State of HBV DNA in HBsAg-negative, anti-HCV-positive hepatocellular carcinoma: existence of HBV DNA possibly as nonintegrated form with analysis by Alu-HBV DNA PCR and conventional HBV PCR

The role of hepatitis B virus (HBV) in carcinogenesis of hepatitis B surface antigen (HBsAg)-negative, anti-hepatitis C virus (anti-HCV)-positive hepatocellular carcinoma (HCC) remains unknown. To investigate the state of HBV DNA in such HCC, HBV DNA was examined by polymerase chain reaction (PCR) between HBV DNA and human Alu sequence (HBV-Alu PCR), which could detect integrated form of HBV DNA only, and by conventional HBV PCR, which could detect both integrated and episomal forms of HBV DNA. In all the 17 HBsAg-positive HCC, HBV DNA was detected by both HBV-Alu PCR method and conventional HBV PCR method. By contrast, in HBsAg-negative, anti-HCV-positive cases, HBV DNA was detected in 10 of 21 (47.6%) by conventional HBV PCR and in none of 21 (0%) by HBV-Alu PCR method. Thus, integrated form of HBV DNA was not found in most HbsAg-negative, anti-HCV-positive HCC in the current study. The role of episomal form of HBV DNA requires further investigation of its involvement in the process of the development of HBsAg-negative, anti-HCV-positive HCC.     

Gain-of-function polymorphism in mouse and human Ltk: implications for the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus (SLE), a complex multigenic disease, is a typical antibody-mediated autoimmune disease characterized by production of autoantibodies against a variety of autoantigens and immune complex-type tissue inflammation, most prominently in the kidney. Evidence suggests that genetic factors predisposing to aberrant proliferation/maturation of self-reactive B cells initiate and propagate the disease. In SLE-prone New Zealand Black (NZB) mice and their F1 cross with New Zealand White (NZW) mice, B cell abnormalities can be ascribed mainly to self-reactive CD5+ B1 cells. Our genome-wide scans to search for susceptibility genes for aberrant activation of B1 cells in these mice showed evidence that the gene, Ltk, encoding leukocyte tyrosine kinase (LTK), is a possible candidate. LTK is a receptor-type protein tyrosine kinase, belonging to the insulin receptor superfamily, and is mainly expressed in B lymphocyte precursors and neuronal tissues. Sequence and functional analyses of the gene revealed that NZB has a gain-of-function polymorphism in the LTK kinase domain near YXXM, a binding motif of the p85 subunit of phosphatidylinositol 3-kinase (PI3K). SLE patients also had this type of Ltk polymorphism with a significantly higher frequency compared with the healthy controls. Our findings suggest that these polymorphic LTKs cause up-regulation of the PI3K pathway and possibly form one genetic component of susceptibility to abnormal proliferation of self-reactive B cells in SLE.     

Successive infection of coxsackievirus B3 and encephalomyocarditis virus: an animal model of chronic myocarditis.

Successive infection of coxsackievirus B3 and encephalomyocarditis virus was investigated as a disease model of chronic myocarditis. Four-week-old C3H/He mice were inoculated with coxsackievirus B3 and then inoculated with encephalomyocarditis virus at 8 weeks old. The hearts were evaluated on histopathological changes compared with those of non-infected mice and mice infected with either virus alone. At 10 weeks old, the hearts of the mice infected successively with both viruses showed co-existence of fibrosis surrounding calcified lesions and marked cellular infiltration with myocardial necrosis. These findings resembled chronic active myocarditis in humans, unlike the lesions due to either virus alone. At 12 weeks old, the hearts of all the infected mice showed fibrosis with scarce cellular infiltration. The successively infected hearts also showed a significantly higher heart weight to body weight ratio than that of the non-infected control mice, and localized wall thinning in the damaged regions. Thus, we conclude that successive infection additively causes myocardial damage that resembles chronic myocarditis and may produce a heart condition similar to dilated cardiomyopathy.     

