Abdul Mannan Baig

In Vitro Efficacies of Clinically Available Drugs against Growth and Viability of an Acanthamoeba castellanii Keratitis Isolate Belonging to the T4 Genotype

ABSTRACT The effects of clinically available drugs targeting muscarinic cholinergic, adrenergic, dopaminergic, and serotonergic receptors; intracellular calcium levels and/or the function of calcium-dependent biochemical pathways; ion channels; and cellular pumps were tested against a keratitis isolate of Acanthamoeba castellanii belonging to the T4 genotype. In vitro growth inhibition (amoebistatic) assays were performed by incubating A. castellanii with various concentrations of drugs in the growth medium for 48 h at 30°C. To determine amoebicidal effects, amoebae were incubated with drugs in phosphate-buffered saline for 24 h, and viability was determined using trypan blue exclusion staining. For controls, amoebae were incubated with the solvent alone. Of the eight drugs tested, amlodipine, prochlorperazine, and loperamide showed potent amoebicidal effects, as no viable trophozoites were observed (>95% kill rate), while amiodarone, procyclidine, digoxin, and apomorphine exhibited up to 50% amoebicidal effects. In contrast, haloperidol did not affect viability, but all the drugs tested inhibited A. castellanii growth. Importantly, amlodipine, prochlorperazine, and loperamide showed compelling cysticidal effects. The cysticidal effects were irreversible, as cysts treated with the aforementioned drugs did not reemerge as viable amoebae upon inoculation in the growth medium. Except for apomorphine and haloperidol, all the tested drugs blocked trophozoite differentiation into cysts in encystation assays. Given the limited availability of effective drugs to treat amoebal infections, the clinically available drugs tested in this study represent potential agents for managing keratitis and granulomatous amoebic encephalitis caused by Acanthamoeba spp. and possibly against other meningoencephalitis-causing amoebae, such as Balamuthia mandrillaris and Naegleria fowleri.

Novel chemotherapeutic strategies in the management of primary amoebic meningoencephalitis due to Naegleria fowleri.

Primary amoebic meningoencephalitis (PAM) due to Naegleria fowleri is a fulminating infection that can result in death within days. The disease prognosis is poor, although early diagnosis and aggressive treatment might increase survival chances [1]. Through contaminated water, N. fowleri enters nostrils and then migrates along the olfactory nerve, through the cribriform plate into the brain [2]. PAM is characterized by parosmia, rapidly progressing to anosmia (with resultant ageusia) as the nerve cells of the olfactory bulbs are destroyed leading to seizures and finally death [3]. Despite advances in antimicrobial chemotherapy and supportive case, the mortality rate associated with PAM has remained over 90% [1–3]. A high mortality is attributed to (1) delayed diagnosis, (2) our incomplete understanding of the pathogenesis and pathophysiology of the disease, (3) lack of availability of safe and effective drugs, and (4) the difficulty in delivering anti-N. fowleri drugs to the brain. So far, only three survivors of PAM have been documented [4]. These patients were treated with amphotericin B, rifampicin, fluconazole, dexamethasone, and phenytoin intravenous and/or intrathecally within first week of the infection [4,5]. A successful prognosis was attributed to early diagnosis (limited deeper tissue involvement), followed by aggressive antimicrobial chemotherapy. Both amphotericin B [5] and miltefosine [6] have been suggested as useful agents against PAM [7]. The aim of this letter is to debate whether an improved therapeutic outcome can

Primary amoebic meningoencephalitis: amoebicidal effects of clinically approved drugs against Naegleria fowleri

Naegleria fowleri is a protist pathogen known to produce fulminating primary amoebic meningoencephalitis (PAM) (Marciano-Cabral & Cabral, 2007; Visvesvara et al., 2007). Although rare, PAM is a serious human disease with a fatality rate of .95 %. It is generally associated with swimming in contaminated freshwater and possibly through nasal irrigation for cleansing (Marciano-Cabral & Cabral, 2007; Visvesvara et al., 2007). PAM cases have recently been observed in Muslims who routinely perform ritual ablution, which involves repeated water exposure to the nostrils for cleansing (Siddiqui & Khan, 2011). These so-called ‘brain-eating amoebae’ invade the nervous system via the nose when contaminated water is deeply inhaled into the nose, and migrate to the brain tissue where severe haemorrhaging and inflammation are caused, resulting in widespread brain tissue destruction (Marciano-Cabral & Cabral, 2007; Visvesvara et al., 2007). As well studied as this protist is, we still do not know of any effective chemotherapeutic interventions. The current treatment regimen involves a mixture of drugs ranging from antimicrobial compounds to experimental anti-cancer drugs (MarcianoCabral & Cabral, 2007; Visvesvara et al., 2007), to provide additive or synergistic effects, but even then the mortality remains very high (~98 %) (Marciano-Cabral & Cabral, 2007; Visvesvara et al., 2007). This is probably due to difficulties in early diagnosis, resulting in delayed initiation of effective chemotherapy, and/or poor penetration of antimicrobial compounds across the blood–brain barrier. With the devastating nature of this disease and the problems associated with its chemotherapy, the overall aim of the present study was to test anti-N. fowleri effects of clinically available drugs in vitro.

