Biomedical subjects

Naveed Ahmed Khan

Publications and source records attributed to Naveed Ahmed Khan.

At least 19 recordsLinked to original sources

PubMed2007

Cellulose biosynthesis pathway is a potential target in the improved treatment of Acanthamoeba keratitis.

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.

Acanthamoeba Keratitis↗

PubMed2007

Effects of human serum on Balamuthia mandrillaris interactions with human brain microvascular endothelial cells.

Balamuthia mandrillaris is a free-living amoeba and a causative agent of fatal granulomatous encephalitis. In the transmission of B. mandrillaris into the central nervous system (CNS), haematogenous spread is thought to be the primary step, followed by blood-brain barrier penetration. The objectives of the present study were (i) to determine the effects of serum from healthy individuals on the viability of B. mandrillaris, and (ii) to determine the effects of serum on B. mandrillaris-mediated blood-brain barrier perturbations. It was determined that normal human serum exhibited limited amoebicidal effects, i.e. approximately 40 % of trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Using human brain microvascular endothelial cells (HBMEC), which form the blood-brain barrier, it was observed that B. mandrillaris exhibited binding (>80 %) and cytotoxicity (>70 %) to HBMEC. However, normal human serum exhibited more than 60 % inhibition of B. mandrillaris binding and cytotoxicity to HBMEC. ELISAs showed that both serum and saliva samples exhibit the presence of anti-B. mandrillaris antibodies. Western blots revealed that normal human serum reacted with several B. mandrillaris antigens with approximate molecular masses of 148, 115, 82, 67, 60, 56, 44, 42, 40 and 37 kDa. Overall, the results demonstrated that normal human serum has inhibitory effects on B. mandrillaris growth and viability, as well as on their binding and subsequent cytotoxicity to HBMEC. A complete understanding of B. mandrillaris pathogenesis is crucial to develop therapeutic interventions and/or to design preventative measures.

Amebicides↗

PubMed2007

FimH-mediated Escherichia coli K1 invasion of human brain microvascular endothelial cells.

Adhesion to brain microvascular endothelial cells, which constitute the blood-brain barrier is considered important in Escherichia coli K1 bacterial penetration into the central nervous system. Type 1 fimbriae are known to mediate bacterial interactions with human brain microvascular endothelial cells (HBMEC). Here, we demonstrate that type 1 fimbriae, specifically FimH adhesin is not only an adhesive organelle that provides bacteria with a foothold on brain endothelial cells but also triggers signalling events that promote E. coli K1 invasion in HBMEC. This is shown by our demonstrations that exogenous FimH increases cytosolic-free-calcium levels as well as activates RhoA. Using purified recombinant mannose-recognition domain of FimH, we identified a glycosylphosphatidylinositol-anchored receptor, CD48, as a putative HBMEC receptor for FimH. Furthermore, E. coli K1 binding to and invasion of HBMEC were blocked by CD48 antibody. Taken together, these findings indicate that FimH induces host cell signalling cascades that are involved in E. coli K1 invasion of HBMEC and CD48 is a putative HBMEC receptor for FimH.

Adhesins, Escherichia coli↗

PubMed2006

Acanthamoeba invasion of the central nervous system.

Pathogenic Acanthamoeba are known to infect the CNS, resulting in fatal granulomatous encephalitis. The mechanisms associated with the pathogenesis remain unclear; however pathophysiological complications involving the CNS most likely include induction of pro-inflammatory responses, invasion of the blood-brain barrier and the connective tissue and neuronal damage leading to brain dysfunction. The routes of entry include the olfactory neuroepithelium pathway and/or lower respiratory tract, followed by haematogenous spread. Skin lesions may provide direct entry into the bloodstream, bypassing the lower respiratory tract. For the haematogenous route, entry of amoebae into the CNS most likely occurs at the sites of the blood-brain barrier. Recent studies have identified several molecular mechanisms associated with Acanthamoeba traversal of the blood-brain barrier and targeting those may help develop therapeutic interventions and/or design preventative strategies.

Acanthamoeba↗

PubMed2006

The capsule plays an important role in Escherichia coli K1 interactions with Acanthamoeba.

