Liver Resident Macrophages (Kupffer Cells) Share Several Functional Antigens in Common with Endothelial Cells.

The identification and specific functions of Kupffer cells (KCs), a liver resident macrophage subpopulation, are still unclear. We compared KCs with peritoneal macrophages using cDNA microarray analysis and found that these cells share some antigens with endothelial cells. KCs highly express VCAM-1 and VEGF receptors (VEGF-Rs) at transcriptional and protein levels. VCAM-1 mediates the functional binding of KCs with lymphocytes and induces KC activation. Among the VEGF receptors, VEGF-R2 and VEGF-R3 were expressed on the KCs, while VEGF-R1 was expressed on other tissue macrophage subsets. VEGF120, a ligand of both VEGF-R1 and VEGF-R2, transduced strong survival and chemotactic signals through the KCs, when compared to PIGF, a VEGF-R1 ligand, indicating that VEGF-R2 plays significant roles in regulating KC activities. Expression of the VEGF-Rs was regulated by TLR4 signalling. These results suggest that the function of KCs is partly regulated by the common antigens shared with endothelial cells.

Complete remission of metastatic clear cell sarcoma with DAV chemotherapy.

We report the first case of metastatic clear cell sarcoma with dramatic response to DAV treatment (DTIC + ACNU + VCR). Clear cell sarcoma of tendons and aponeuroses, or malignant melanoma of soft parts, is a rare tumour that occurs predominantly in the extremities of young adults. It tends to recur locally or metastasize and the prognosis is poor. Although the importance of surgery has been established, the role of adjuvant chemotherapy has yet to be determined. DAV should be considered as a first-line palliative treatment in disseminated disease as well as adjuvant therapy after surgery of primary clear cell sarcoma.

Perceptions of schizophrenic patients and their therapists: application of the semantic differential technique to evaluate the treatment relationship.

This study is an attempt to evaluate the treatment relationship with schizophrenic patients by examining the patients' and their therapists' perceptions of themselves and each other, which are hypothesized to reflect features of the relationship. One hundred fifty-eight schizophrenic patients and 11 psychiatrists who maintained a supportive relationship with the patients as a therapist estimated their perceptions using the semantic differential (SD) technique with 17 adjective pairs. Eight composite scales with sufficient internal consistency were constructed from the estimations. The interrelationship among the perceptual elements, which was represented by correlation analysis of the composite scale scores, seemed consistent with our clinical experience. A factor-analytic study of the scales yielded 3 orthogonal factors that could be assumed to characterize the treatment relationship. The patient-therapist cooperation factor indicated the degree of trust between the two participants, supposedly the affective or relational aspect of the therapeutic alliance. The therapist passivity factor reflects the therapist's passive role-taking and the clinical stability of the patient. The patient strength factor was related to the condition-related and characterological strength of the patient. It is demonstrated that the estimations performed by patients and therapists are valid and useful for evaluation of the treatment relationship in the current status.

Reduced transcription and progeny virus production of hepatitis B virus containing an 8-bp deletion in basic core promoter.

We have demonstrated previously the presence of an 8-bp deletion mutant, spanning from nt. 1768 to nt. 1775 in the basic core promoter region of hepatitis B virus (HBV) in patients with anti-HBe positive asymptomatic phase before developing acute exacerbation after immunosuppressive treatment. The transcription and progeny virus production activities of the mutant were examined by transfection of the recombinant plasmid [pUC Del(2)] containing the head-to-tail dimer DNA of the mutant into HepG2 cells. The amounts of hepatitis B surface antigen (HBsAg) and HBe antigens secreted into the culture medium were markedly reduced. Southern blotting of DNAs extracted from the culture medium also showed reduced mutant activity to produce progeny virus. Northern blotting and RNase protection assay of RNAs extracted from transfected cells demonstrated that the transcription of both precore mRNA and pregenome RNA was reduced significantly compared to that of wild-type HBV. The promoter activity examined by transfection of the CAT plasmid containing deletion mutant DNA was much lower than that of wild type. Co-transfection experiments, however, of the CAT plasmid containing wild-type DNA with pUC Del(2) reduced CAT activity induced by wild-type, suggesting that truncated X protein produced by the mutant does not possess a sufficient transactivating activity. Gel shift assay using HepG2 nuclear extract and a probe containing four TA-rich regions in CP and various competitors suggested that the lack of the third TA-rich region was responsible for the transcription reduction of precore mRNA and pregenome RNA. The possible mechanisms are discussed.

