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PubMed1956

The effect of enucleation on the DPN level of ameba.

1. Amebae contain DPN at levels of from 1 to 4 x 10(-13) moles per cell. 2. Following enucleation, nucleate and enucleate halves continue to have equal DPN contents over the six day period studied. Similarly, starving whole amebae maintain their DPN level over this period. 3. No reduced DPN could be detected in these aerobic animals. This remained true for whole amebae and for nucleate and enucleate halves over 5 days of starvation. 4. A method is described for the preparation and rapid separation of nucleate and enucleate ameba halves, based on a response of amebae to light.

Amoeba↗
PubMedDate not supplied

Epidemiology of free-living ameba infections.

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred worldwide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.

Acanthamoeba↗
PubMedDate not supplied

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum.

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.

Amebiasis↗
PubMedDate not supplied

In-vitro activity of miltefosine and voriconazole on clinical isolates of free-living amebas: Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri.

The anticancer agent miltefosine and the antifungal drug voriconazole were tested in vitro against Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri. All three amebas are etiologic agents of chronic (Balamuthia, Acanthamoeba) or fulminant (Naegleria) encephalitides in humans and animals and, in the case of Acanthamoeba, amebic keratitis. Balamuthia exposed to <40 microm concentrations of miltefosine survived, while concentrations of >or=40 microM were generally amebacidal, with variation in sensitivity between strains. At amebastatic drug concentrations, recovery from drug effects could take as long as 2 weeks. Acanthamoeba spp. recovered from exposure to 40 microM, but not 80 microM miltefosin. Attempts to define more narrowly the minimal inhibitory (MIC) and minimal amebacidal concentrations (MAC) for Balamuthia and Acanthamoeba were difficult due to persistence of non-proliferating trophic amebas in the medium. For N. fowleri, 40 and 55 microM were the MIC and MAC, respectively, with no trophic amebas seen at the MAC. Voriconazole had little or no inhibitory effect on Balamuthia at concentrations up to 40 microg/ml, but had a strong inhibitory effect upon Acanthamoeba spp. and N. fowleri at all drug concentrations through 40 microg/ml. Following transfer to drug-free medium, Acanthamoeba polyphaga recovered within a period of 2 weeks; N. fowleri amebas recovered from exposure to 1 microg/ml, but not from higher concentrations. All testing was done on trophic amebas; drug sensitivities of cysts were not examined. Miltefosine and voriconazole are potentially useful drugs for treatment of free-living amebic infections, though sensitivities differ between genera, species, and strains.

Acanthamoeba↗
PubMedDate not supplied

Amebae resembling Entamoeba gingivalis in the genital tracts of IUD users.

A total of 2,252 vaginopancervical (Fast) smears were studied as a continuation of an ongoing program to further investigate the occurrence of amebae in the genital tracts of women using intrauterine devices (IUDs). Of the 947 IUD users, 1% harbored trophic amebae. No amebae, however, were found in any of the 1,164 smears from non-IUD wearers. In all instances the amebae were associated with Actinomyces. Both organisms disappeared promptly after removal of the IUD. In preparations stained according to the Papanicolaou, iron-hematoxylin and Gomori-Wheatley trichrome methods, the amebae could be diagnosed as belonging to the genus Entamoeba. These trophozoites failed to stain with fluorescein-labeled anti-Entamoeba-histolytica serum. Although on the basis of the presently available evidence these trophozoites cannot be assigned with certainty to any known species, they appear to share many morphologic characteristics with Entamoeba gingivalis.

Actinomyces↗
PubMed1999

Persistence and multiplication of obligate anaerobe bacteria in amebae under aerobic conditions.

After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.

Journal Article↗
PubMed1993

Entamoeba histolytica: an explanation for the reported conversion of "nonpathogenic" amebae to the "pathogenic" form.

The reported conversion of "nonpathogenic" Entamoeba histolytica isolates to the "pathogenic" form during attempted axenization of the amebae is highly controversial. After failing to obtain conversions ourselves we concluded that the simplest explanation for the published observations would be contamination of nonpathogenic cultures with pathogenic amebae. To address this possibility we used a method based on analysis of stable DNA polymorphisms that allows the positive identification of individual pathogenic isolates. The DNA patterns obtained using the "converted" amebae proved to be identical to those of reference isolates present in the laboratories at the time of conversion. We also found that very few cells need be transferred for a pathogenic contaminant to become established in a nonpathogenic culture. Cross-contamination fully explains the conversion phenomenon and thus recognition of nonpathogenic and pathogenic amebae as the distinct species Entamoeba dispar Brumpt 1925 and E. histolytica Schaudinn 1903 (Emend. Walker 1911), respectively, is upheld.

Animals↗
PubMed1985

Dictyostelium amebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP.

Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.

Cell Aggregation↗
PubMed1990

Cytoplasmic pH of Dictyostelium discoideum amebae during early development: identification of two cell subpopulations before the aggregation stage.

Development of the cellular slime mold Dictyostelium discoideum is initiated by the removal of nutrients, and results in formation of a mature fruiting body composed of two cell types, the stalk and spore cells. A considerable body of evidence supports the hypothesis that cytoplasmic pH may be an essential regulator of the choice to differentiate in either the prestalk or prespore pathway. We have devised methods for measurement and analysis of intracellular pH in developing Dictyostelium amebae in order to assess directly the potential role of cytoplasmic pH in regulating the pathway of differentiation. The intracellular pH of single D. discoideum amebae during development and in intact slugs has been measured using the pH-sensitive indicator pyranine in a low light level microspectrofluorometer. We have used the ATP-mediated loading method to introduce pyranine into these cells. Cells loaded by the ATP method appear healthy, have no detectable defects in development, and exhibit a similar population distribution of intracellular pH to those loaded by sonication. The intracellular pH of populations comprised of single amebae was found to undergo a transient acidification during development resulting in a bimodal distribution of intracellular pH. The subpopulations were characterized by fitting two gaussian distributions to the data. The number of cells in the acidic intracellular pH subpopulation reached a maximum 4 h after initiation of development, and had returned to a low level by 7 h of development. In addition, a random sample of single amebae within a slug had a median intracellular pH of 7.2, nearly identical to the median pH (7.19) of similarly treated vegetative cells. No gradient of intracellular pH along the anterior to posterior axis of the slug was detected. Our data demonstrate the existence of two distinct subpopulations of cells before the aggregation stage of development in Dictyostelium, and offers support for the hypothesis that changes in intracellular pH contribute to development in D. discoideum.

Adenosine Triphosphate↗
PubMed1979

Recovery of soil Amebas from the nasal passages of children during the dusty harmattan period in Zaria.

Following a fatal case of primary amebic meningoencephalitis during the dusty harmattan period in an 8-month-old child in whose case Naegleria fowleri was recovered both from the cerebrospinal fluid and from material from the nose in absence of a history of swimming, it was hypothesized that dust during the harmattan might harbor amebic cysts, which may be inhaled by human beings and cause infection. A preliminary survey was thus carried out to examine the nasal passages of children for the presence of soil amebas during the harmattan. In all, 50 children were evaluated for the presence of soil amebas. Positive cultures for the soil amebas were obtained from 12 children (24%). Four species of amebas were isolated singly or in combination with other species. Pathogenic Naegleria fowleri, proved pathogenic for mice, were cultured from specimens from two children.

Amebiasis↗
PubMed1977

Ultrastructural observations of experimental Naegleria meningoencephalitis in mice: intranuclear inclusions in amebae and host cells.

Primary amebic meningoencephalitis was experimentallly produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality with average time of onset of symtoms of death occurring on the 7-8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.

Amebiasis↗
PubMed2002

Cultivation of pathogenic and opportunistic free-living amebas.

Free-living amebas are widely distributed in soil and water, particularly members of the genera Acanthamoeba and NAEGLERIA: Since the early 1960s, they have been recognized as opportunistic human pathogens, capable of causing infections of the central nervous system (CNS) in both immunocompetent and immunocompromised hosts. Naegleria is the causal agent of a fulminant CNS condition, primary amebic meningoencephalitis; Acanthamoeba is responsible for a more chronic and insidious infection of the CNS termed granulomatous amebic encephalitis, as well as amebic keratitis. Balamuthia sp. has been recognized in the past decade as another ameba implicated in CNS infections. Cultivation of these organisms in vitro provides the basis for a better understanding of the biology of these amebas, as well as an important means of isolating and identifying them from clinical samples. Naegleria and Acanthamoeba can be cultured axenically in cell-free media or on tissue culture cells as feeder layers and in cultures with bacteria as a food source. Balamuthia, which has yet to be isolated from the environment, will not grow on bacteria. Instead, it requires tissue culture cells as feeder layers or an enriched cell-free medium. The recent identification of another ameba, Sappinia diploidea, suggests that other free-living forms may also be involved as causal agents of human infections.

Acanthamoeba↗
PubMed1990

Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals.

Amebae belonging to the order Leptomyxida are regarded as innocuous soil organisms incapable of infecting mammals. We report here the isolation of a leptomyxid ameba from the brain of a pregnant baboon (Papio sphinx) that died of meningoencephalitis at the San Diego Zoo Wild Animal Park. By using rabbit anti-leptomyxid serum in the immunofluorescence assay, we have identified the leptomyxid ameba in the brain sections of a number of human encephalitic cases from around the world as well as a few cases of meningoencephalitis in animals in the United States, which suggests that the leptomyxid amebae are potential etiologic agents of fatal meningoencephalitis in humans and animals.