Enkephalin fibers synapse on cholinergic neurons in the rat sacral intermediolateral nucleus: a double-immunostaining at the light and electron microscopic levels

Relationships between leucine-enkephalin fibers and cholinergic neurons in the rat sacral intermediolateral nucleus were examined by light and electron microscopy using double-immunostaining method. Cholinergic neurons in the sacral intermediolateral nucleus were labeled by a rat-mouse monoclonal antibody to choline acetyltransferase and stained bluish green with 5-bromo-4-chloro-3-indolyl-beta-D- galactoside reaction products using beta-galactosidase as a marker. On the same sections, leucine-enkephalin fibers were labeled by a rabbit polyclonal antiserum to leucine-enkephalin and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the sacral intermediolateral nucleus. In the same region, leucine-enkephalin-like immunoreactive cells. In the electron microscope, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products were in the form of coarse electron dense deposits in the choline acetyltransferase-like immunoreactive structures and could be distinguished from the much finer grained diaminobenzidine reaction products. Choline acetyltransferase-like immunoreactive neurons received synaptic inputs from leucine-enkephalin fibers-like immunoreactive terminals. These findings suggest that leucine-enkephalin fibers may affect the activity of cholinergic parasympathetic preganglionic neurons.     

Vagal afferents interact with substance P-immunoreactive structures in the nucleus of the tractus solitarius: immunoelectron microscopy combined with an anterograde degeneration study

The morphological features of substance P-immunoreactive (SP-IR) structures in the nucleus of the tractus solitarius (NTS) were examined by immunoelectron microscopy combined with an anterograde degeneration study. Vagal afferents were allowed to degenerate by resecting the nodose ganglion two days prior to the examination. SP-IR axon terminals in the ipsilateral NTS were often found to make a synaptic contact with non-reactive dendrites in contact with degenerated terminals. SP-IR terminals also made contact with degenerated terminals, or SP-IR cells in contact with degenerated terminals. These findings suggest a close relationship between SP neuronal structures and vagal afferents in the NTS.     

Inflammatory markers, especially the mechanism of increased CRP

Acute phase proteins are synthesized mainly in the liver cells, induced by various inflammatory cytokines which are produced by activated macrophages/monocytes at the inflammatory sites. C-reactive protein is a principal acute phase protein, and increased most significantly upon various inflammations. False negative results may be recognized in the patients with viral infections, collagen diseases such as SLE, PSS, dermatomyositis, ulcerative colitis, Sj?gren's syndrome, leukemia, cerebral infarction, etc.     

Expression of blood group-related antigens and Helix pomatia agglutinin in malignant pleural mesothelioma and pulmonary adenocarcinoma

In an attempt to differentiate malignant pleural mesothelioma from pulmonary adenocarcinoma by histochemical and immunohistochemical means, the glycoconjugate profiles of five reactive mesothelial lesions, 29 mesotheliomas (20 epithelial, three biphasic, and six fibrous types), and 38 well-differentiated pulmonary adenocarcinomas (34 papillary, two tubular, and two bronchioloalveolar types) were tested with ABH blood group-related antigens (BGR-Ag) antibody and Helix pomatia agglutinin (HPA) which agglutinates human type A erythrocytes. Formalin-fixed, paraffin-embedded sections were stained by the avidin-biotin-peroxidase complex method. Reactive mesothelial lesions and malignant mesothelioma of the pleura were not stainable with BGR-Ag antibody or HPA, irrespective of the blood group type. In pulmonary adenocarcinoma, however, the test with BGR-Ag antibody showed a high positive rate with the compatible blood group type, especially in type O cases (83%). Using HPA, reactions of adenocarcinoma with types A and AB also demonstrated high positive results (94% and 100%, respectively), but even with types B and O positive reactions occurred in 80% and 33% of cases, respectively. The findings suggest that positive reactions with either BGR-Ag antibody or HPA can be indicative of pulmonary adenocarcinoma.     

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.