Eu-Hian Yap

Recent advances in Blastocystis hominis research: hot spots in terra incognita

Despite being discovered more than 80 years ago, progress in Blastocystis research has been gradual and challenging, due to the small number of laboratories currently working on this protozoan parasite. To date, the morphology of Blastocystis hominis has been extensively studied by light and electron microscopy but all other aspects of its biology remain little explored areas. However, the availability of numerous and varied molecular tools and their application to the study of Blastocystis has brought us closer to understanding its biology. The purpose of this review is to describe and discuss recent advances in B. hominis research, with particular focus on new, and sometimes controversial, information that has shed light on its genetic heterogeneity, taxonomic links, mode of transmission, in vitro culture and pathogenesis. We also discuss recent observations that B. hominis has the capacity to undergo programmed cell death; a phenomenon similarly reported for many other unicellular organisms. There are still many gaps in our knowledge of this parasite. Although there is a growing body of evidence suggesting that B. hominis can be pathogenic under specific conditions, there are also other studies that indicated otherwise. Indeed, more studies are warranted before this controversial issue can be resolved. There is an urgent need for the identification and/or development of an animal model so that questions on its pathogenesis can be better answered. Another area that requires attention is the development of methods for the transfection of foreign/altered genes into B. hominis in order to facilitate genetic experiments.

Full-length cDNA sequence of dengue type 1 virus (Singapore strain S275/90)☆

The complete nucleotide sequence and the deduced amino acid sequence of the genome of dengue virus type 1 (Singapore strain S275/90) were determined from cDNA clones. The single-stranded, positive-sense RNA is 10,718 nucleotides in length and contains a single long open reading frame of 10,188 nucleotides encoding a polyprotein of 3396 amino acids. The genomic size and organization were found to be similar to that of other dengue virus serotypes. Both the nucleotide and deduced amino acid sequences were compared with the partial sequence of DEN1 (Nauru Island) and complete sequences of DNE2 (Jamaica), DEN3 (H87), and DEN4 (Dominica) virus genomes.

DNA Polymorphism Revealed by Arbitrary Primers Polymerase Chain Reaction Among Blastocystis Strains Isolated from Humans, a Chicken, and a Reptile

DNA polymorphisms of different strains of Blastocystis isolated from humans, a chicken, and a reptile were examined by an arbitrary primer PCR method. Two strains of Blastocystis hominis isolated from humans in the USA and Japan yielded nearly identical PCR products. However, one strain of B. hominis (isolated from a human in Singapore) yielded quite different PCR products. Blastocystis sp. isolated from a chicken yielded PCR products similar to those of the former two strains, while Blastocystis lapemi, isolated from a reptile, shared no bands with any of the other isolates. These results indicate the possibility that our isolate from the chicken is a zoonotic strain, and that there is intraspecific variation of Blastocystis hominis.

KINETICS OF INTERFERON-GAMMA SECRETION AND ITS REGULATORY FACTORS IN THE EARLY PHASE OF ACUTE GRAFT-VERSUS-HOST DISEASE

Increased serum levels of interferon‐γ (IFN‐γ) have been observed in acute graft‐versus‐host disease (GVHD). Recent in vitro studies have demonstrated that interleukin‐12 (IL‐12) and interleukin‐18 (IL‐18) synergistically up‐regulate IFN‐γ secretion. In this communication, we investigated the factors relevant to IFN‐γ secretion in acute GVHD. A murine model of acute GVHD was established by injecting donor spleen cells into severe combined immunodeficiency (SCID) mice. A series of specimens, including sera, livers and spleens derived from the GVHD mice, were investigated with histological examination, enzyme‐linked immunosorbent assay (ELISA), flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). IFN‐γ secretion increased in serum 3 days after spleen cell transfer, peaked on day 7, and then gradually decreased close to the baseline level by day 35. A synchronized increase of activated T cells and mRNA expression of IL‐12, IL‐18 and their respective receptors was observed after spleen cell transfer. However, only the kinetic expression pattern of IL‐12 receptor (IL‐12R) β2 chains was closely correlated with that of IFN‐γ, while IL‐12 dropped to the baseline level earlier than IFN‐γ. Therefore, IFN‐γ expression in the early phase of acute GVHD is a mono‐peak and self‐restricted pattern. Its secretion is closely related with T‐cell activation, the presence of IL‐12, IL‐18 and their respective receptors. However, the limiting factors for IFN‐γ secretion seem to be IL‐12 and IL‐12R β2 chains.

Blocking L‐selectin and α4‐integrin changes donor cell homing pattern and ameliorates murine acute graft versus host disease

L‐selectin, LFA‐1 and α4 integrins play important roles in the homing of naïve T cells into peripheral lymphoid tissues. L‐selectin‐ or LFA‐1‐deficient lymphocytes cannot effectively home to lymph nodes (LN), and antibody blockade of α4 integrins also hinders lymphocytes homing. The present study was initiated to explore whether it is feasible to ameliorate acute graft‐versus‐host disease (aGVHD) by modulating the homing process of donor cells in the recipient in a mouse model. Using a fluorescence labeling method, we found that two monoclonal antibodies directed at L‐selectin and α4 integrins, respectively, when used in combination, could delay half of the donor C57BL / 6J mouse spleen cells homing into the LN of recipient BALB / c mouse 15 h after injection. Spleen cells (1 × 107) derived from C57BL / 6J (H‐2b) mice were injected into each C.B‐17 SCID recipient mouse (H‐2d) with or without prior incubation with 10 μg each of the two antibodies. T cell repopulation in the blood was observed in both groups of mice at a comparable level 14 days after injection of the donor cells. Eight control mice started to show aGVHD signs 7 – 14 days after the injection, and all died by day 31. However, among the ten mice that received the antibody‐treated donor cells, two died before day 29, four survived between 36 and 78 days, and the remaining four survived more than 150 days, with two of them aGVHD free. It is apparent that the temporarily reduced lymphocyte homing into LN reduced the alloreactivity of the donor T cells, thus providing a simple way of modifying aGVHD. This novel approach may shed light on the prevention of aGVHD associated with clinical bone marrow transplantation.

