinoids that are elicited in response to Acanthamoeba. Furthermore, the collective results suggest that the CB2R plays a critical immune functional role in the CNS.

Cannabinoids inhibit LPS-inducible cytokine mRNA expression in rat microglial cells

The effect of cannabinoids on the induction of cytokine mRNA by rat microglial cells was examined. Exposure of neonatal rat cortical microglial cells to the exogenous cannabinoid δ9‐tetrahydrocannabinol (THC) resulted in reduced amounts of lipopolysaccharide (LPS)‐induced mRNAs for IL‐1α, IL‐1β, IL‐6, and TNF‐α. Of these cytokine mRNAs, the response of that for IL‐6 was exquisitely sensitive to THC. Similarly, exposure of microglial cells to the putative endogenous cannabinoid anandamide before LPS treatment resulted in a decrease in cytokine mRNA levels, but not to the same extent as that caused by THC; however, when methanandamide, the non‐hydrolyzable analog of anandamide was tested, its ability to inhibit cytokine mRNA expression was comparable to that of THC. Exposure of microglial cells to either of the paired enantiomers CP55,940 or CP56,667 resulted in similar inhibition of LPS‐induced cytokine mRNA expression. A comparable inhibitory outcome was obtained when the paired enantiomers levonantradol and dextronantradol were employed. Neither the CB1‐selective antagonist SR141716A nor the CB2‐selective antagonist SR144528 was able to reverse the inhibition of cytokine mRNA expression by levonantradol. The CB2 antagonist, however, when administered alone augmented the production of cytokine mRNAs. Collectively, these studies demonstrate that cannabinoids can modulate levels of cytokine mRNA in rat microglial cells; however, the inhibition of cytokine mRNA expression is apparently not mediated through either the CB1 or CB2 cannabinoid receptors. GLIA 29:58–69, 2000.

Emerging role of the cannabinoid receptor CB2 in immune regulation: therapeutic prospects for neuroinflammation.

There is now a large body of data indicating that the cannabinoid receptor type 2 (CB2) is linked to a variety of immune events. This functional relevance appears to be most salient in the course of inflammation, a process during which there is an increased number of receptors that are available for activation. Studies aimed at elucidating signal transduction events resulting from CB2 interaction with its native ligands, and of the role of exogenous cannabinoids in modulating this process, are providing novel insights into the role of CB2 in maintaining a homeostatic immune balance within the host. Furthermore, these studies suggest that the CB2 may serve as a selective molecular target for therapeutic manipulation of untoward immune responses, including those associated with a variety of neuropathies that exhibit a hyperinflammatory component.

Synthetic cannabinoid WIN55,212-2 inhibits generation of inflammatory mediators by IL-1β-stimulated human astrocytes

Activated glial cells have been implicated in the neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators have been proposed to play a role in glial cell‐related brain damage; e.g., free radicals such as nitric oxide (NO), cytokines, and chemokines. Our laboratory has been interested in the effect of psychoactive drugs and their derivatives on the production of these mediators. Cannabinoids have been shown to possess immunomodulatory as well as psychoactive properties. We previously have shown that interleukin (IL)‐1β‐stimulated human astrocytes, but not microglia, produce NO. In this study, we investigated the effects of the synthetic cannabinoid WIN55,212‐2 on the production of several key inflammatory mediators by human fetal astrocytes activated by IL‐1β. Expression of the cannabinoid receptors CB1 and CB2 was detected on human astrocytes. WIN55,212‐2 (10−5 M) potently inhibited inducible NO synthase (iNOS) and corresponding NO production by IL‐1β‐stimulated astrocytes. The CB1 and CB2 receptor‐specific antagonists SR141716A and SR144528, respectively, partially blocked this suppressive effect. In addition, treatment of astrocytes with WIN55,212‐2 downregulated in a concentration‐dependent manner IL‐1β‐induced tumor necrosis factor (TNF)‐α release. Treatment with WIN55,212‐2 also inhibited production of the chemokines CXCL10, CCL2 and CCL5 by IL‐1β‐activated astrocytes. These findings indicate that WIN55,212‐2 inhibits the production of inflammatory mediators by IL‐1β‐stimulated human astrocytes and suggest that comparable agents may have therapeutic potential for the management of brain inflammation.

Endocannabinoid signaling at the periphery: 50 years after THC

In 1964, the psychoactive ingredient of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC), was isolated. Nearly 30 years later the endogenous counterparts of THC, collectively termed endocannabinoids (eCBs), were discovered: N-arachidonoylethanolamine (anandamide) (AEA) in 1992 and 2-arachidonoylglycerol (2-AG) in 1995. Since then, considerable research has shed light on the impact of eCBs on human health and disease, identifying an ensemble of proteins that bind, synthesize, and degrade them and that together form the eCB system (ECS). eCBs control basic biological processes including cell choice between survival and death and progenitor/stem cell proliferation and differentiation. Unsurprisingly, in the past two decades eCBs have been recognized as key mediators of several aspects of human pathophysiology and thus have emerged to be among the most widespread and versatile signaling molecules ever discovered. Here some of the pioneers of this research field review the state of the art of critical eCB functions in peripheral organs. Our community effort is aimed at establishing consensus views on the relevance of the peripheral ECS for human health and disease pathogenesis, as well as highlighting emerging challenges and therapeutic hopes.

The Increasing Importance of Acanthamoeba Infections1

Abstract Free-living amebae belonging to the genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis, a chronic progressive disease of the central nervous system, and of amebic keratitis, a chronic eye infection. Granulomatous amebic encephalitis occurs more frequently in immunocompromised patients while keratitis occurs in healthy individuals. The recent increased incidence in Acanthamoeba infections is due in part to infection in patients with acquired immune deficiency syndrome, while that for keratitis is due to the increased use of contact lenses. Understanding the mechanism of host resistance to Acanthamoeba is essential since the amebae are resistant to many therapeutic agents. Studies in our laboratory as well as from others have demonstrated that macrophages from immunocompetent animals are important effector cells against Acanthamoeba. We have demonstrated also that microglial cells, resident macrophages of the brain, elicit cytokines in response to A. castellanii. Neonatal rat cortical microglia from Sprague-Dawley rats co-cultured with A. castellanii produced mRNA for the inflammatory cytokines, interleukin 1α, interleukin 1β, and tumor necrosis factor α. In addition, scanning and transmission electron microscopy revealed that microglia ingested and destroyed A. castellanii in vitro. These results implicate macrophages as playing an effector role against Acanthamoeba and suggest immune modulation as a potential alternative therapeutic mode of treatment for these infections.

Drugs and immunity: cannabinoids and their role in decreased resistance to infectious disease.

Marijuana, Cannabis sativa, elicits a variety of effects in experimental animals and humans. Delta-9-tetrahydrocannabinol (THC) is the major psychoactive component in marijuana. This substance has been shown, also, to be immunosuppressive and to decrease host resistance to bacterial, protozoan, and viral infections. Macrophages, T lymphocytes, and natural killer cells appear to be major targets of the immunosuppressive effects of THC. Definitive data which directly link marijuana use to increased susceptibility to infection in humans currently is unavailable. However, cumulative reports indicating that THC alters resistance to infection in vitro and in a variety of experimental animals support the hypothesis that a similar effect occurs in humans.