Trophozoites of Entamoeba histolytica epigenetically silenced in several genes are virulence-attenuated.

The human intestinal parasite Entamoeba histolytica causes amoebic colitis and amoebic liver abscesses. Three classes of amoebic molecules have been identified as the major virulence factors, the Gal/GalNAc inhibitable lectin that mediates adherence to mammalian cells, the amoebapores which cause the formation of membrane ion channels in the target cells and the cysteine proteinases which degrade the matrix proteins, the intestinal mucus and secretory IgA. Transcriptional silencing of the amoebapore (Ehapa) gene occurred after transfection of trophozoites with a plasmid containing a segment of the 5' upstream region of the gene. Transcriptional silencing of the Ehap-a gene continued even after the removal of the plasmid and the cloned amoebae were termed G3. Transfection of G3 trophozoites with a plasmid construct containing the cysteine proteinase (EhCP-5) gene and the light subunit of the Gal- lectin (Ehlgl1) gene, each under the 5' upstream sequences of the amoebapore gene, caused the simultaneous epigenetic silencing of expression of these two genes. The resulting trophozoites, termed RB-9, were cured from the plasmid and they do not express the three types of virulent genes. The RB9 amoeba are virulence attenuated and are incapable of killing mammalian cells, they can not induce the formation of liver abscesses and they do not cause ulcerations in the cecum of experimental animals. The gene-silenced amoebae express the same surface antigens which are present in virulent strains and following intra peritoneal inoculation of live trophozoites into hamsters they evoked a protective immune response. Further studies are needed to find out if RB-9 trophozoites could be used for vaccination against amoebaisis.

Characteristics and outcomes of paediatric rhegmatogenous retinal detachment treated by segmental scleral buckling plus an encircling element.

PURPOSE: To evaluate the outcome of paediatric rhegmatogenous retinal detachment treated by segmental scleral buckling plus an encircling element. METHODS: A retrospective review was carried out of 15 consecutive paediatric patients (16 eyes). RESULTS: Thirteen of the 15 patients were male (87%), 2 (13%) were female. Their average age was 10.9 years (range 6-18 years; median 10 years). Retinal detachment was caused by trauma in 9 of 16 eyes (56%), high myopia in 5 of 16 eyes (31%) and had an unknown cause in 2 of 16 eyes (13%). Diagnosis was delayed by more than 1 month in 10 of 16 eyes (63%). The visual acuity was 6/60 or worse and the macula was detached on presentation in 13 of 16 eyes (81%). Two of 16 eyes (13%) had mild proliferative vitreoretinopathy. Final reattachment was achieved in all cases. Of 11 eyes with a follow-up of 6 months or more, there was improvement in visual acuity in 7 (63%), and a best-corrected visual acuity of 6/20 or better in 5 (46%). CONCLUSION: Paediatric rhegmatogenous retinal detachment is characterised by a delay in diagnosis and a high degree of macular involvement on presentation. Anatomical reattachment with segmental scleral buckling plus an encircling element was successful in all eyes, and improvement of visual acuity was achieved in one-half of the eyes which had a follow-up of 6 months or more.

Inhibition of gene expression in Entamoeba by the transcription of antisense RNA: effect of 5' and 3' regulatory elements.

Down regulation of gene expression by antisense RNA is one of the ways to investigate the specific contribution of certain components to the physiology and activities of a cell. A successful inhibition of gene expression in Entamoeba trophozoites was achieved in stable transfectants by using hybrid plasmid constructs containing promotors that produce transcripts which do not bind to polysomes. Different promotors were found to be required for Entamoeba histolytica or Entamoeba dispar. In E. histolytica one of the two copies (g34) of the gene coding for ribosomal protein L21 was previously found to be transcribed but not translated. Inhibition of gene expression was obtained by placing in a transfection vector, the amoebapore A gene, in its antisense orientation, under the control of the g34 promotor. Transfectants of E. histolytica were shown to accumulate antisense transcripts and inhibit amoebapore synthesis. In contrast, transfectants with plasmid constructs in which the amoebapore gene was placed under the control of the gLE3 promotor of RP-L21, which is known to be translated, did not accumulate antisense transcript or inhibit gene expression. Maximal inhibition of amoebapore expression was obtained when the antisense construct also included the 5' and 3' untranslated regions of the amoebapore gene. In E. dispar the opposite situation was found, plasmid constructs containing the promotor regions of the gLE3 copy, which were shown to be poorly translated, were more efficient in inhibiting the synthesis of a 30 kDa surface-specific antigen than a construct with the g34 promotor element.

