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A simian cell line, Si-IIA, harboring Epstein-Barr-virus (EBV)-related herpesvirus (Si-IIA-EBV), produces malignant lymphoma in rabbits when administered by intravenous inoculation. In this study, we analyzed the Si-IIA-EBV genome and compared it with human EBV and herpesvirus macaca fascicularis 1 (HVMF 1), which is associated with B-cell lymphoma developing in SIV-infected immunosuppressed monkeys. DNA from Si-IIA-EBV was amplified by the polymerase chain reaction using three different primer pairs complementary to human EBV (B95-8) DNA; two of the primer pairs covered part of the long internal repeat 1 region (IR 1) and the third covered part of the BRRF 1 region. Direct sequencing of the three PCR products revealed that Si-IIA-EBV DNA had about 82% nucleotide homology to the human EBV DNA in all three regions and 92.4% homology to HVMF1 in the IR1 region. The blotting pattern by Southern blot analysis was different between Si-IIA-EBV and human EBV.

1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches). 2. Levcromakalim produced a concentration-dependent outward current at a holding potential of -50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations. 3. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min. 4. The peak amplitude of the levcromakalim (100 microM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide. 5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 microM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used. 6. In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 microM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM. 7. In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly. 8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening. 9. In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner. 10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 microM levcromakalim in inside-out patches. 11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.

The effects of nicorandil on ionic currents recorded from single smooth muscle cells of pig proximal urethra were investigated using patch-clamp techniques. Tension measurement was also performed to study the effects of nicorandil on the resting tone of pig urethra. Nicorandil produced a concentration-dependent sustained outward current that was suppressed by glibenclamide at -50 mV and was carried selectively by K+. In cell-attached configuration, nicorandil activated a 43-pS K+ channel that was reversibly inhibited by 10 microM glibenclamide. This glibenclamide-sensitive 43-pS K+ channel (KGS) "ran down" after excision of the membrane patch. In inside-out configuration, the application of either 1 mM Mg-ATP or 1 mM nucleotide diphosphate reactivated the KGS. In symmetrical 140 mM K+ conditions, 300 microM nicorandil and 300 microM levcromakalim activated a 2.14-pA K+ channel that exhibited the same amplitude and similar channel-opening kinetics. Methylene blue (10-100 microM), a soluble guanylate cyclase inhibitor, did not inhibit the opening of the nicorandil-induced KGS. The KGS was not activated by either sodium nitroprusside (10-100 microM) or 8-bromo guanosine 3':5'-cyclic monophosphate (1 mM). Nicorandil caused a concentration-dependent relaxation of the urethral resting tone but was less potent than levcromakalim. The relaxation induced by 10 microM nicorandil was partially inhibited by glibenclamide (1-10 microM) and also by methylene blue (10-100 microM). These results indicate that two independent nicorandil-induced relaxation mechanisms may be present in pig urethra.

Recently, clonal EBV-DNA and/or EBV-encoded small RNA(EBER1) have been detected in some gastric carcinomas. We reported the first observation of EBV infection in gastric glands with intestinal metaplasia, and characterized the EBV-infected lymphocytes which infiltrated in gastric mucosa. To determine the cellular location of EBV, EBER1 in situ hybridization(ISH) with an EBER1 oligonucleotide probe was applied to paraffin sections of the non-neoplastic gastric mucosa in 80 cases of gastric carcinoma and 49 cases of gastric ulcer. Not only was EBER1 expression detected in the nucleus of gastric cancer cells(5 cases) but also in non-neoplastic gastric epithelial cells(3 cases) and in infiltrated lymphocytes(40 cases). A single or a few shedding non-neoplastic epithelial cells in 2 cases of EBV-associated gastric cancer showed EBER1 expression. In one case, EBER1 was observed in all of the epithelial cells of a few gastric glands with intestinal metaplasia, suggesting that the stem cells of the metaplastic gastric glands were infected with EBV. EBV DNA was also detected in the EBER1-positive metaplastic glands scratched from the paraffin section by a single cell PCR method with a BamHI W primer pair. However, immunohistochemical examination showed that these metaplastic glands lacked expression of EBNA2 and LMP-1. The observation of these rare EBV-infected metaplastic glands reinforces the pre-transformation EBV infection hypothesis for EBV-associated gastric carcinoma. The double staining using ISH and immunohistochemistry revealed that EBER1 positive lymphocytes showed a B cell marker of L26(< 50%) but not T cell markers of UCHL1 and OPD4.

