Immunolocalization of cytoskeletal components in the tegument of the 3-week-old juvenile and adult Fasciola gigantica.

Components of three cytoskeletal elements, namely, microtubule, intermediate and actin filaments have been localised in the tegument of the 3-week-old juvenile and adult Fasciola gigantica by means of immunofluorescence and immunoperoxidase techniques, using mouse monoclonal anti-alpha-tubulin, anti-cytokeratin antibodies and biotinylated-phalloidin, respectively. The immunostaining with the above probes were also performed in adult Schistosoma mansoni for comparison. The presence of tubulin, indicative of microtubules, was demonstrated in the tegumental cell bodies, their cytoplasmic processes, and the basal layer of the tegumental syncytium of F. gigantica. While in S. mansoni, tubulin appeared as vertical lines stretching across the whole thickness of the syncytium. Cytokeratin, representing one type of intermediate filaments, was detected in the tegumental cell bodies, their cytoplasmic processes, tegumental syncytium and spines of F. gigantica. In contrast, cytokeratin was evident only in the syncytium of S. mansoni, but not in the spines. Phalloidin, which could bind to actin, a subunit of microfilament, was detected in the tegumental cell bodies, their processes, and the microtrabecular network which form the scaffold of the tegumental syncytium of F. gigantica. In S. mansoni, actin was localized in similar tissues except the syncytium was not stained while spines exhibited intense staining. In F. gigantica, the presence of microtubules and actin filaments in the tegumental cells, their processes and in the syncytium could mediate the movement of secretory granules from the cell bodies towards the basal as well as the apical layer of the tegument. Cytokeratin filaments may serve to reinforce the integrity of the tegumental syncytium as well as the spines.

Biochemical and histopathological effects of glyphosate herbicide on Nile tilapia (Oreochromis niloticus).

In Oreochromis niloticus that had been exposed for 3 months to sublethal concentrations (5 and 15 ppm) of the commercial glyphosate herbicide (C(3)H(8)NO(5)P) Roundup, the organs exhibited varying degrees of histopathological change. In the gills filament cell proliferation, lamellar cell hyperplasia, lamellar fusion, epithelial lifting, and aneurysm were observed. In the liver there were vacuolation of hepatocytes and nuclear pyknosis. Kidney lesions consisted of dilation of Bowman's space and accumulation of hyaline droplets in the tubular epithelial cells. The structural damages could be correlated to the significant increase (p

Histopathological alterations of white seabass, Lates calcarifer, in acute and subchronic cadmium exposure.

Histopathological alterations to white seabass, Lates calcarifer aged 3 months in acute and subchronic cadmium exposure were studied by light and scanning electron microscopy. The 96-h LC50 values of cadmium to L. calcarifer was found to be 20.12 +/- 0.61 mg/l and the maximum acceptable toxicant concentration (MATC) was 7.79 mg/l. Fish were exposed to 10 and 0.8 mg/l of Cd (as CdCl,H2O) for 96 h and 90 days, respectively. The study showed that gill lamellae and kidney tubules were the primary target organs for the acute toxic effect of cadmium while in the subchronic exposure, the toxic effect to gills was less than that of kidney and liver. Gill alterations included edema of the epithelial cells with the breakdown of pillar cell system, aneurisms with some ruptures, hypertrophy and hyperplasia of epithelial and chloride cells. The liver showed blood congestion in sinusoids and hydropic swelling of hepatocytes, vacuolation and dark granule accumulation. Lipid droplets and glycogen content were observed in hepatocytes at the second and third month of subchronic exposure. The kidney showed hydropic swelling of tubular cell vacuolation and numerous dark granule accumulation in many tubules. Tubular degeneration and necrosis were seen in some areas.

V3 sequence diversity of HIV-1 subtype E in infected mothers and their infants.