Antisense inhibition of amoebapore expression in Entamoeba histolytica causes a decrease in amoebic virulence.

Amoebapores have been proposed to be a major pathogenicity factor of the protozoan parasite Entamoeba histolytica, which is responsible for the killing of target cells. These 77-residue peptides are structural and functional analogues of NK-lysin and granulysin of porcine and human cytotoxic lymphocytes. Inhibition of amoebapore gene expression in amoebae was obtained following transfection with a hybrid plasmid construct (pAP-R2) containing the Neo resistance gene and the gene coding for amoebapore A, including its 5' and 3' untranslated region (UTR) sequences, in reverse orientation under a promoter (g34) taken from one of the E. histolytica ribosomal protein (RP-L21) gene copies. Transfectants of virulent E. histolytica strain HM-1:IMSS, in which the expression of amoebapore was inhibited by approximately 60%, were significantly less pathogenic. Cytopathic and cytolytic activities of viable trophozoites against mammalian nucleated cells, as well as lysis of red blood cells, were markedly inhibited. Moreover, trophozoite extracts of pAP-R2 transfectant displayed lower pore-forming activity and were less potent in inhibiting bacterial growth compared with controls. Notably, liver abscess formation in hamsters by the pAP-R2 transfectant was substantially impaired. These results demonstrate for the first time that amoebapore is one of the pathogenicity factors by which trophozoites of E. histolytica exert their remarkable cytolytic and tissue destructive activity.

Analysis by enzyme-linked immunosorbent assay and PCR of human liver abscess aspirates from patients in China for Entamoeba histolytica.

Aspirates of liver abscesses were analyzed for Entamoeba histolytica. PCR detected a gene encoding a 30-kDa protein in all samples but detected the ribosomal DNA gene in only 14 (33.3%) samples. Enzyme-linked immunosorbent assay detected antigen in 41 (97.6%) samples. PCR analysis of a strain-specific antigen (SSG) revealed that abscesses were caused by various strains.

Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc lectin.

Entamoeba histolytica virulence is related to a number of amebic components (lectins, cysteine proteinases, and amebapore) and host factors, such as intestinal bacterial flora. Trophozoites are selective in their interactions with bacteria, and the parasite recognition of glycoconjugates plays an important role in amebic virulence. Long-term monoxenic cultivation of pathogenic E. histolytica trophozoites, strains HK-9 or HM-1:IMSS, with Escherichia coli serotype O55, which binds strongly to the Gal/GalNAc amebic lectin, markedly reduced the trophozoites' adherence and cytopathic activity on cell monolayers of baby hamster kidney (BHK) cells. Specific probes prepared from E. histolytica lectin genes as well as antibodies directed against the light (35-kDa) and heavy (170-kDa) subunits of the Gal/GalNAc lectin revealed a decrease in the transcription and expression of the light subunit in trophozoites grown monoxenically with E. coli O55. This effect was not observed when E. histolytica was grown with E. coli 346, a mannose-binding type I pilated bacteria. Our results suggest that the light subunit of the amebic lectin is involved in the modulation of parasite adherence and cytopathic activity.

Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.

Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.

A new method for isolation and differentiation of native Entamoeba histolytica and E. dispar cysts from fecal samples.

A new method for the purification of protozoan cysts from feces was established, allowing to isolation of native cysts. The procedure consists of two sucrose-density gradients and enzymatic digestion of cellulose particles by cellulase and can be accomplished in a few hours. The cyst fractions were differentiated into Entamoeba histolytica and E. dispar using the DNA probes P145 and B133 and a dot-blot test.

Inhibition of expression of the lysine-rich 30 kDa surface antigen of Entamoeba dispar by the transcription of its antisense RNA.