To investigate whether the lymphocyte homing receptors, adhesion molecules regulating normal lymphocyte traffic, influence the dissemination of lymphoma cells, 24 lymphoma/leukemia cell lines were inoculated into SCID mice subcutaneously, and the correlation between the expression of the adhesion molecules and the metastatic potential of the cell lines was examined. Among the six adhesion molecules examined (LFA-1, ICAM-1, CLA, VLA-4, L-selectin and CD44), L-selectin increased the incidence of lymph node metastasis, and CD44 expression was related to both lymph node and organ (hematogenous) metastasis. A monoclonal antibody to the standard form of CD44 (CD44s), Hermes-3, inhibited the local growth and remote metastasis of CD44+ cell lines. Thus, it is concluded that at least CD44s expression is important in both lymphatic and hematogenous metastasis.

Reed-Sternberg cells (RS cells) of Hodgkin's disease (HD) are frequently infected with Epstein-Barr virus (EBV) and express EBV-encoded nonpolyadenylated RNA transcripts (EBER)-1. EBV latency has been classified into three distinct forms: Latency I, expressing only one of the latent proteins, EBV nuclear antigen (EBNA)-1, latency II, coexpressing EBNA-1 and LMPs, and latency III, expressing all latent viral proteins. RS cells express LMP-1 in addition to EBNA-1 and are considered to be EBV latency II frequently encountered in nasopharyngeal carcinoma. We examined 13 cases of EBV-infected HD by combined EBER-1 in situ hybridization and immunostaining for LMP-1. All of the RS cells expressed EBER-1, but a substantial number of EBER-1+ RS, cells were negative for LMP-1. The percentage of LMP-1+ RS cells out of EBER-1+ RS cells varied from 7% to 100% (average 69%). In this study, we showed that all EBV-infected RS cells were not restricted to latency II, and some belonged to latency I.

Effects of synthesized calciseptine (CaS), found naturally in the venom of the black mamba, on voltage-dependent Ca2+ channels in smooth muscle cells of the guinea-pig portal vein were investigated. In the whole-cell voltage-clamp configuration, extracellular application of CaS (>/= 10 nM) inhibited the inward current in a concentration- and voltage-dependent manner at a holding potential of -90 mV. The Ca2+ current recorded at a high holding potential (-50 mV) was approximately 8 times more sensitive to CaS than that at a more negative holding potential (-90 mV). CaS (50 nM) shifted to the left the steady-state inactivation curve obtained by using single 8-s conditioning pulses of various amplitudes. When CaS (>/= 200 nM) was present in the pipette, the Ca2+ current remained for the duration of the experiments (more than 60 min) in the whole-cell configuration. Two different Ca2+ channel conductances are present in this tissue (25-pS and 12-pS channels). Both channels are blocked by dihydropyridine (DHP) derivatives, but have different sensitivities. In the cell-attached condition, CaS hardly changed the activity of either unitary Ca2+ channel current. To prevent the "run down" of the Ca2+ channels in cell-free conditions, we added cardiac cytosol, a supernatant from homogenized cardiac cells and an endogenous Ca2+ channel activating factor, in the pipette. The unitary Ca2+ channel currents were then recorded using the outside-out membrane patch configuration. Application of CaS (1 microM) in the bath completely blocked the open events of the 25-pS Ca2+ channel. CaS (10 nM) in the bath reduced the mean open time and channel availability, resulting in a decrease in the open probability of the 25-pS channel currents without affecting the amplitude of the single-channel conductance. CaS also reduced the open probability (though less potently) and channel availability of the 12-pS Ca2+ channel without a change in its amplitude. From these results, we conclude that CaS has inhibitory effects on the voltage-dependent Ca2+ current that are similar to those of DHP derivatives and that it acts from the outside of the membrane.

Background: The Epstein-Barr virus (EBV) has been frequently detected in lymphoid malignancies. However, EBV infection in the nonneoplastic cells of lymphoid malignancies has not been extensively studied., Methods: Four hundred nine cases of lymphoid malignancies including 377 non-Hodgkin's lymphoma (NHL) and 32 Hodgkin's disease (HD) were examined for EBV infection by EBER-1 in situ hybridization (EBER-ISH), immunostaining against LMP-1, Epstein-Barr nuclear antigen 2 (EBNA2) and ZEBRA, and Southern hybridization using a BamHIW fragment as a probe. Double staining with EBER-ISH and immunostaining against CD20, CD45RO, and LMP-1 was performed in selected cases., Results: Although EBER-1-positive cells (EPCs) were detected in 49 of 276 B-cell lymphomas, 31 of 100 T-cell lymphomas, 1 of 1 natural killer-cell lymphoma, and 17 of 32 HDs, almost all of the tumor cells were exclusively EBER-1-positive in the 10 NHL cases. Some EPCs were of different cell lineages than the tumor cells in 15 of the 26 NHLs examined by double staining. LMP-1, EBNA2, and ZEBRA were detected in 22, 4, and 3 cases, respectively. In 4 LMP-1-positive HDs, double staining revealed that some EBER-1-positive Reed-Sternberg cells were negative for LMP-1, EBV genomic DNA was detected in 8 of the 39 examined cases., Conclusions: T-cell lymphomas contained EPCs more frequently than B-cell lymphomas. Nonneoplastic lymphocytes were infected with EBV more frequently than lymphoma cells. Rowe's latency II may be unstable in lymphoid malignancies. Some NHLs, especially T-cell lymphoma, may provide favorable conditions for EBV infection of nonneoplastic lymphocytes.