Amebiasis↗
PubMed1988

Transcriptional regulation of coordinate changes in flagellar mRNAs during differentiation of Naegleria gruberi amebae into flagellates.

The nuclear run-on technique was used to measure the rate of transcription of flagellar genes during the differentiation of Naegleria gruberi amebae into flagellates. Synthesis of mRNAs for the axonemal proteins alpha- and beta-tubulin and flagellar calmodulin, as well as a coordinately regulated poly(A)+ RNA that codes for an unidentified protein, showed transient increases averaging 22-fold. The rate of synthesis of two poly(A)+ RNAs common to amebae and flagellates was low until the transcription of the flagellar genes began to decline, at which time synthesis of the RNAs found in amebae increased 3- to 10-fold. The observed changes in the rate of transcription can account quantitatively for the 20-fold increase in flagellar mRNA concentration during the differentiation. The data for the flagellar calmodulin gene demonstrate transcriptional regulation for a nontubulin axonemal protein. The data also demonstrate at least two programs of transcriptional regulation during the differentiation and raise the intriguing possibility that some significant fraction of the nearly 200 different proteins of the flagellar axoneme is transcriptionally regulated during the 1 h it takes N. gruberi amebae to form visible flagella.

Animals↗
PubMed1984

Pathogenic free-living amebae. Immunocytologic demonstration and species identification.

This report describes methods for preparation, immunologic marking, and staining of amebae in formalin-fixed brain tissue, and in exudates, discharges and body fluids. The suggested procedures provide slide preparations of unknown specimens together with known amebae from cultures as controls, all on a single slide for each antiserum. This allows for cytologic studies of host cells, morphology of the amebae present, and specific immune marking of the amebae by use of immune sera and protein A Staphylococcus. Such preparations may also be used for immunoperoxidase and immunofluorescence. The need for the utilization of direct microscopic examination by the methods suggested here, or comparable ones, is discussed.

Agglutination Tests↗
PubMed2006

The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates.

Differentiation of Naegleria amebae into flagellates was used to examine the interaction between actin, actomyosin and microtubules in defining cell shape. Amebae, which lack microtubules except during mitosis, differentiate into flagellates with a fixed shape and a complex microtubule cytoskeleton in 120 min. Based on earlier models of ameboid motility it has been suggested that actomyosin is quiescent in flagellates. This hypothesis was tested by following changes in the cytoskeleton using three-dimensional reconstructions prepared by confocal microscopy of individual cells stained with antibodies against actin and tubulin as well as with phalloidin and DNase I. F-actin as defined by phalloidin staining was concentrated in expanding pseudopods. Most phalloidin staining was lost as cells rounded up before the onset of flagellum formation. Actin staining with a Naegleria-specific antibody that recognizes both F- and G-actin was confined to the cell cortex of both amebae and flagellates. DNase I demonstrated G-actin throughout all stages. Most of the actin in the cortex was not bound by phalloidin yet was resistant to detergent extraction suggesting that it was polymerized. The microtubule cytoskeleton of flagellates was intimately associated with this actin cortex. Treatment of flagellates with cytochalasin D produced a rapid loss of flagellate shape and the appearance of phalloidin staining while latrunculin A stabilized the flagellate shape. These results suggest that tension produced by an actomyosin network is required to maintain the flagellate shape. The rapid loss of the flagellate shape induced by drugs, which specifically block myosin light chain kinase, supports this hypothesis.

Actins↗
PubMed2004

Cannabinoid-mediated exacerbation of brain infection by opportunistic amebae.

Recent reports indicate a higher frequency of brain infections with opportunistic amebae of the genus Acanthamoeba among immune compromised individuals, including AIDS patients. We have demonstrated, using a murine model of Granulomatous Amebic Encephalitis (GAE), that the major psychoactive and immune suppressive component in marijuana delta-9-tetrahydrocannabinol (THC) exacerbates infection by these amebae. Mice administered THC and infected with Acanthamoeba exhibited dose-related higher mortalities than infected vehicle controls. The greater severity of disease for THC-treated mice was accompanied by decreased accumulation of macrophage-like cells at focal sites of infection in the brain. Furthermore, THC administration resulted in decreased levels of mRNA for the pro-inflammatory cytokines interleukin-1 alpha, interleukin-1 beta, and tumor necrosis factor alpha for neonatal rat microglia co-cultured with Acanthamoeba. These results indicate a potential for marijuana to alter the capacity of brain macrophage-like cells to mount a full complement of immune responsiveness to brain infection by opportunistic amebae.

Acanthamoeba↗
PubMed1986

New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates.

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).

Amoeba↗