The gene coding for the 30 kDa lysine rich surface antigen (Ed-Ag) that is present on membrane surfaces of Entamoeba dispar trophozoites has been characterized. A specific monoclonal antibody MAb 318-28 prepared against this antigen reacts with all E. dispar strains tested, but not with any of the antigens of E. histolytica. In order to understand the function of this antigen, we constructed two plasmids, pEdA-9 and pEdA-Rev, in which the antigen-coding sequence was introduced into the pEhAct-Neo shuttle vector in the direct and opposite orientation, respectively. When E. dispar trophozoites were transfected with pEdA-9, only a slight increase was observed in the expression of the antigen. However, when E. dispar trophozoites were transfected with pEdA-Rev, the expression of the native 30 kDa antigen was significantly inhibited. This inhibition was proportional to the level of resistance of the E. dispar culture to the neomycin derivative G418. Cytopathic assays detected only a slight difference between untransfected, pEdA-9 transfected and pEdA-Rev transfected trophozoites.

Molecular cloning of a 30-kilodalton lysine-rich surface antigen from a nonpathogenic Entamoeba histolytica strain and its expression in a pathogenic strain.

A monoclonal antibody (MAb), 318-28, that specifically reacts with a 30-kDa antigen present on membrane surfaces of all nonpathogenic (NP) Entamoeba histolytica strains tested and which did not react with pathogenic (P) strains was used for the isolation of the cDNA coding for this antigen from an expression library of an NP E. histolytica strain. The deduced amino acid composition was rich in lysine residues (14.5%), with some sequence similarity to a polyadenylate-binding protein. Southern and Northern (RNA) blot analyses, as well as amplifications of DNA segments by PCR, indicate that a very similar gene (identity of 96.5%) exists in P strains of E. histolytica. Unexpectedly, the NP-specific antigen was also identified by MAb 318-28 on the surfaces of a cloned, xenically cultivated and well-characterized P strain (BNI:0591) that was recently isolated from a human liver abscess. Binding of the MAb, both to the cell surfaces and to Western blots (immunoblots), was abolished, however, upon axenization of the BNI:0591 cultures. Oligonucleotide primers, designed to anneal only to specific DNA sequences of the NP 30-kDa protein gene copy, amplified a DNA segment from P strain BNI:0591 which was identical in sequence to that of the NP 30-kDa protein gene. Our findings indicate that a P strain of E. histolytica can possess and express, under certain growth conditions, an antigen that is usually detected only in NP strains.

Deformed optic chiasm and colloid cyst in a patient with visual disturbances.

Colloid cysts of the third ventricle are rare intracranial lesions which comprise 0.5 to 1% of all intracranial tumors. We describe a patient with a third ventricle colloid cyst who presented with non specific visual deterioration.

Spontaneous resolution of proliferative vitreoretinopathy.

A case of proliferative vitreoretinopathy that spontaneously regressed, leading to reattachment of the retina, is presented. The importance of such a spontaneous resolution in terms of understanding the pathogenesis, and planning a treatment, is discussed.

Cloning and partial characterization of an antigen detected on membrane surfaces of non-pathogenic strains of Entamoeba histolytica.

A novel and unique mAb (318-28) which specifically interacts with a 60 kDa antigen that is found only on the surfaces of non-pathogenic (NP) strains of E. histolytica has been recently characterized. The antigen appears to be present also on (NP) cyst forms of amebas but was not detected on any of the various (P)-strains tested. It was also not found on other Entamoeba species such as Moshkovskii, Laredo, Huff, Coli, Gingivalis, or Invadens. Clinical trails for the differentiation of (P) and (NP) amebas directly from stools using this mAb are in progress. Cloning of the gene encoding for the (NP)-specific antigen was achieved after screening with mAb 318-28 a lambda gt11 expression library of NP strain SAW 1734R. No sequence homology to any known protein was found in data bases.

Intraocular lens in a fighter aircraft pilot.

A pseudophakic pilot of the Israeli air force flying an F-15 (Eagle) aircraft was followed up for three years. He experienced about 100 flying hours, 5% of the time under high g stress. The intraocular lens did not dislocate and no complications were observed. It seems that flying high performance fighter aircraft is not contraindicated in pseudophakic pilots.