Escherichia coli K1 is shown to bind to, associate with, invade and survive inside Acanthamoeba, but the precise mechanisms associated with these events are unclear. We have previously shown that outer membrane protein A and lipopolysaccharide are critical bacterial determinants involved in E. coli K1 interactions with Acanthamoeba. Using an isogenic K1 capsule-deletion mutant (lacking the neuDB genes cluster that is necessary for the production of cytoplasmic precursors to the exopolysaccharide capsule), we observed that the capsule modulates and enhances E. coli K1 association and survival inside Acanthamoeba. The capsule-deletion mutant exhibited significantly reduced association compared with the wild type strain, E44. Similarly, the K1 capsule-deletion mutant exhibited limited ability for invasion/uptake by and survival inside Acanthamoeba. Next, we determined whether E. coli K1 survive inside Acanthamoeba during the encystment process and that viable bacteria can be isolated from the mature cysts. Using encystment assays, our findings revealed that E. coli K1, but not its capsule-deletion mutant, exhibit survival inside Acanthamoeba cysts. We believe this is the first demonstration that the K1 capsule plays an important role in E. coli K1 interactions with Acanthamoeba.

Acanthamoeba↗

PubMed2006

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris.

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.

Acanthamoeba↗

PubMed2006

Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis.

BACKGROUND: Granulomatous amoebic encephalitis due to Acanthamoeba is often a fatal human disease. However, the pathogenesis and pathophysiology of Acanthamoeba encephalitis remain unclear. In this study, the role of extracellular Acanthamoeba proteases in central nervous system pathogenesis and pathophysiology was examined. RESULTS: Using an encephalitis isolate belonging to T1 genotype, we observed two major proteases with approximate molecular weights of 150 KD and 130 KD on SDS-PAGE gels using gelatin as substrate. The 130 KD protease was inhibited with phenylmethylsulfonyl fluoride (PMSF) suggesting that it is a serine protease, while the 150 KD protease was inhibited with 1, 10-phenanthroline suggesting that it is a metalloprotease. Both proteases exhibited maximal activity at neutral pH and over a range of temperatures, indicating their physiological relevance. These proteases degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue, as shown by the degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as casein and haemoglobin. The proteases were purified partially using ion-exchange chromatography and their effects were tested in an in vitro model of the blood-brain barrier using human brain microvascular endothelial cells (HBMEC). Neither the serine nor the metalloprotease exhibited HBMEC cytotoxicity. However, the serine protease exhibited HBMEC monolayer disruptions (trypsin-like) suggesting a role in blood-brain barrier perturbations. CONCLUSION: Overall, these data suggest that Acanthamoeba proteases digest ECM, which may play crucial role(s) in invasion of the brain tissue by amoebae.

Acanthamoeba↗

PubMed2006

Escherichia coli interactions with Acanthamoeba: a symbiosis with environmental and clinical implications.

The ability of Acanthamoeba to feed on Gram-negative bacteria, as well as to harbour potential pathogens, such as Legionella pneumophila, Coxiella burnetii, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes and Mycobacterium avium, suggest that both amoebae and bacteria are involved in complex interactions, which may play important roles in the environment and in human health. In this study, Acanthamoeba castellanii (a keratitis isolate belonging to the T4 genotype) was used and its interactions with Escherichia coli (strain K1, a cerebrospinal fluid isolate from a meningitis patient, O18 : K1 : H7, and a K-12 laboratory strain, HB101) were studied. The invasive K1 isolate exhibited a significantly higher association with A. castellanii than the non-invasive K-12 isolate. Similarly, K1 showed significantly increased invasion and/or uptake by A. castellanii in gentamicin protection assays than the non-invasive K-12. Using several mutants derived from K1, it was observed that outer-membrane protein A (OmpA) and LPS were crucial bacterial determinants responsible for E. coli K1 interactions with A. castellanii. Once inside the cell, E. coli K1 remained viable and multiplied within A. castellanii, while E. coli K-12 was killed. Again, OmpA and LPS were crucial for E. coli K1 intracellular survival in A. castellanii. In conclusion, these findings suggest that E. coli K1 interactions with A. castellanii are carefully regulated by the virulence of E. coli.

Acanthamoeba castellanii↗

PubMed2006

Balamuthia mandrillaris exhibits metalloprotease activities.

Balamuthia mandrillaris is a recently identified protozoan pathogen that can cause fatal granulomatous encephalitis. However, the pathogenesis and pathophysiology of B. mandrillaris encephalitis remain unclear. Because proteases may play a role in the central nervous system (CNS) pathology, we used spectrophotometric, cytopathic and zymographic assays to assess protease activities of B. mandrillaris. Using two clinical isolates of B. mandrillaris (from human and baboon), we observed that B. mandrillaris exhibits protease activities. Zymographic assays revealed major protease bands of approximate molecular weights in the region of 40-50 kDa on sodium dodecyl sulfate-polyacrylamide gels using gelatin as substrate. The protease bands were inhibited with 1,10-phenanthroline, suggesting metallo-type proteases. The proteolytic activities were observed over a pH range of 5-11 with maximum activity at neutral pH and at 42 degrees C. Balamuthia mandrillaris proteases exhibit properties to degrade extracellular matrix (ECM), which provide structural and functional support to the brain tissue. This is shown by degradation of collagen I and III (major components of collagenous ECM), elastin (elastic fibrils of ECM), plasminogen (involved in proteolytic degradation of ECM), as well as other substrates such as casein and gelatin but not haemoglobin. However, these proteases exhibited a minimal role in B. mandrillaris-mediated host cell death in vitro using human brain microvascular endothelial cells (HBMECs). This was shown using broad-spectrum matrix metalloprotease inhibitors, GM 6001 and GM 1489, which had no effect on B. mandrillaris-mediated HBMEC cytotoxicity. This is the first demonstration that B. mandrillaris exhibits metalloproteases, which may play important role(s) in the ECM degradation and thus in CNS pathology.