Biological action of keratinocyte growth factor in BeWo cells, a human choriocarcinoma cell line.

Previously, we reported that the expression of keratinocyte growth factor (KGF) is enhanced in secretory phase endometrial and decidual cells in early pregnancy as compared with the expression of KGF in proliferative phase endometrial cells, in humans. In order to clarify the role of KGF in embryo-endometrial interaction, we analyzed the in vitro effect of KGF on the human chorionic gonadotropin (hCG) secretion and on DNA synthesis in chorionic villi which are in close contact with the endometrium/decidua in the early stage of pregnancy. In this study, we used the BeWo cell line, a human choriocarcinoma cell line that possesses the biological features of secreting various placental hormones including hCG. Furthermore, we investigated the expression of KGF receptor (KGF-R) in these cells. KGF significantly stimulated hCG secretion in cultured BeWo cells but did not affect [3H]-thymidine incorporation. KGF-R mRNA was detected in BeWo cells by reverse transcriptase-polymerase chain reaction. These results suggest that the expression of KGF, which is induced in endometrial/decidual cells by progesterone, plays an important role in the embryo-endometrial/ decidual interaction by stimulating hCG secretion rather than affecting cell proliferation.

Assessment of the severity of coronary artery stenosis by the ratio of the regional washout rate determined by adenosine triphosphate stress Tl-201 SPECT.

BACKGROUND: Adenosine triphosphate stress thallium-201 single-photon emission computed tomography (ATP SPECT) is useful for diagnosis of coronary artery disease, but its usefulness for evaluating the severity of coronary artery stenosis has not been established. METHODS AND RESULTS: We performed region-of-interest analysis of short-axis images obtained by ATP SPECT in 31 patients with single-vessel disease (>50% stenosis of the luminal diameter). We selected the lowest and highest washout rates (WR) among the anterior, lateral, and inferior WRs and calculated the ratio of the lowest WR to the highest WR (WR ratio = 0.925+/-0.027 in 14 control subjects). ATP SPECT showed positive results in 29 (94%) of 31 patients. The severity of coronary artery stenosis was inversely correlated with the WR ratio (r = -0.703, P < .0001). The sensitivity and specificity of a WR ratio < or = 0.660 for the diagnosis of severe coronary stenosis (> or =80% stenosis) were 83% and 80%, respectively. CONCLUSIONS: Results suggest that ATP SPECT may be useful for assessment of the severity of coronary artery stenosis in patients with single-vessel disease.

Gene expression of double-stranded RNA-dependent protein kinase in the human endometrium and decidua.

To clarify the biological significance of double-stranded RNA-dependent protein kinase (PKR), an interferon (INF)-inducible substance, we investigated (1) PKR gene expression and the (2) effect of IFN-gamma on PKR gene expression in human endometrium. By Northern blot analysis, PKR mRNA was detected as a 2.5 kb band in human endometrium throughout the menstrual cycle and decidua in early pregnancy. The addition of IFN-gamma to culture medium increased the PKR mRNA level in a dose-dependent manner in cultured endometrial stromal cells. These results suggest that IFN-gamma, which is reported to have an inhibitory effect on cell proliferation, plays an important role in human endometrial function by mediating PKR gene expression.

Sequential analyses of the mutations in the core upstream and precore regions of hepatitis B virus genome in anti-HBe positive-carriers developing acute exacerbation.