Combined drug therapy in the management of granulomatous amoebic encephalitis due to Acanthamoeba spp., and Balamuthia mandrillaris.

Granulomatous amoebic encephalitis (GAE) is caused by two protist pathogens, Acanthamoeba spp., and Balamuthia mandrillaris. Although rare, it almost always results in death. In the present study, amoebae were treated with various combinations of clinically-approved drugs, targeting vital cellular receptors and biochemical pathways. The results revealed that among the seven different combinations tested, three proved highly effective against both Acanthamoeba castellanii as well as B. mandrillaris at a concentration of 100μM. These combinations included (i) prochlorperazine plus loperamide; (ii) prochlorperazine plus apomorphine; and (iii) procyclidine plus loperamide. In viability assays, none of the drug-treated amoebae emerged as viable trophozoites, suggesting irreversible amoebicidal effects. Four combinations of drugs tested showed varied potency against A. castellanii and B. mandrillaris at 100μM. The combination of haloperidol and loperamide was highly effective against A. castellanii at 100μM, but potent effects against B. mandrillaris were observed only at 250μM. Digoxin and amlodipine were effective against A. castellanii and B. mandrillaris at 100μM and 250μM, respectively. In contrast, the combination of apomorphine and haloperidol was effective against B. mandrillaris and A. castellanii at 100μM and 250μM, respectively. At 100μM, the combination of procyclidine and amiodarone was effective against neither A. castellanii nor B. mandrillaris. In this case, amoebicidal properties were observed at 750μM for A. castellanii, and 950μM for B. mandrillaris. As these drugs are used clinically against non-communicable diseases, the findings reported here have the potential to be tested in a clinical setting against amoebic encephalitis caused by A. castellanii and B. mandrillaris.

Cloned Microglias with Novel Delivery System in Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic inflammatory neurological disease of the Central Nervous System (CNS), characterized by demyelination and activation of microglia. Mitochondrial mutations and dysfunctions in microglial cells are thought to contribute to the detrimental effects of neuroinflammation seen in MS. The Somatic Nuclear Transfer (SCNT) technology offers a more practical mode of therapy in MS, this method would attempt to dilute and/or progressively replace the mutated and activated microglia with cloned Olfactory Ensheathing Cells (OEC) with remyelinating and scavenging properties which would attempt to limit the progression of MS. Applying SCNTderived Embryonic Stem (ES) cells based therapy by cloning Olfactory Ensheathing Cells (OEC), engineered with an autologous nuclear component of the recipient OEC with a healthy donor oocyte. The inner cell mass of the subsequently developed blastocyst would be the source to generate the radial microglia to be used for cell therapy in MS. The novel proposed transcribrial route device offers a painless mode of cell transplantation to the brain. This mode of generating cloned glia and its transplantation to the brain is expected to replace the mutated and activated microglia of the patients with MS and use the regenerative and remyelinating and scavenging properties of the OEC’s, as has been seen in clinical trials in patients with spinal cord injuries. The use of SCNT to develop isogenic ES cellbased therapies for the prevention and treatment of MS associated with mtDNA mutations may open a new avenue of designer’s targeted cell therapy unique for the patients with MS. The proposed “transcribrial device” to access the brain can be an advantageous route of delivery of cloned cells to the brain.

Recommendations for the management of Acanthamoeba keratitis

Acanthamoeba is the causative agent of a painful, sight-threatening keratitis. The first case of Acanthamoeba keratitis was reported in 1972 in the USA and soon after, it was reported in the UK in 1974 (Nagington et al., 1974). Ulcerative keratitis due to Acanthamoeba is often associated with the improper use of contact lenses, but it is also reported in non-contact lens wearers (Sharma et al., 2000). At present, treatment involves hourly topical application of a mixture of anti-Acanthamoeba drugs including 0.02 % polyhexamethylene biguanide or chlorhexidine for up to a week. The hourly drops may be reduced after 48 h to alleviate the epithelial toxicity, but treatment continues for up to a year (Clarke et al., 2012).

Evidence of a M1-muscarinic GPCR homolog in unicellular eukaryotes: featuring Acanthamoeba spp bioinformatics 3D-modelling and experimentations

Abstract Acetylcholine affects the target cellular function via muscarinic and nicotinic cholinergic receptors that are seen to exist in humans. Both the cholinergic receptors are G-protein coupled receptors (GPCRs) that perform cardinal functions in humans. Anti-muscarinic drugs, particularly the ones that target M1 subtype (mAChR1), have consistently shown to kill unicellular pathogenic eukaryotes like Acanthamoeba spp. As the M1 receptor subtype has not been reported to be expressed in the above protist, the presence of an ancient form of the M1 muscarinic receptor was inferred. Bioinformatic tools and experimental assays were performed to establish the presence of a ligand-binding site. A search for sequence homology of amino acids of human M1 receptor failed to uncover an equivalent ligand-binding site on Acanthamoeba, but structural bioinformatics showed a hypothetical protein L8HIA6 to be a receptor homolog of the human mAChR1. Immunostaining with an anti-mAChR1 antibody showed cellular staining. Growth assays showed proliferation and lethal effects of exposure to mAChR1 agonist and antagonist respectively. With the recent authentication of human mAChR1 structure and its addition to the database, it was possible to discover its structural analog in Acanthamoeba; which could explain the effects of anticholinergics observed in the past on Acanthamoeba spp. The discovery of a receptor homolog of human mAChR1 on Acanthamoeba with future studies planned to show its expression and binding to cholinergic agonist and antagonist would help clarify its role in the biology of this protist pathogen.

