Role of caspase 8 as a determinant in chemosensitivity of p53-mutated head and neck squamous cell carcinoma cell lines.

We studied factors that control chemoresistance to 6 head and neck squamous cell carcinoma cell lines carrying p53 mutations. Cell lines were chosen, based on the presence of EGFR amplifications, the presence of H-ras mutations, and the absence of either. WST-1 viability assays showed that, in response to etoposide, Ca922 was most sensitive, HOC313 most resistant, and HSC6 and the others moderately sensitive. A similar tendency was shown by further analyses with cisplatin, 5-fluorouracil, LY294002, and combined treatment with LY294002 and TNF-related apoptosis-inducing ligand (TRAIL). Although both Ca922 and HOC313 had activating mutations upstream of Akt signaling, the constitutive phosphorylation of Akt at S473 was observed in chemosensitive Ca922, but not in chemoresistant HOC313, suggesting that constitutive Akt phosphorylation was not the primary determinant for chemoresistance in these cell lines. Further, by the combined treatment with LY294002 and TRAIL, apoptosis was induced in Ca922 and HSC6 but not in HOC313. Interestingly, caspase 8 was not detected in HOC313, while it was cleaved in the other 2 cell lines. Further, in Ca922 and HSC6 but not in HOC313, caspase 8 inhibitor restored loss of viability induced either with LY294002 and TRAIL or even with etoposide alone. These findings suggest that caspase 8 played an important role in chemoresistance against genotoxic drugs.

Multiple organ dysfunction in congenital murine toxoplasmosis.

Infection of gravid Nya: NYLAR (NYLAR), C57BL/6J (C57), and BALB/c mice with Toxoplasma gondii, on gestation day 7, resulted in fetal resorptions, abortions, and stillbirths. Fetal wastage (estimated) was 35 and 40% in the NYLAR and C57 strains and 55% in the BALB/c strain. Postnatally, pups were cachectic and growth-retarded, with some developing hind limb weakness, petechial lesions on ears and tail, and a blood-tinged nasal exudate. Only 13 of 97 BALB/c pups, 14 of 41 C57 pups, and 46 of 153 NYLAR pups survived the first month of life. At necropsy, swollen, blotched livers, enlarged spleens, pallid kidneys and pulmonary hemorrhages were observed. Cysts of T. gondii were detected in every pup, via press-smears of brain. Histologic examination revealed mineralizing cavitations, ventricular deformations, and periventricular edema in the central nervous system; extensive liver pathology marked by hepatocellular necrosis and calcification, sinusoidal dilatation, and giant cell granulomas; congestion of the spleen with blurring of red and white compartments and cavitations in the white pulp; and tubule and glomerular necrosis and calcification in the kidney. The pulmonary hemorrhages and dermal petechial lesions may reflect a bleeding diathesis due to hepatic insufficiency. The pathogenesis of congenital toxoplasmosis, in the 3 strains of mice, appears due to microvascular dysfunction characterized by dysregulation of hemostasis, perfusion failure, and multiple organ dysfunction, rather than to parasite-mediated cytopathology. We suggest that hematogenous dissemination and endothelial invasion by the parasite induced a systemic inflammatory response syndrome (i.e; toxoplasmic sepsis) leading to the microvascular dysfunction.

VH3 gene usage in neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit.

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V kappa 1 family, in which the closest V kappa germ line gene was 02/012 or L5, with the J kappa 1, J kappa 2, J kappa 4, or J kappa 5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

ANALYSIS OF PATHOGENICITY OF ENTAMOEBA HISTOLYTICA BY POLYMERASE CHAIN REACTION / 中国寄生虫学与寄生虫病杂志

DNA from five isolates of Entamoeba histolytica were examined for their pathogenicity by polymerase chain reaction. Three isolates SH-3,SH-6,SH-8 were isolated from patients with acute amoebic dysentery, whereas SH-5 and SH-7 were isolated from asymptomatic cyst passers. Gel electrophoresis of PCR products showed that primers P11 , P12 for pathogenic strains could amplify genomic DNA extracted from SH-8 , and primers P13, P14 for non-pathogenic strains could amplify genomic DNA extracted from SH-3, SH-5, SH-6 and SH-7. Furthermore, zymodeme analysis and the reactivity of McAb 4G6, which recognizes the 30 kDa antigen of pathogenic E. histolytica indicated that only SH-8 was pathogenic, while the others were nonpathogenic. The results of the genotypic analysis by PCR were in accord with the phenotypic properties.It is suggested that there are differences in genomic DNA between pathogenic and non-pathogenic strains. PCR is a highly sensitive and specific method for genomic DNA analysis of E. histolytica.