An in vitro study on thalassemic erythroid precursors in liquid culture.

Thalassemia is an inherited hematological disorder which can generally be classified according to the affected globin imbalance (alpha- or beta-globin) into two main types, i.e. alpha-thalassemia and beta-thalassemia, respectively. There is a wide range of cellular abnormalities associated with thalassemic erythrocytes such as hypochromia, microcytosis, reduced cellular deformability and membrane oxidative damage. The red cell abnormalities lead to premature destruction with marrow erythroid hyperplasia and ineffective erythropoiesis. The abnormalities in thalassemic red blood cells have been found along the erythroid differentiation pathway other than the mature stage as previously shown in bone marrow erythroid precursors and in reticulocytes, the penultimate stage of erythroid differentiation. However, there is a lag in our understanding of the more primitive erythroid stages due to the difficult and hazardous marrow aspiration and heterogeneity of cells derived. We have utilized a novel method of Two-Phase Liquid Culture (TPLC) of beta-thalassemia/HbE erythroid precursors instead of conventional semisolid culture. This type of liquid culture can given higher cell yield with quite synchronous cell differentiation stages and easily be applied for other cellular analytical techniques. The peripheral blood mononuclear cells (PBMC) obtained from non-splenectomized and splenectomized beta-thalassemia/HbE patients were first cultured in medium supplemented with 5637 conditioned medium for a 6-day period (phase I) and then transferred to medium supplemented with recombinant human erythropoietin to allow the terminal differentiation of erythroid precursors (phase II). During the phase I or II, the cultured cells were periodically sampled to determine the cell number, cytocentrifuged on glass slides and stained with Wright stain for morphological assessment of their differentiation stages and analyzed flow cytometrically by staining with fluoresceinated anti-transferrin receptor (anti-CD71) and R-phycoerythrin-conjugated anti-glycophorin A. After assessment by flow cytometry, the remaining stained cells were cytocentrifuged on glass slides and photographed by a fluorescent microscope and a laser scanning confocal microscope. The results of morphological assessment, flow cytometric analysis and microscopic pictures will be presented.

Dengue-3 (16562) PGMK 33 vaccine: neurovirulence, viremia and immune responses in Macaca fascicularis.

Investigation of monkey neurovirulence of dengue-3 viruses (DEN-3, 16562) was undertaken to provide an evaluation of the relative safety of virus strain attenuated for potential use of live virus vaccine. Ten flavivirus-negative, cynomolgus monkeys (Macacafascicularis) were used in the test. The animals were inoculated intrathalamically, intraspinally and intramuscularly with DEN-3 PGMK 33 attenuated live virus vaccine (6 monkeys): parent virus (2) and control cell culture fluid (2). Blood samples were collected on days 0, 1, 2, 4, 6, 8, 10, 12 and 21 for virus isolation and days 0 and 21 or 22 for serologic testing. One monkey with DEN-3 (16562) PGMK 33 candidate vaccine had detectable viremia on day 10. By day 21, all recipients of PGMK 33 and both monkeys with DEN-3 parent virus developed serum neutralizing antibodies to DEN-3 titers ranged from 56-320. The monkeys showed no evidence of illness and none died of dengue infection. Histopathological examination of tissue collected on day 21 or 22 revealed only minimal neurovirulence lesions as scored by the routine grading system. No differences were observed between the DEN-3 parent and vaccine viruses and it is concluded that neither virus is neurovirulent for cynomolgus monkeys.

Scanning electron microscopic study of the tegumental surface of the hybrid schistosome between Schistosoma mekongi and S. japonicum-like (Malaysian).