Primary Amoebic Meningoencephalitis: Neurochemotaxis and Neurotropic Preferences of Naegleria fowleri

Naegleria fowleri causes one of the most devastating necrotic meningoencephalitis in humans. The infection caused by this free-living amoeba is universally fatal within a week of onset of the signs and symptoms of the disease called primary amoebic meningoencephalitis (PAM). In all the affected patients, there is always a history of entry of water into the nose. Even though the diagnostic and treatment protocols have been revised and improved, the obstinate nature of the disease can be gauged by the fact that the mortality rate has persisted around ∼95% over the past 60 years. Some of the unanswered questions regarding PAM are is there a neurochemical basis of the chemotaxis of N. fowleri to the brain? What immune evasion means occurs preceding the neurotropic invasion? What is the contribution of the acute inflammatory response in the fatal cases? Can a combination of anti-amoebic drugs with antagonism of the acute inflammation help save the patients life? As prevention remains the most valuable safeguard against N. fowleri, a quicker diagnosis, better understanding of the pathogenesis of PAM coupled with testing of newer and safer drugs could improve the chances of survival in patients affected with PAM.

Granulomatous Amoebic Encephalitis: Ghost Response of an Immunocompromised Host?

Naegleria fowleri, Acanthamoeba and Balamuthia mandrillaris spp. are known to cause fatal amoebic encephalitis. Here, I attempt to draw attention to these cases, which are reported as ‘granulomatous amoebic encephalitis’ (GAE), and their occurrence in immunocompromised individuals and patients with AIDS. GAE, like any other granulomatous inflammation, can occur only in the presence of ample numbers of CD4 Tlymphocytes. Extensive reviews of manuscripts published over a period of 50 years on this topic and cytokine studies and/or morphological evidence provided in peer-reviewed published studies were evaluated in detail by independent resources to analyse the granulomatous inflammatory evidence provided to justify the title of GAE in this group of patients. The evidence given in support of GAE did not appear to be convincing enough in the majority of published studies, and in particular its occurrence in patients with AIDS and other immunocompromised states was not justified. The distinction between the early development of type IV hypersensitivity reactions and granuloma/ granulomatous inflammation was found to be vague and inconclusive. It is therefore recommended that this terminology is used only when all the diagnostic criteria have been met, and use of a term such as ‘granulomatoid’ is suggested in cases where there remains an ambiguity in the morphological appearance of the lesions, especially in AIDS and related diseases.

Antibiotic Effects of Loperamide:Homology of human targets of Loperamide with targets in Acanthamoeba spp

BACKGROUND Loperamide is an anti-diarrheal drug prescribed for non-infectious diarrhea. The drug is an opioid receptor agonist, blocker of voltage-dependent calcium channel (Cav) and calmodulin (CaM) inhibitor on human cells. Loperamide has been reported to exert anti-amoebic effects against pathogenic strains of Acanthamoeba castellanii. OBJECTIVES The precise mode of antibiotic action, cellular target homology with human counterparts and the pattern of cell death induced by loperamide in Acanthamoeba castellanii remain to be established. Additionally, we attempt to establish the presence a primitive Cav in Acanthamoeba castellanii. METHODS Bioinformatics, 3D structural modelling, ligand binding predictions and apoptotic/ amoebicidal assays were used in this study to answer the above queries. Amino acid sequences and structural models were compared between human and A. castellanii proteins that are involved in the regulation of calcium (Ca+2) homeostasis. RESULTS Our results show that A. castellanii expresses similar, to near identical types of primitive calcium channels Cav Ac and CaM that are well known targets of loperamide in humans. The growth assays showed anti-amoebic effects of loperamide at different doses, both alone and in combinations with other Ca+2- CaM inhibitors. The synergistic actions of loperamide with haloperidol showed to be more amoebicidal than when either of them used alone. Imaging with Annexin V, Acridine orange and Propidium iodide showed apoptosis in A. castellanii at a dose of 100 µg/ml and necrosis at higher doses of 250 µg/ml. CONCLUSION Though, Acanthamoeba does not express a homolog of the human mu-opioid receptor, but does shows evidence of the homologs for other known human targets of loperamide that are involved in Ca+2 uptake and Ca+2 signal transduction pathways. This suggests optimization of similar drug interactions with these targets may be useful in developing new approaches to control the growth of this parasite and possibly the diseases caused by it.