To elucidate genetic characteristics of HIV-1 subtype E involved in vertical transmission, V3 regions of HIV-1 subtype E isolated from 17 infected mothers (M1-M17) and their infants (I1-I17) at 1 month after birth were sequenced after cloned into pCRII vectors. At least three clones of each sample were collected. All mothers were asymptomatic and had been infected through a heterosexual route. Nine infants (I9-I17) showed mild symptomatic and immunosuppression within the first year of life. The interpatient nucleotide distance of mothers and infants in this group (0.065+/-0.008) were of greater diversity than those of a nonimmunosuppression group (0.039+/-0.006) by a significant amount (Fischer's exact test, p = .003). The substitution with asparagine (N) at threonine (T) at position 13 and aspartic acid (D) at position 29 of the V3 sequence were significantly associated with nonimmunosuppression in the first year of life (F-test, p = 0.003). Either a single or multiple viral variants could transmit from mothers to their infants.

PIP: At least 1.5 million children worldwide are infected with HIV-1. Most HIV-infected children obtained the virus from their mother either in utero, at delivery, or postpartum through breast-feeding. Since the V3 loop of HIV is an important determinant for viral neutralization and cellular tropism, mutations in the V3 region could possibly affect mother-to-child transmission. Serum specimens from 17 HIV-1-seropositive mother-child pairs being treated at the pediatric clinic of Siriraj Hospital, Bangkok, in 1994 and 1995, were studied to better understand the genetic characteristics of HIV-1 subtype E involved in vertical transmission. The V3 regions of HIV-1 subtype E isolated from the subjects at 1 month after birth were sequenced after being cloned into pCRII vectors, with at least 3 clones of each sample collected. All mothers were asymptomatic and had been infected through a heterosexual route. 9 infants were mildly symptomatic and had evidence of immunosuppression during their first year of life. The nucleotide sequences of asymptomatic infants were significantly closer to maternal sequences than those of the AIDS cases. The data suggest that 1 or 2 genotypes from the mother were selected, transmitted to the infant, and then became diverse. The substitution with asparagine at threonine at position 13 and aspartic acid at position 29 of the V3 sequence were significantly associated with nonimmunosuppression during the first year of life.

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.

Langerhans cell density and serological changes following intradermal immunisation of mice with dengue 2 virus.

After the introduction of the dengue-2 (16681) virus by intradermal (i.d.) injection into the footpads of mice, Langerhans cells (LCs) increased in numbers within 24 h at the site of injection and neutralising antibody developed. On comparing the i.d. and intramuscular (i.m.) routes, antibody was produced more rapidly and at higher levels when the virus was injected by the i.d. route. Subsequent re-challenge by the i.d. route produced an even more rapid serological response with all mice producing significant neutralising titres within 12 h. Numbers of ATPase-positive LCs varied with time. A significant sharp drop in LC densities in the early post-injection phase directly correlated with the increased numbers of dendritic cells in the superficial dermis and interfollicular sinuses of draining lymph nodes (LN). Immunofluorescence showed the presence of viral antigen in the footpad epidermis and draining LN within minutes or within 2 h of challenge, respectively.

Liver injury due to iron overload in thalassemia: histopathologic and ultrastructural studies.

The livers of 30 cases of thalassemia (19/beta-thal/HbE, seven thal/HbH and four beta-thal major) were studied histopathologically and electron microscopically, in an effort to define the morphologic alterations due to iron overload. The results of light and electron microscopy were similar in most cases. Iron accumulation and fibrosis were the common features found in these patients, except that thal/HbH exhibited lesser hepatic damage. The degrees of iron deposition and fibrosis were found to be higher in splenectomized and cirrhotic than non-splenectomized and non-cirrhotic patients. The subcellular changes were swollen mitochondria, with the presence of an electron dense matrix and ruptured mitochondrial membrane. Proliferation of smooth endoplasmic reticulum (ER) and dilated rough ER was observed. Increases in lysosomal hemosiderin in hepatocytes and in Kupffer cells were demonstrated. The possible ways by which the iron compounds or free radicals mediated membrane damage are mentioned. The pattern of liver cell damage is similar to that of viral hepatitis.