Hybridization experiments between the two non-sibling species of schistosomes, Schistosoma mekongi in man and S. japonicum-like (Malaysian) in rodents, were carried out. Two laboratory-bred snail species, Tricula aperta (beta race), the snail host of S. mekongi and Robertsiella kaporensis, the snail host of S. japonicum-like (Malaysian), were used for the production of cercariae. Cross mating between S. mekongi and S. japonicum-like (Malaysian) were achieved in the laboratory by the usual procedure of exposing snails to single miracidia of each species, then exposing mice to cercariae emanating from two snails only, each infected with a different species. Hybrid eggs and miracidia were used to infect snails of both species. The resultant F1 cercariae were used to infect mice. It was shown in this study that the attempt to cross these two species of schistosomes could be achieved in the laboratory, but the results provided very low yield of hybrid worms and eggs. F1 hybrid adult worms from S. mekongi male and S. japonicum-like (Malaysian) female were obtained and examined for the microtopography of the tegument by scanning electron microscopy. The tegumental surface of the hybrid male schistosome resembled the male parent, S. mekongi, with a few characters which resembled the male, S. japonicum-like (Malaysian). The surface tegument of the hybrid male worm was characterized by the presence of highly-branched and perforated ridges interspersed with a large number of papillae all over the body surface with the heaviest concentration on the middle portion of the body. There were four types of papillae present; the pleomorphic papillae; the cratered papillae, with or without cilia; the hemispherical sensory papillae with cilia; and the fungiform papillae. Spines were absent on the body surface except in the oral and ventral suckers and in the gynecophoral canal. The tegument lining the gynecophoral canal was characterized by the presence of low ridges with scattered papillae with small number of short spines in the posterior portion of the canal. In contrast to the male, the female hybrid worm had numerous spines all over the body surface with the most concentration in the posterior region. Among the spines were low perforated ridges. Two types of papillae were present in the female hybrid; the cratered papillae, with or without cilia, and the hemispherical papillae.

The neurovirulence of flaviviruses in crab-eating monkeys (Macaca fascicularis).

The neurovirulent properties of attenuated dengue-2 and yellow fever (YF) vaccines, dengue-2 (DEN-2) and Japanese encephalitis (JE) viruses were studied in crab-eating monkeys (Macaca fascicularis). Number of central nervous system sites (as proportion affected) with neurovirulence (NV) lesions were compared. The results indicate that these monkeys reliably developed NV-lesion when inoculated with either JE or YF vaccine viruses (87%). NV-lesions occurred in a minority when inoculated with DEN-2 vaccine virus, were of minimal severity (9%), were probably biologically insignificant, and were of equal or less severity than lesions produced by its parental virus (10%).

Blood value changes in flavivirus-inoculated Macaca fascicularis monkeys.

Blood values were analysed in eighteen cynomolgus monkeys on pre-and post-neurovirulence testing of dengue-2 and yellow fever vaccine viruses, dengue-2 parental and Japanese encephalitis viruses. Certain changes between blood chemistry, hematology and serology were observed and briefly discussed.

A case comparison of acquired immune deficiency syndrome (AIDS) in homosexual males with spindle-endothelial cell abnormalities and with recrudescent melioidosis.

The AIDS syndrome includes cases of biopsy-proven Kaposi's sarcoma in persons under 60 years of age, or biopsy- or culture-proven Pneumocystis carinii pneumonia, or either of the life-threatening opportunistic infections in young previously healthy persons with no underlying cause of immunodeficiency (Center for Disease Control criteria). Here we described the first case of AIDS with early Kaposi's sarcoma-like lesions in homosexual male drug addict and have compared the clinical and laboratory findings with those of another homosexual male having recrudescent melioidosis due to Pseudomonas pseudomallei.

Scanning electron microscopic study of the tegumental surface of adult Schistosoma sinensium.

The SEM study of tegumental surface of adult Schistosoma sinensium reveals that the male tegument lacks tubercles or bosses; instead it is corrugated with small pits or perforated ridges. On the dorsal surface, spines are present whose number and size progressively increase towards the posterior end of the body. In addition, there are three types of papillae interspersed among the ridges and spines. The first type of papillae has crater-like holes surrounded by a circular doughnut-shaped elevation; some are ciliated and others are non-ciliated. They are generally found on the dorsal and ventral surfaces. The second is sensory papillae which are hemispherical in shape bearing apical cilia. They are found to be concentrated around the oral sucker and on the posterior end of the worm. The third is fungiform papillae without cilia which are found on the posterior end. There are short spines present on the tegument lining the gynecophoral canal of the male worm. The tegument of the female S. sinensium is corrugated with ridges on the ventral surface. Small spines are present on the anterior portion of the dorsal surface. They become larger and increased in number towards the posterior end of the worm. The three types of papillae are present but they are much fewer and less developed than those in the male worm.