A survey of Blastocystis in reptiles.

A total of 28 species of reptiles were investigated forBlastocystis using light microscopy and in vitro culture in biphasic egg slant medium.Blastocystis species were detected in 8 (28.6%) of these 28 species in 3 tortoises (Geochelone elephantopus, G. elegans andG. carbonaria), 3 snakes (Boiga dendrophilla, Python reticulatus andElaphe radiata), 1 crocodile (Crocodylus porosus) and 1 iguana lizard (Cyclura cornuta). The reptilianBlastocystis appeared to be morphologically similar toB. hominis.

A Blastocystis species from the sea-snake, Lapemis hardwickii (Serpentes: Hydrophiidae)

Observations were made on Blastocystis isolated from the sea-snake, Lapemis hardwickii. Exponential growth of the organism was observed between 2 and 4 days of culture. Vacuolated, amoeboid and granular forms were observed in cultures, similar to B. hominis. The optimal growth temperature for the sea-snake Blastocystis was 24 degrees C compared with 37 degrees C for B. hominis. The karyotypic patterns of B. hominis and the sea-snake Blastocystis were studied in the clamped homogeneous electric field (CHEF) technique and found to be different. Based on the above differences, the sea-snake Blastocystis was designated as Blastocystis lapemi sp. nov.

Passive protection studies in mice with monoclonal antibodies directed against the non-structural protein NS3 of dengue 1 virus.

Antibody-mediated enhancement of dengue virus replication is thought to be a mechanism contributing to the pathogenesis of dengue haemorrhagic fever and dengue shock syndrome. Enhancement is associated with antibodies to structural components of the virus. To circumvent the problem of immune enhancement, studies to identify protective antigens of dengue virus have involved non-structural proteins. Passive and active protection against lethal dengue virus infection in mice have been demonstrated with the non-structural protein NS1. In this study, the dengue virus non-structural protein NS3 was examined in passive protection studies with monoclonal antibodies prepared against NS3 of dengue 1 virus (Hawaiian). Five monoclonal antibodies that were authenticated to be reactive to NS3 were used to immunize 13- to 14-day old mice intraperitoneally. Thereafter, the mice were challenged intracerebrally with 100 LD50 of neurotropic dengue 1 virus and the survival indices of the mice were calculated. Significant decreases in survival indices (P less than 0.05), indicating increases in survival times were observed with four of five monoclonal antibodies tested. Monoclonal antibodies to NS3 of dengue 1 virus are able to increase the survival time of mice challenged with a lethal dose of dengue 1 virus, although the mechanism remains to be defined.

Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing

Abstract The growth of axenic reptilian isolates of Blastocystis in Iscove’s modified Dulbecco’s medium (IMDM) was studied and the morphology of the parasite was examined by phase-contrast microscopy. The chromosomal patterns of these reptilian isolates of Blastocystis were examined by pulsed-field gel electrophoresis (PFGE) and compared with those of B. hominis and B. lapemi, a sea snake Blastocystis. IMDM with 10% horse serum supported excellent growth of the reptilian Blastocystis isolates. The parasites from all the isolates were predominantly vacuolar, but multivacuolar and amoeboid forms were also seen. Amoeboid forms with rather elongate pseudopodia were also observed. There were some differences in size, morphology, and growth characteristics in the different reptilian isolates. The karyotypic patterns of the Blastocystis isolates from tortoise, iguana, and python were distinctly different from one another and from those obtained with B. hominis and B. lapemi. On the basis of the above-mentioned differences in chromosomal patterns, the tortoise, iguana, and python isolates are described as new species, viz., B. geocheloni sp. nov. from Geochelone carbonaria (red-footed tortoise), B. cycluri sp. nov. from Cyclura cornuta (rhino iguana), and B. pythoni sp. nov. from Python reticulatus (reticulated python).

In vitro response of Blastocystis hominis against traditional Chinese medicine.

This is the first inn vitro study on the activity of 20 kinds of crude extracts of traditional Chinese medicine (TCM) on the intestinal parasite, Blastocystis hominis using the criteria of living cell count (LCC) and living cell rate (LCR). LCC and LCR were applied as observation indicators, the former as a fixed-quantity and the latter as a fixed-quality method. LCR calculated percentage rate of living cells using eosin-brilliant cresyl blue staining which could differentiate between living cells and dying or dead cells. There were five extracts with no inhibitory activity, thirteen with moderate inhibition and two with high inhibition. The crude extracts of Coptis chinensis (CC) and Brucea javanica (BJ) were found to be most active against B. hominis. The active concentration of CC was 100 micrograms/ml. The active concentration of BJ was 500 micrograms/ml. The active concentration of metronidazole (MD) was 10 micrograms/ml and this was taken as an active standard drug for B. hominis.