The nucleotide sequences of the core upstream and precore regions (371 nucleotide length, nt. 1604-1974) of hepatitis B virus (HBV) were analysed sequentially in three subjects who were positive serologically for anti-HBe and had acute clinical exacerbation after immunosuppressive treatment. These patients were asymptomatic HBV carriers before therapy. The results revealed that the mutant with an 8-bp deletion (nt. 1768-1775) located in the basic core promoter region was dominant in the asymptomatic HBV carrier phase in two of three subjects. After exacerbation, however, such mutant clones possessing 8-bp deletion disappeared or decreased in number and were replaced by the clones possessing a precore stop codon mutation G to A (nt. 1896) or by the clones possessing additional contiguous point mutations A to T (nt. 1762) and G to A (nt. 1764) and a new point mutation C to T (nt. 1653). Possible relationships between acute exacerbation of liver function accompanied by mutation and the transition of the dominant clones were discussed.

[Trial of home anti-cancer chemotherapy with infusion of 5-FU and low-dose cisplatin].

We tried home anti-cancer chemotherapy for patients with advanced or recurrent cancer of the digestive system, using two disposable balloon pumps connected to an implantable drug delivery system via central venous line. There were 33 patients under 75 years old, including 20 cases of gastric cancer, 9 cases of colorectal cancer, 2 cases of cholangiocarcinoma and 2 cases of esophageal cancer enrolled in this study. The protocol was combined chemotherapy with continuous intravenous infusion of 5-FU (300 mg/body/day) and low-dose intravenous injection of cisplatin (5 mg/body/day) in 10-day courses for two weeks, and it was repeated 3 times for 6 weeks. Because of side effects such as nausea, vomiting and bone marrow suppression, treatment was discontinued in 12 cases with peritoneal cancer infiltration. In two of 10 with estimable disease, the reduction of the metastatic lymph node was observed, but no effect was shown in the colorectal metastatic liver tumor. Thanks to the portability of the pump with this method, the patient need not undergo hospitalization. Moreover, there is no renal dysfunction or other major side effects, quality of life is not compromised and a return to family and social life is possible. Thus, if the patient cannot take the oral nutrition, it is easy to start home hyper-alimentation.

[How to palliate the symptoms of terminally ill patients at home].

We reported the kind of symptoms and how they could be palliated in terminally ill patients at home based on our experience of about 9 years. Cancer pain, which was the most frequent symptom, appeared in 67 among 126 patients receiving home care, and it could be effectively controlled with morphine; no patient returned to the hospital because of aggravation of pain. Very few patients stayed in the hospital and never returned home due to uncontrollable pain. Home parenteral infusion was done for 63 patients who were unable to eat or drink because of peritonitis carcinomatosa or cancer cachexia. High fever in the tumor mass was controlled by glucocorticoid hormone, and ascites was drained continuously when the patients suffered from abdominal distension. From analysis of the cases in which home care was interrupted or those in which patients were unable to transfer to home care, symptoms that were difficult to palliate at home were nausea caused by bowel obstruction, acute symptoms (bleeding, disturbance of consciousness, and so on), and dyspnea. But if the patients and family are eager for home care and an adequate medical support system is in place, home care may be possible despite these symptoms.

Changes in the distribution of cells expressing tumour-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.

A monoclonal antibody (MAb), c143, that recognizes a tumour-associated antigen that is "upregulated" on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. An immunohistochemical examination of neoplastic tissue and superficial cervical lymph nodes from 14 animals with EBL revealed three morphologically definable stages of change in the structure of lymph nodes, associated with the distribution of c143-positive cells: (1) the presence of c143-positive cells at the marginal sinus with no apparent changes in lymph node structure; (2) the presence of positive cells extending into and distorting the architecture of the lymph node, with clear evidence of proliferation before overt changes (enlargement of lymph nodes) were evident; and (3) the presence of positive cells throughout the lymph node with total disruption of lymph node structure when clinical signs of lymph node enlargement were evident. The results indicated that the bovine leukaemia virus-transformed lymphocytes or neoplastic cells in peripheral blood accumulate in the marginal sinus area at the earliest stages, and subsequently proliferate and infiltrate into follicles, leading to the development of clinical signs of lymphosarcoma.

Immunohistologic studies on subpopulations of lymphocytes in cattle with enzootic bovine leukosis.