Identification of different types of lymphoblasts in patients with acute lymphoblastic leukemia.

Identification of different types of lymphoblasts in acute lymphoblastic leukemia were studied with light microscopy, SEM and TEM.

Scanning electron microscopy of the tegumental surface of adult Schistosoma spindale.

The tegumental surfaces of adult male and female of Schistosoma spindale were studied by scanning electron microscopy. In general, the body surface of the male appears to be fairly uniform from anterior end to posterior end. It is characterized by the presence of transverse ridges and papillae of various types. These papillae are distributed fairly regularly over the whole body surface of the worm. The tegument lining the gynecophoral canal of the male worm is covered with numerous spines interspersed with papillae, some without cilia and some with crater-like holes in the centres and apical cilia. The tegument of the female worm is covered with smooth and perforated ridges and sensory bulbs with apical nodules.

Scanning electron microscopy of adult Schistosoma incognitum.

The integumental surfaces of adult Schistosoma incognitum were studied by scanning electron microscopy. The anterior part of the male which bears oral and ventral suckers is spongy in appearance due to the presence of ridges, while the middle and posterior parts are covered with numerous bosses with variable numbers of spines. Surfaces between bosses possess tegumental papillae. Bosses disappear at the edge of the gynecophoral canal which is composed of ridges. They are replaced by variable forms of papillae. The lining of the gynecophoral canal is composed of ridges and papillae, free of spines, with small number of sensory bulbs. The female tegument is relatively smooth and appears to be pitted with minute spines around the oral and ventral suckers. The posterior end of the body, in the vicinity of the excretory pore, is composed of prominent anteriorly directed spines with trabeculae and sensory bulbs.

Electron microscopic study of the vascular endothelial cell in dengue hemorrhagic fever.

Electron microscopic studies were performed from skin biopsies taken from patients aged between 1-14 years suffering from dengue hemorrhagic fever. Several capillaries showed marked distortion, but severely damaged vessels were not observed in this study. In many cases, however, swelling of a single endothelial cell was noted. This was characterized by a rarefaction of the cytoplasm and formation of the plasma membrane which extruded into lumen and narrowed it. In many vessels examined, these blebs became detached from the endothelium and were found free within the capillary lumen. Myelin figures were often observed in such vessels. Mitochondria in the swollen endothelial cells often showed contraction of the inner compartments. Although, most of the endothelial junctional complexes were intact, several gap formations, in vascular wall were observed. Vacuolation of the cytoplasm and increase in the pinocytotic vesicles in endothelial cells which represented transport of plasma fluids from the capillary to the pericapillary space were quite evident. In no case did there seem to be marked alternation of the basement membrane of the capillary. The morphological alterations observed in this study only suggest the non-specific response of the dermal capillaries. These findings were similar to the findings observed in capillaries after being subjected to heat or ischemia.

Scanning and transmission electronmicroscopy of Mekong Schistosoma eggs and adults.

Eggs and adult males and females of Mekong Schistosoma were studied by scanning and transmission electronmicroscopy. The observation of the eggs by the scanning and light microscopy revealed fine shell fenestration and a prominent knoblike spine. There are marked differences between the surface structures of male and female as studied by scanning and transmission electronmicroscopy. The surface of the male schistosome is moderately rough while that of the female is relatively smooth. SEM reveals certain basic features such as spines in the oral sucker, minute spines and folds in the gynecophoral canal of the male, and general features of male and female tegumental surfaces. The observations of the cross sections of adult schistosomes by transmission technique revealed certain features such as spines or ridges, and mucin droplets on the surface, the smooth muscles lining the integument, the mucin-producing cells and numerous lipid droplets in the body of the Schistosoma.