Immunobiological diagnosis of tropical ocular diseases: Toxocara, Pythium insidiosum, Pseudomonas (Burkholderia) pseudomallei, Mycobacterium chelonei and Toxoplasma gondii.

Of the sera obtained from 18 patients with ocular diseases comprising ocular larva migrans (OLM), Toxoplasma gondii, Mycobacterium chelonei, Pythium insidiosum bacteria and tumour, 3 sera were positive for Toxocara antibody at the titre over 1/1600 ELISA. All 3 of these sera came from males with unilateral grayish fundi. We demonstrated the value of the direct immunofluorescent assay (DIFA) on vitreous specimens from 7 cases of Toxoplasma retinitis. One vitreous specimen under electronmicroscopic examination revealed bradyzoites of T. gondii. In addition, a few cases of keratoconjunctivitis/ corneal ulcer with endophthalmitis, due to P. insidiosum, Ps. pseudomallei and Mycobacterium chelonei, were also diagnosed.

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.

Enhanced hepatotoxicity of aflatoxin B1 in the rat by ethanol: ultrastructural changes.

The effect of ethanol pretreatment on aflatoxin B1 (AFB1)-induced ultrastructural alteration in hepatocytes was investigated in male Wistar rats. Pretreatment with 4 oral doses of ethanol (4.0 g/kg body wt. each) at 48, 45, 24 and 21 h prior to a single intraperitoneal administration of AFB1 (2.0 mg/kg body wt.) produced an alteration of fine structure more extensive than that observed after administration of AFB1 or ethanol alone. The organelles most affected were the mitochondria, endoplasmic reticulum and nucleus. The degree of nuclear edema, rough endoplasmic reticulum dilatation and mitochondrial swelling in hepatocytes from rats treated with ethanol and AFB1 was much greater than that of hepatocytes from rats treated with AFB1 or ethanol alone. Thus, ethanol may play a specific role in the potentiation of hepatic injury induced by AFB1 at the mitochondrial, endoplasmic reticular and nuclear level.

The influence of pre- and postnatal caloric intake on colon carcinogenesis.

Mother rats were allowed to litter under conventional conditions. They were fed a complete, semipurified diet during gestation, and at time of littering the numbers of pups were reduced to either eight per litter or four per litter in two additional groups. At weaning, all rats were continued on the same diet that their mothers had consumed. One group of those reduced to four per litter at birth was allowed to continue to eat ad libitum while the other group, reduced to four per litter, was pair fed to the ad libitum eight per litter group. The group reduced to four per litter at birth and allowed to eat ad libitum during postnatal life gained the most weight and were heaviest at the termination of the study. This group also had the greater incidence and frequency of colon tumors when exposed to dimethylhydrazine (DMH). The group pair fed to rats fed conventional diets, eight rats per litter, had an incidence and frequency of tumors between the other two groups. These data demonstrate that early exposure to excess calories increased risk for cancer and that early and late excess caloric intake further increased risk. Thus, pre- and perinatal caloric intake may have a significant influence on susceptibility to cancer later in life. Mechanisms are only speculative but may include differences in metabolism and modulation of hormonal balance.

Minimal dose and time protection by lindane (gamma-isomer of 1,2,3,4,5,6 hexachlorocyclohexane) against liver tumors induced by aflatoxin B1.

This study was carried out in order to investigate the minimal exposure to lindane (LD, 99.72% gamma isomer of 1,2,3,4,5,6 hexachlorocyclohexane), a chlorinated hydrocarbon insecticide, required to protect against liver tumor induced by aflatoxin B1 (AFB1). Materials fed to Buffalo strain rats were as follows: LD 100 ppm; AFB1 1 ppm, LD 100 ppm plus AFB1 1 ppm; and control basal diet. The experimental animals were clinically observed and then serially killed at 1, 3, 5, 10, 15 and 82 weeks. Concurrent administration of LD with AFB1 to rats for more than 3 weeks totally inhibited the incidence of AFB1-induced hepatocellular carcinomas by week 82. Only 1 of 20 rats (5%) fed the same regimen for 1 week developed liver tumors. Animals given 1 ppm AFB1 singly for 15 weeks had a high liver tumor incidence (31.5%). No animals developed liver tumors in LD-treated and control groups. LD may inhibit AFB1-induced liver tumors by stimulating hepatic metabolism and excretion of AFB1 so that less carcinogen is available to liver tissue.