1. The effects of levcromakalim (BRL 38227) on ionic currents recorded from pig proximal urethra were investigated by use of tension measurement and patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, and cell-attached configuration). 2. Levcromakalim (1 microM) caused a relaxation in the resting tone. This levcromakalim-induced relaxation was inhibited by the pretreatment with 1 microM glibenclamide. 3. The resting membrane potential recorded from single cells in current-clamp mode was-36.1 +/- 4.4 mV (n = 5). 4. Levcromakalim induced a concentration-dependent hyperpolarization with a maximum (at > or = 10 microM) close to the theoretical equilibrium potential of potassium (EK). The membrane hyperpolarization caused by 1 microM levcromakalim (24.7 +/- 5.8 mV, n = 4) was abolished by 1 microM glibenclamide. 5. Levcromakalim (100 microM) caused an outward K current in whole-cell recordings which was unaffected by iberiotoxin (300 nM) but abolished by glibenclamide (10 microM). 6. In cell-attached patches, levcromakalim activated a 43 pS K channel which was inhibited by the application of glibenclamide. 7. The metabolic poison, cyanide (CN), also activated a 43 pS K channel which was suppressed by the application of 10 microM glibenclamide. 8. These results indicate that levcromakalim and metabolic inhibition activate the same 43 pS K channel in pig proximal urethra. The resultant urethral hyperpolarization might reduce the usefulness of K channel openers in the treatment of detrusor instability, but be of value in treating outflow obstruction.

To elucidate the latent state and reactivation of Epstein-Barr virus (EBV) in non-neoplastic lymphoid lesions, we investigated 144 non-neoplastic lymphoid lesions by in situ hybridization (ISH) to detect the expression of EBV-encoded small RNAs (EBER)-1 and BCRF-1 and by immunostaining for latent membrane protein (LMP)-1 and ZEBRA. ISH for EBER-1 detected EBER-1-positive cells (EPC) in 31 of the 144 examined lesions (22%). EPC were detected in 4 of 49 cases of nonspecific lymphoid hyperplasia, in 16 of 20 abscess-forming granulomatous lymphadenitis (AFGL), 5 of 25 Kikuchi's disease, and in 3 of 3 infectious mononucleosis. LMP-1 was expressed in 6 of 124 non-neoplastic lymphoid lesions (4.8%). LMP-1-positive cells were observed in 6 of the 31 EBER-1-positive cases (19%). EPC were detected significantly more frequently in LMP-1- and ZEBRA-positive specimens than in the LMP-1- and ZEBRA-negative specimens. BCRF-1 was expressed in 4 of 11 cases examined: 2 of 3 AFGL, 1 of 2 Kikuchi's disease, and in the 1 case of atypical lymphoid hyperplasia. This study suggests that Epstein-Barr virus is prevalent and can be reactivated in the lymph nodes effaced by destructive inflammation, such as AFGL. Such inflammation may provide a local milieu that is conducive for EBV to enter the lytic cycle.

Background: Little is known about the clonal heterogeneity of non-Hodgkin's lymphoma between presentation and relapse, although several such reports have been published on acute lymphoblastic leukemia., Methods: We examined five patients with B-cell lymphoma who relapsed more than 5 years after initial presentation. Formalin fixed, paraffin embedded tissues were analyzed for clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction (PCR) and sequencing of the PCR products. Four specimens retained the original histologic type, but one showed histologic transformation from diffuse large cell lymphoma to follicular small cleaved cell lymphoma., Results: Although the size of the PCR products looked identical on the gel between presentation and relapse in all patients, only three of the four specimens that retained the original type had identical gene rearrangements at both presentation and relapse. One of these four and the fifth specimen showed novel gene rearrangements., Conclusions: This study suggests that late relapse lymphoma may present as a new clone. Sequencing of the PCR products is important in the evaluation of clonal heterogeneity.

