Somphong Sahaphong

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.

Fasciola gigantica and Schistosoma mansoni: Vaccine potential of recombinant glutathione S-transferase (rFgGST26) against infections in mice

Recombinant Fasciola gigantica glutathione S-transferase (rFgGST26) was expressed in Escherichia coli. This protein had 86% and 56% sequence identity with 26 kDa GST from Fasciola hepatica and Schistosoma mansoni, respectively. Polyclonal antibody raised in ICR mice against rFgGST26 recognized immunoblotted 26 kDa native GSTs from F. gigantica and S. mansoni. rFgGST26 was used as a vaccine in combination with Freunds adjuvant to evaluate the induction of immune responses and protection against F. gigantica and S. mansoni infection in mice. Mice were immunized via subcutaneous (s.c.), intramuscular (i.m.) or intradermal (i.d.) routes. Strong protection (77-84%) against F. gigantica was observed in all routes. Immunization via s.c. route induced immune response with IgG1 isotype predominating, while i.m. and i.d. routes resulted in mixed IgG1/IgG2a immune responses. Passive intraperitoneal transfer of IgG1 predominating antisera from s.c. rFgGST26-immunized donors to naive recipient mice resulted in 47% protection against F. gigantica infection. This suggests that the mechanism of resistance depends on the presence of specific antibody against rFgGST26. Immunization with rFgGST26 via i.m. and i.d. routes resulted in significant cross protection (55%) against S. mansoni infection in the i.d. route with mixed IgG1/IgG2a response with IgG1 isotype predominating. This indicated that rFgGST26 is a good vaccine candidate against F. gigantica in mice and could also provide cross protection against S. mansoni.

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

The livers of 30 cases of thalassemia (19/β-thal/HbE, seven thal/HbH and four β-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.

Histopathological effects of triphenyltin hydroxide on liver, kidney and gill of Nile tilapia (Oreochromis nilotica)

The histopathological effects of triphenyltin hydroxide (TPTH) on the liver kidney and gill of Nile tilapia (Oreochromis nilotica) one month old was studied by light microscopy. Two concentrations of TPTH were used: 1 mg l -1 and 3 mg 1 -1 . The fish were sacrificed at the end of one, two, three and four months. The results showed that the hepatocytes underwent a variety of changes from congestion and dilatation of sinusoidal space, pallor of cytoplasm, vacuolation and accumulation of hyaline droplets. Subcapsular and scattered focal necrosis was also observed. In the kidney, hydropic degeneration and accumulation of hyaline droplets in the tubular epithelial cells were noted. In addition, a congestion of peritubular capillaries and detachment of tubular epithelial cells were observed. In more severe case there was a collapse of glomerular capillary tuft with a widening of the Bowmans capsule. There were some changes in the gill filaments and lamellae, namely hyperplasia of the covering gill epithelium, congestion of gill capillaries and vessels, and aneurysmal formation of gill lamellar capillaries. These alterations were time-and dose-dependent.

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.

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.

Fasciola gigantica: The in vitro effects of artesunate as compared to triclabendazole on the 3-weeks-old juvenile

The in vitro effect of artesunate (ATS) on the 3-week-old juveniles of Fasciola gigantica was compared with triclabendazole (TCZ) by incubating the parasites in M-199 medium containing the drugs at concentrations of 20, 40, and 80 μg/ml for 1, 3, 6, 12, and 24h. The anthelmintic activities of these drugs were evaluated based on the relative motility value (RM) and the alterations of the tegument as observed by scanning (SEM) and transmission (TEM) electron microscopy. The RM values of TCZ-treated flukes decreased significantly from 6 to 24h for all dosages. For ATS-treated flukes, RM value decreased markedly from 12 to 24h, but the rates of decline were less than TCZ at the same doses. When observed by SEM, the tegument showed similar sequence of morphological changes after treatments with both drugs, comprising of swelling of tegumental ridges, followed by blebbing and later rupturing of the blebs, leading to erosion and lesion, and disruption of the tegument. When examined by TEM, ultrastructural changes in the tegument and associated structures after treatments with TCZ and ATS were similar which comprised of swelling, blebbing of the tegument, dilation of basal infoldings, and depolymerization of the microtrabecular network. After a longer incubation time, the tegument was completely sloughed off and the tegument cell bodies became necrotic. Additionally, in ATS-treated flukes, mitochondria showed severe swelling, rupturing of outer membrane, and their interior filled with flocculent materials.

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.

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.

Defective spectrin dimer self‐association in thalassemic red cells

The relative proportions of spectrin tetramer and dimer forms extrated from red cell membranes in a low ionic strength buffer at 4°C were determined for 15 normal subjects, 27 subjects with α‐thalassemia (7 α‐thalassemia trait, 9 Hb H disease (α‐thal 1/α‐thal 2) and 11 Hb H with Hb Constant Spring (CS), 23 subjects with β‐thalassemia (6 β‐thalassemia trait, 5 homozygous β‐thalassemia, 11 β°‐thalassemia with Hb E and 1 β +‐thalassemia with Hb E), 6 subjects with Hb E (2 homozygous and 4 carriers) and 1 subject with combined α‐thal 1/Hb CS and Hb E (AE Barts disease). In all subjects (except carriers of Hb E and 1 splenectomized case of β°‐thal/Hb E) spectrin dimer forms were elevated when compared to levels in normal controls, but there were no significant differences between carrier and disease forms. Conversion of spectrin dimers to tetramers at 30° C was reduced in the thalassemic subjects with disease but was within normal range for thalassemic carriers.