Detection of small inhibitory effects of acetylsalicylic acid (ASA) by platelet impedance aggregometry in whole blood.

Our investigations have demonstrated on 10 volunteers receiving either 500 mg or 100 mg acetylsalicylic acid (ASA) that a low collagen concentration (1 microgram/ml) can best detect the aggregation defect caused by ASA. With the impedance aggregometry the mean inhibition reaches 82% and 52% with 500 mg and 100 mg ASA, respectively. Collagen at higher concentration (3 micrograms/ml) as well as ADP 10 and 25 mumol/l are less sensitive, less than 25% inhibition was recorded. These results suggest that a 1 microgram/ml concentration of collagen is adequate for the control of the ASA effect up to 6 days after intake of 100 mg. Furthermore, the von Willebrand factor (vWF) dependent platelet aggregation induced by 0.6 and 1.0 mg/ml ristocetin was clearly diminished after ASA. Therefore, a ristocetin screening test in whole blood for vWF disorder is possibly distorted when the test is performed within 6 days from ASA administration.

Dengue-4 vaccine: neurovirulence, viraemia and immune responses in rhesus and cynomolgus monkeys.

A dengue 4 (DEN-4, strain 1036-PDK 48) vaccine attenuated by passage in primary dog kidney cells was tested using rhesus (Macaca mulatta) and cynomolgus (M. fascicularis) monkeys to determine its safety, potency, and immunogenicity. 14 rhesus monkeys were divided into 3 groups: group 1, 2 animals given control culture fluid; group 2, 2 animals given DEN-4 parental virus; group 3, 10 animals given DEN-4 vaccine virus. 10 cynomolgus were similarly grouped, but group 3 contained 6 monkeys. No significant neurovirulence was observed with parental or with DEN-4 virus passaged in primary dog kidney (PDK) cells. Both cynomolgus monkeys inoculated with DEN-4 vaccine virus developed minimal (V-1) and mild (V-2) neurovirulence-type lesions in the central nervous system, which were nondestructive in both species. All parental and vaccine viruses produced moderate to high neutralizing antibody titres. Only parental virus produced viraemia, in 2 cynomolgus monkeys. Because of its safety and avirulence in monkeys, PDK 48 is recommended for human trial.

Electron microscopic study of liver tissue from 30 thalassemic patients.

Electron microscopic study of liver tissue from 30 thal patients in advanced stages has been described. In all cases, regardless of the type of hemoglobin, electron microscopic observations gave identical results. Significant findings are ferroacidophilic bodies, ferroacidophilic degeneration of hepatocytes, interhepatocyte collagen, hemosiderin and ferritin, paracrystalline accumulations of ferritin molecules, and liver cell ballooning. The ultrastructures of FAB, FAD, and balloon cells were similar to those seen in viral hepatitis, but no viral particles were found.

Reducing effects of rodent malaria on hepatic carcinogenesis induced by dietary aflatoxin B1.

The present study was undertaken to evaluate the effects of Plasmodium berghei infection on the development of liver tumors induced in male Buffalo rats by aflatoxin B1 (AFB1). Intraperitoneal (i.p.) injection of 10(6) parasitized red blood cells (pRBC) into the rat 12 days prior to administration of 2 ppm dietary AFB1 for 10 weeks diminished hepatocellular carcinoma (HCC) induction compared to that observed in rats given AFB1 alone at weeks 60-82. No animals in a control group developed HCC lesions, while only 1 of 22 rats treated with P. berghei alone developed a neoplastic nodule at week 82. These data suggest a reducing effect of P. berghei on the development of liver tumors induced by AFB1 in male Buffalo rats.

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.