The distribution of subpopulations of lymphocytes in lymph nodes and tumors from cattle with enzootic bovine leukosis (EBL) was examined by immunohistochemistry using a panel of monclonal antibodies against leukocyte differentiation molecules of EBL. The lesions in lymph nodes could be divided into three types based on the extent of infiltration and proliferation of neoplastic cells with provirus and differential expression of leukocyte differentiation molecules. The number of B-B2+, sIgM+ cells was reduced in frequency in follicles during the neoplastic cell proliferation. CD4- and CD8-positive alpha/beta T cells and gamma/delta T cells positive for WC1 (workshop cluster designation) were also reduced in frequency in areas infiltrated with neoplastic cells. Almost all neoplastic cells were B-B2- and IgM-positive. However, there were a few B-B2- and/or IgM-negative cells or cells stained faintly in all cases. WC1+ cells were not observed in tumor tissues. However, CD4+ and CD8+ cells were observed throughout tumor tissues, suggesting a role for these cells in tumor immunity.

Mutations in the core promoter/enhancer II regions of naturally occurring hepatitis B virus variants and analysis of the effects on transcription activities.

The regulatory regions for transcription and replication of several hepatitis B virus (HBV) genomes from 19 patients having various forms of HBV infection were sequenced. Predominant mutations were found to occur naturally in nucleotide positions 1762 (A to T) and 1764 (G to A) in chronic hepatitis patients and in asymptomatic carriers after seroconversion, but were not observed in HBeAg-positive healthy carriers. Since these positions were located in the basic core promoter and the overlapping enhancer II regions situated within the core upstream region, transcriptional activity was examined by chloramphenicol acetyltransferase assay to determine if there was a possible difference between the mutant and wild-type HBV. However, no significant difference was detected upon comparison of the promoter and enhancer activities between mutant and wild-type HBV.

Pathogenesis of highly virulent infectious bursal disease virus infection in intact and bursectomized chickens.

The pathogenesis of highly virulent infectious bursal disease (IBD) virus (IBDV) infection was studied using 6-week-old intact and 5-week-old bursectomized chickens inoculated with highly virulent strain 90-11 or reference strain I. Chickens inoculated with 10(0.7) EID50 of strain 90-11 showed neither clinical signs nor lesions during the 4-day observation period. In contrast, birds inoculated with 10(2.7) or 10(4.7) EID50 developed severe clinical IBD, as well as gross and histologic lesions, typical of IBD, and produced IBDV antigen demonstrable by immunostaining in the bursa of Fabricius (BF), thymus, spleen and bone marrow from day 2 post-inoculation (PI) onwards. The antigen was also detected by the agar-gel precipitation and latex microsphere agglutination tests in a bursal suspension of these birds from day 2 or day 3 PI on. Birds inoculated with 10(6.1) EID50 of strain I developed only slight clinical signs at day 4 PI. Their lesion- and antigen-scores in the BF were almost the same as those in virulent strain-infected chickens, but lesion- and antigen-scores in the other organs were negligible. Bursectomized chickens inoculated with strain 90-11 did not develop clinical IBD despite the presence of infection that was evidenced by histologic lesions in the thymus and spleen as well as IBDV antigen demonstrable by immunostaining in these organs.

Analysis of upstream region of hepatitis B virus core gene using in vitro transcription system.

Transcription of the core (C) gene of hepatitis B virus DNA (HBV-DNA) was studied by an in vitro transcription system using nuclear extracts of human liver cell (HepG2) and non-liver cell (HeLa) origins. RNA polymerase II-dependent run-off transcription of 3.5-kb (C) mRNA was observed in both nuclear extracts; but the efficiency was much higher in the HepG2 nuclear extract. Analysis of run-off transcripts using upstream deletion mutants of HBV-DNA demonstrated that there are two transcription start sites located at approximately nucleotide numbers (nt) 1,797 +/- 5 and 1,815 +/- 5. This analysis also suggested that the minimum core promoter sequence and a cis-acting and liver-specific element for C mRNA transcription are located in the downstream region from -80 and -110 (HincII site) of transcription start sites, respectively. DNA-binding protein assays using synthetic double-stranded oligonucleotide probes corresponding to three regions in the upstream region (nt from 1,401 to 1,760) of transcription start sites revealed that there are some liver cell-specific and non-specific DNA-binding proteins in both nuclear extracts. The amount of those proteins was generally higher in the HepG2 nuclear extract. However, no obvious correlation was observed in the present study between the presence of DNA-binding proteins and transcription activity of nuclear extracts in our system. The possible causes of this discrepancy are discussed.