Amebiasis↗

PubMed2006

Acanthamoeba: biology and increasing importance in human health.

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and is well recognized to produce serious human infections, including a blinding keratitis and a fatal encephalitis. This review presents our current understanding of the burden of Acanthamoeba infections on human health, their pathogenesis and pathophysiology, and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be targets for therapeutic interventions and/or the development of preventative measures.

Acanthamoeba↗

PubMed2006

Use of in vitro assays to determine effects of human serum on biological characteristics of Acanthamoeba castellanii.

Normal human serum inhibits Acanthamoeba (encephalitis isolate) binding to and cytotoxicity of human brain microvascular endothelial cells, which constitute the blood-brain barrier. Zymographic assays revealed that serum inhibits extracellular protease activities of acanthamoebae. But it is most likely that inhibition of specific properties of acanthamoebae is a consequence of the initial amoebicidal-amoebistatic effects induced by serum. For example, serum exhibited amoebicidal effects; i.e., up to 50% of the exposed trophozoites were killed. The residual subpopulation, although viable, remained static over longer incubations. Interestingly, serum enhanced the phagocytic ability of acanthamoebae, as measured by bacterial uptake. Overall, our results demonstrate that human serum has inhibitory effects on Acanthamoeba growth and viability, protease secretions, and binding to and subsequent cytotoxicity for brain microvascular endothelial cells. Conversely, Acanthamoeba phagocytosis was stimulated by serum.

Acanthamoeba castellanii↗

PubMed2005

Balamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelial cells via a phosphatidylinositol 3-kinase-dependent pathway.

Balamuthia mandrillaris is an emerging protozoan parasite that can cause fatal granulomatous encephalitis. Haematogenous spread is a likely route prior to entry into the central nervous system (CNS), but it is not clear how circulating amoebae cross the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, we determined HBMEC inflammatory response to B. mandrillaris and the underlying mechanisms associated with this response. We demonstrated that HBMEC incubated with B. mandrillaris released significantly higher levels of interleukin-6 (IL-6) (>400 pg/ml) as compared with less than 50 pg/ml in HBMEC incubated alone. Western blotting assays determined that B. mandrillaris specifically activates phosphatidylinositol 3-kinase (PI3K). By using LY294002, a PI3K inhibitor, as well as by using HBMEC expressing dominant-negative PI3K, we have identified PI3K as an important mediator of B. mandrillaris-mediated IL-6 release. We conclude that B. mandrillaris induces HBMEC signalling pathways, which lead to IL-6 release. This is the first time PI3K has been shown to play a crucial role in B. mandrillaris-mediated IL-6 release in HBMEC.

Animals↗

PubMed2005

Mechanisms associated with Acanthamoeba castellanii (T4) phagocytosis.

Using fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba is studied. This assay is based on the quenching effect of trypan blue on FITC-labelled E. coli. Only intracellular E. coli retain their fluorescence, which are easily discriminated from non-fluorescent adherent bacteria. Acanthamoeba uptake of E. coli is significantly reduced in the presence of genistein, a protein tyrosine kinase inhibitor. In contrast, sodium orthovanadate (protein tyrosine phosphatase inhibitor) increases bacterial uptake by Acanthamoeba. Treatment of Acanthamoeba with cytochalasin D (actin polymerization inhibitor) abolished the ability of Acanthamoeba to phagocytose E. coli suggesting that tyrosine kinase-mediated signaling may play a role in Acanthamoeba phagocytosis. In addition, we showed that phosphatidylinositol 3-kinase (PI3K) plays an important role in Acanthamoeba uptake of E. coli. Role of mannose-binding protein in Acanthamoeba phagocytosis is discussed further.

Acanthamoeba castellanii↗

PubMed2005

Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells.

Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and can cause human infections. The life cycle of Acanthamoeba consists of an infective trophozoite form. However under harsh environmental conditions trophozoites differentiate into a double-walled, metabolically inactive and resistant cyst form. Research in Acanthamoeba has mostly focussed on the infective trophozoite form and its pathogenic mechanisms. In this study, we used Acanthamoeba isolates belonging to T1, T2, T3, T4, T7 genotypes and studied their cysts properties. We determined that food deprivation stimulates encystment in Acanthamoeba isolates belonging to T1, T2, T3, T4 and T7 genotypes in a sodium dodecyl sulfate (SDS)-resistant manner. In addition, increase in osmolarity triggered encystment in T1, T2, T3, T4 isolates (SDS-resistant) but T7 failed to encyst (SDS-labile). Adhesion assays revealed that Acanthamoeba cysts belonging to T1, T2, T3, T4, and T7 genotypes exhibited no and/or minimal binding (<5%) to the host cells. Fluorescein-labelled lectins showed that all Acanthamoeba isolates tested exhibited binding to concanavalin A, indicating the expression of mannosyl- and/or glucosyl-residues. Role of cysts in the transmission of infection is discussed further.

Acanthamoeba↗

PubMed2005

Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood-brain barrier.

OBJECTIVES: Granulomatous amoebic encephalitis (GAE) is a serious human infection with fatal consequences, however, the pathogenic mechanisms associated with this disease remain unclear. Several lines of evidence suggest that haematogenous spread is a prerequisite for Acanthamoeba encephalitis but it is not clear how circulating amoebae cross the blood-brain barrier to gain entry into the central nervous system. Objectives of this study were to determine the effects of Acanthamoeba on the permeability of an in vitro blood-brain barrier model and factors contributing to these changes. METHODS: Using human brain microvascular endothelial cells, an in vitro blood-brain barrier model was constructed in 24-well Transwell plates. Acanthamoeba (GAE isolate belonging to T1 genotype) or its conditioned media were used to determine permeability changes. Zymography assays were performed to characterise Acanthamoeba proteases. In addition, the ability of Acanthamoeba to bind brain microvascular endothelial cells was determined using adhesion assays. RESULTS: We observed that Acanthamoeba produced an increase of more than 45% in the blood-brain barrier permeability. Acanthamoeba-conditioned media exhibited similar effects indicating Acanthamoeba-mediated blood-brain barrier permeability is contact-independent. Prior treatment of conditioned media with phenylmethyl sulfonyl fluoride (PMSF, serine protease inhibitor), abolished permeability changes indicating the role of serine proteases. Of interest, methyl alpha-d-mannopyranoside inhibited Acanthamoeba binding to human brain microvascular endothelial cells but had no effect on Acanthamoeba-mediated blood-brain barrier permeability. Zymography assays revealed that Acanthamoeba produced two major proteases, one of which was inhibited by PMSF (serine protease inhibitor) and the second with 1,10-phenanthroline (metalloprotease inhibitor). CONCLUSIONS: We have for the first time shown that Acanthamoeba produces human brain microvascular endothelial cells permeability, which can be blocked by PMSF. A metalloprotease of approx. molecular weight of 150 kDa is produced by A. castellanii (GAE isolate belonging to T1 genotype) and its role in the disease is suggested.

Acanthamoeba castellanii↗

PubMed2005

Acanthamoeba genotype T4 from the UK and Iran and isolation of the T2 genotype from clinical isolates.

The majority of the keratitis-causing Acanthamoeba isolates are genotype T4. In an attempt to determine whether predominance of T4 isolates in Acanthamoeba keratitis is due to greater virulence or greater prevalence of this genotype, Acanthamoeba genotypes were determined for 13 keratitis isolates and 12 environmental isolates from Iran. Among 13 clinical isolates, eight (61.5%) belonged to T4, two (15.3%) belonged to T3 and three (23%) belonged to the T2 genotype. In contrast, the majority of 12 environmental isolates tested in the present study belonged to T2 (7/12, 58.3%), followed by 4/12 T4 isolates (33.3%). In addition, the genotypes of six new Acanthamoeba isolates from UK keratitis cases were determined. Of these, five (83.3%) belonged to T4 and one was T3 (16.6%), supporting the expected high frequency of T4 in Acanthamoeba keratitis. In total, the genotypes of 24 Acanthamoeba keratitis isolates from the UK and Iran were determined. Of these, 17 belonged to T4 (70.8%), three belonged to T2 (12.5%), three belonged to T3 (12.5%) and one belonged to T11 (4.1%), confirming that T4 is the predominant genotype (S2=4.167; P=0.0412) in Acanthamoeba keratitis.

Acanthamoeba↗

PubMed2005

Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism.

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase.

Acanthamoeba castellanii↗

PubMed2005

Escherichia coli K1 RS218 interacts with human brain microvascular endothelial cells via type 1 fimbria bacteria in the fimbriated state.

Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

Adhesins, Escherichia coli↗