Light and electron microscopic analyses of immediate and late tissue damage caused by radiofrequency ablation in porcine liver

Radiofrequency ablation (RFA) is an effective procedure for localized hepatocellular carcinoma. Contrast-enhanced CT depicts the ablated area as a hypoattenuated area without hepatic blood flow; however, light microscopy does not show obvious necrosis in the ablated area. We evaluated liver tissue changes after RFA by light microscopy and electron microscopy. The normal livers of three anesthetized pigs were coagulated using RFA after laparotomy. The liver was examined immediately, and 1 week after operation by light and electron microscopy. After RFA, the liver parenchyma surrounding the needle electrode was brown in color and surrounded by a red marginal zone separate from the normal liver parenchyma. Hematoxylin-eosin staining of the central area did not show cell necrosis, and the structures of liver sinusoids, liver cell cord and the nuclei of hepatocytes were preserved. However, electron microscopic examination of tissue immediately after RFA showed destruction of mitochondria of hepatocytes and fixation of sinusoidal cells. One week later, there was a large quantity of debris in the enlarged sinusoids, in addition to irreversible destruction of hepatocyte organelles. RFA of the porcine liver causes hepatocyte damage. This damage was not evident by light microscopy but clearly identified by electron microscopy.     

Increased release of glucuronoxylomannan antigen and induced phenotypic changes in Trichosporon asahii by repeated passage in mice

Clinically important fungi such as Candida albicans and Cryptococcus neoformans are known to undergo phenotypic changes after repeated subculture or passages in vivo. However, there are no reports describing this phenomenon in Trichosporon species. This study investigated whether in-vivo passages of environmental isolates of Trichosporon asahii in mice changes their phenotype; three environmental isolates and 14 clinical isolates (from deep-seated infections) were used. The shape of the colony and cell type were observed, and the titre of glucuronoxylomannan (GXM) antigen and concentration of (1-->3)-beta-D-glucan were measured for each isolate. Changes in these features were also examined after three passages of the environmental isolates in mice. The shape of colonies and cell types were clearly different in environmental and clinical isolates. Furthermore, the clinical isolates released significantly higher levels of GXM antigen than environmental isolates (titre: log2 9.4 SD 0.7 versus log2 5.4 SD 1.4). The phenotype of passaged isolates was significantly different from the original environmental isolates with respect to the morphology of colonies and cell type and GXM release (titre: log2 10.0 SD 0.7 versus log2 5.4 SD 1.4). These results suggest that the phenotypic changes in T. asahii occur as a result of in-vivo passages. This process may allow a proportion of the fungal population to escape eradication by the host immune system, as GXM antigen is considered to protect the fungi against phagocytosis by polymorphonuclear leucocytes and monocytes in vivo.     

Production of tumor necrosis factor, interleukin 1, and interferon-gamma by peripheral blood mononuclear cells from patients with primary biliary cirrhosis

Since patients with primary biliary cirrhosis (PBC) have evidence of abnormal function of the immune system, we evaluated production of various cytokines by peripheral blood mononuclear cells (PBMCs) and monocytes from patients with this disease, using an enzyme-linked immunosorbent assay. The mean amounts of production of tumor necrosis factor alpha(TNF alpha), interleukin 1 beta (IL1 beta), and interferon-gamma (IFN-gamma) by PBMCs from patients with PBC tended to be increased in cultures in the presence of stimulating agents in comparison with controls, but there was no significant difference because of a wide scatter of results. Monocytes from PBC patients also tended to produce higher amounts of TNF alpha and IL1 beta than control monocytes did, although the percentage of monocytes in PBMCs was similar in PBC and controls. A significant correlation was found between TNF alpha production and IL1 beta production in PBC patients. The number of TNF alpha or IFN-gamma positive infiltrating mononuclear cells detected by immunohistochemical staining in liver biopsy sections correlated with the production of these cytokines by PBMCs in vitro. However, cytokine production did not correlate with serum biochemical or hepatic histologic findings, except for serum alkaline phosphatase values. In patients with type B chronic active hepatitis, IL1 beta and IFN-gamma production was similar to controls, while TNF alpha production tended to be enhanced. Thus the cytokines studied here may play some role in the pathogenesis of PBC.     

First trimester prenatal diagnosis of haemophilia A using factor VIII gene probe

Accurate first-trimester prenatal diagnosis was achieved in a Japanese haemophilia A family by the use of a restriction fragment length polymorphism (RFLP) located within the F.VIII gene. Since the pregnant woman's heterozygosity for BclI polymorphism in F.VIII/intron 18 (F8A) probe was informative, chorionic villus sampling (CVS) was performed at 9 weeks of gestation. Restriction analysis showed that the fetus was heterozygous for the BclI site and had received a normal paternal X chromosome (0.9 kb) and a normal maternal X (1.2 kb). Therefore, we concluded that the fetus was a non-carrier female. Pregnancy went to term and woman gave birth to an apparently healthy female. At one week after birth a coagulation study confirmed that the newborn infant is not a carrier. The first-trimester prenatal diagnosis of haemophilia A is possible by CVS due to a RFLP in the F.VIII gene.     