A rapid quantitative method for detecting infectious bursal disease virus using polystyrene latex microspheres.

A monoclonal antibody (mAb) to infectious bursal disease virus (IBDV) was bound to polystyrene latex microspheres. The microspheres agglutinated with extracts of bursae and sera from chickens infected with all strains or isolates of IBDV tested. Agglutination appeared within a 10-min reaction time. The assay could detect a 10(3.7) to 10(4.5) mean embryo infective dose (EID50) of the virus in 0.01 ml and the titer of the assay was 10- to 40-times higher than that of the agar gel precipitin test.

Correlation of serum antibody titers against hepatitis C virus core protein with clinical features by western blot (immunoblot) analysis using a recombinant vaccinia virus expression system.

In order to study the relationships among the clinical features of hepatitis C patients, the presence of hepatitis C virus (HCV) RNA in their blood, and their serum antibody titers against the core protein of virus and to study the antibody levels in asymptomatic HCV carriers, a recombinant vaccinia virus containing a core protein gene was constructed. The recombinant virus expressed a protein with a molecular mass of 22 kDa in RK-13 cells as determined by Western blot (immunoblot) analysis. By using the cell lysate of virus-infected cells and serially diluted serum samples, core antibody titers in the groups of patients in the chronic hepatitis phase and in the convalescent phase as well as in asymptomatic carriers were determined by enhanced chemiluminescence Western blot analysis. Almost all patients in the chronic phase were shown to have high antibody titers of more than 1:500,000 and with no exception had of HCV RNA in their sera. On the other hand, patients who had recovered naturally and were in the convalescent phase were shown to have significantly lower antibody titers, and the antibody was not detected in the lowest serum dilution of 1:500 in 43% of these patients (three of seven total patients). Antibody levels of patients who showed a good response to interferon treatment decreased to intermediate levels between those of patients in the chronic phase and those of patients in convalescent phase. The antibody titers in asymptomatic carriers varied considerably from 1:500,000 to 1:500, and 41% (11 of 27 total individuals) of these carriers showed a high titer equivalent to that of those in the chronic phase. Core antibody was detected consistently in the individuals in whom HCV RNA was detected. This system for core antibody might be useful for identifying the stage of an apparent HCV infection.

Resistance of mice vaccinated with rabies virus internal structural proteins to lethal infection.

Mice were vaccinated with recombinant vaccinia virus (rVac) expressing the glycoprotein (G), nucleoprotein (N), phosphoprotein (NS) or matrix protein (M) of rabies virus and their resistance to peripheral lethal infection with street rabies virus was examined. Mice vaccinated with rVac-G or rVac-N developed strong antibody responses to the corresponding proteins and essentially all mice survived challenge infection. Mice vaccinated with rVac-NS or rVac-M developed only a slight antibody response, however, a significant protection (59%) was observed in the rVac-NS-vaccinated mice, whereas rVac-M-vaccinated mice were not protected. No anti-G antibodies were detected in the sera of mice which has been vaccinated with rVac-N or rVac-NS and survived challenge infection. Passive transfer of anti-N monoclonal antibodies (MAbs) recognizing an epitope located on amino acids 1-224 of the protein prior to challenge resulted in significant protection, although the protection was not complete even with a high amount of antibodies. In contrast, none of the mice given MAbs recognizing an epitope of amino acids 247-415 or F(ab')2 fragments from a protective MAb IgG were protected. Administration of anti-CD 8 MAb to rVac-N-vaccinated mice showed no significant effect on protection. Our observations suggest that a considerable part of the protection achieved by the vaccination with rVac-N can be ascribed to the intact anti-N antibodies recognizing an epitope located on amino acids 1-224 of the protein.