Induction of interferon-inducible protein-10 and monokine induced by interferon-gamma from human endothelial cells infected with Influenza A virus

Primary human umbilical vein endothelial cells (HUVECs) were infected with Influenza virus A/Aichi/2/68 (H3N2) in order to determine the role of endothelial cells in mediating inflammation induced upon virus infection. Structural proteins of the virus and mRNA of the M2 protein were detected in the infected cells, indicating that virus infection had occurred in HUVECs. The Influenza A virus-infected HUVECs showed elevated levels of gene expression of interferon (IFN)-inducible protein (IP)-10 and monokine induced by IFN-gamma (Mig), while heat-, formalin- and diethyl ether-inactivated viruses did not enhance the IP-10 and Mig gene expression. The results thus indicate that infection of live Influenza A virus is responsible for elevation of IP-10 and Mig gene expression. The elevation of IP-10 and Mig gene expression in infected HUVECs was not accompanied by the elevation of IFN-gamma gene expression, indicating that the elevation of IP-10 and Mig gene expression was independent of the IFN-gamma pathway.     

Down-regulation of antigen-specific antibody production by TCR antagonist peptides in vivo

The efficacy of TCR antagonist peptides in inhibition of antigen-specific antibody production and T cell responses in vivo was evaluated. Among amino acid-substituted analogs of a peptide corresponding to residues 119 - 133 of bovine beta-lactoglobulin (p119 - 133), pR124Q and pD129S, prepared by substitution of Gln and Ser for Arg(124) and Asp(129), respectively, have been shown to display TCR antagonist activity for three out of four distinct p119 - 133-specific T cell clones and for polyclonal T cells derived from p119 - 133-immunized C57BL / 6 mice. Both pD129S and pR124Q inhibited in vivo priming and subsequent activation of T cells by p119 - 133 when co-injected with p119 - 133 into mice, as shown by the decreased proliferation of T cells in response to p119-133 in vitro. pD129S significantly inhibited production of anti-p119 - 113 antibodies of IgG1, IgG2b and IgE isotype in vivo when co-injected into mice together with p119 - 133 at the time of the first immunization. However, pR124Q was totally ineffective in inhibition of the antibody responses. Anti-p119 - 133 antibodies from p119 - 133-immunized mice could bind to pR124Q but not to pD129S, suggesting that the difference in cross-reactivity is responsible for the different effect of these two peptides on specific antibody production. Our findings demonstrate that a single TCR antagonist peptide can inhibit antigen-specific polyclonal antibody production when this antagonist peptide does not cross-react with the antibody elicited in response to an antigenic peptide.     

Recombinant human interleukin 1 alpha is cytotoxic for and increases surface expression of HLA-A,B,C antigens of a human hepatoma cell line, PLC/PRF/5

Our study was undertaken to determine whether human recombinant interleukin 1 alpha (rIL 1 alpha) has any effect on the proliferation and expression of HLA-A,B,C antigens of human liver cell lines. The addition of rIL 1 alpha reduced the cell number of the human hepatoma cell line, PLC/PRF/5. This effect was determined to be cytotoxic, but not growth inhibitory, rIL 1 alpha did not change the number of Chang cell or SK-Hep-1 at a concentration as, high as 25,000 U/ml. rIL 1 alpha enhanced the expression of HLA-A,B,C antigens on PLC/PRF/5, but had no effect on Change cell or SK-Hep-1. Receptor binding studies showed that 125I-rIL 1 alpha bound to PLC/PRF/5 in a specific and saturable manner, but did not bind to Chang cell or SK-Hep-1. Scatchard plot analysis of the binding to PLC/PRF/5 revealed a single type of high affinity binding site with an apparent dissociation constant of approximately 5 x 10(-5) M and the presence of approximately 150 binding sites per cell. These findings suggest that IL 1 alpha may play a role in host defense against some hepatomas as cytotoxic factor and may be an enhancer of expression of HLA-A,B,C antigens on tumor cells.     

Effect of a spider toxin (JSTX) on excitatory postsynaptic current at neuromuscular synapse of spiny lobster

1. We studied the blocking properties of a spider (Nephila clavata) toxin (JSTX) purified from venom on the spiny lobster neuromuscular junction. 2. When a small amount of JSTX was applied to the neuromuscular junction, the excitatory postsynaptic potential (EPSP) was partially suppressed. The amplitude of EPSPs remained at a steady level for several hours during the washing of the preparation, showing that the action of JSTX is irreversible. 3. We recorded the excitatory postsynaptic current (EPSC) from synaptic site using a macro-patch electrode. The amplitude of EPSC increased linearly with hyperpolarization of the membrane potential in the presence and absence of JSTX. 4. The decay phase time constant of EPSC and spontaneous EPSC was decreased by hyperpolarizing the membrane potential both in the absence and in the presence of JSTX. The relationship between the decay time constant and the membrane potential was not modified by JSTX. 5. It is suggested that JSTX irreversibly blocks EPSC by acting on the site that is apart from the ionic channel of the glutamate receptor molecule.