Malignant lymphomas, which were usually of T-cell type, were induced in 10 of 13 (77%) male rabbits (Japanese white, 8/10; New Zealand white, 2/3) inoculated i.v. with HTLV-II-transformed simian (Cynomolgus) leukocyte cell line (Si-IIA) cells. Of 7 rabbits injected with cell-free pellets from Si-IIA cultures, 5 also developed malignant lymphoma (15-28 days). Lymphoma development was completely inhibited by inactivation of cell-free pellets from Si-IIA culture with ethyl ether and was almost suppressed by neutralization of the cell-free pellets with anti-Si-IIA sera. Herpesvirus particles were discovered very rarely in Si-IIA cells, in addition to C-type virus particles, by electron microscopy. Si-IIA cells were positive for Epstein-Barr-virus (EBV)-associated nuclear antigen (EBNA) by immunofluorescence (IF) test. Antibody response to viral capsid antigen of EBV was also detected in sera from rabbits inoculated with Si-IIA. EBV-encoded RNA-1 (EBER-1) was demonstrated in Si-IIA, the tumor tissues and all rabbit tumor cell lines by in situ hybridization. EBV DNA was also detected in Si-IIA and rabbit lymphoma cell lines by polymerase chain reaction (PCR) and Southern blotting. However, EBV DNA was amplified only by some primers complementary to human EBV sequence (B95-8), but not by other primers. Integration of HTLV-II provirus genome could not be detected in Si-IIA-induced rabbit tumor cells. Moreover, no lymphoma was induced by inoculation of HTLV-IIC and MOT (other HTLV-II-producing human cell lines), B95-8(EBV-producing cell line) or TALL-1 and peripheral leukocytes from normal Cynomolgus (controls). Neither Herpesvirus saimiri nor H. ateles (simian oncogenic viruses) were detected in Si-IIA cells by IF test. These data suggest that the high rate of lymphoma induction in rabbits may not be caused by HTLV-II, human EBV (B95-8) or well-known simian oncogenic viruses, but by EBV-related herpesvirus derived from Si-IIA cells or HTLV-IIA cells, with which Si-IIA was established. The availability of this animal model promises to clarify the role of EBV in human lymphoma and provides a means of studying prophylactic and therapeutic regimens.

CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class I mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one call line but was not promoted by the treatment with anti-HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95-mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.

Right-sided cardiac malignant fibrous histiocytoma (MFH) is extremely rare, and to the authors' knowledge only three cases have been reported. In this study, a case of MFH in the right ventricle, the septum, and the pulmonary valves and artery in a 47 year old male is described. The tumor showed typical pathological features of MFH, such as cellular pleomorphism, storiform pattern and abundant mitoses. Immunohistochemical and electron microscopical findings were compatible with MFH, and excluded the possibility of leiomyosarcoma and angiosarcoma. Whole body examination, including Gallium scintigram, localized the primary site to the heart. The details of this case are presented with a review of the reported cases of cardiac MFH.

A new human lymphoma cell line derived from pulmonary non-Hodgkin's lymphoma that developed in a renal transplant recipient was established from the patient's pleural effusion and designated PTLC-1. PTLC-1 grew aggressively in suspension, forming very loose clumps with a doubling time of about 18.9 h. The morphological, chromosomal, and immunophenotypic characteristics of the patient's tumor cells and PTLC-1 cells were very similar. PTLC-1 showed a monoclonal rearrangement of IgH gene and was highly tumorigenic in athymic nude mice. In situ hybridization, Southern blot hybridization and polymerase chain reaction demonstrated the presence of Epstein-Barr virus (EBV) genome in the patient's tumor and PTLC-1. PTLC-1 has been maintained in culture for over 60 months. Since EBV has been implicated in the pathogenesis of post-transplant lymphoma, this new cell line should serve as a useful experimental model for studying the etiology and biology of lymphoma developing in organ transplant recipients.

Biotinylated bauhinia purpurea agglutinin (BPA) was studied, using the ABC method, in different thyroid gland conditions (26 follicular adenomas, 39 papillary carcinomas, 15 follicular carcinomas, and 10 normal thyroids), to determine whether specific lectin binding patterns were developed during malignant transformation that could enable the distinction of carcinoma from adenoma. In normal thyroids, BPA was very rarely and faintly reactive with follicular cells. In neoplastic conditions, BPA binding profiles for follicular adenoma were essentially identical to those of normal follicles, whereas BPA unequivocally reacted with follicular carcinoma and papillary carcinoma. BPA reacted more strongly with cells of papillary structures than with those forming solid nests and follicles. In papillary carcinoma, BPA binding was observed mostly in the apical surface and cytoplasm of carcinoma cells, whereas a diffuse cytoplasmic binding pattern was predominant in follicular carcinoma. Neuraminidase treatment had little or no effect on either normal or adenomatous follicular epithelium, whereas in follicular carcinoma, the number of positive cells and the staining intensity was increased. These findings suggest that BPA would be useful for the differential diagnosis of papillary and follicular carcinomas, and less consistently so, for differentiating follicular carcinoma from follicular adenoma. Peanut agglutinin with similar sugar specificity was not reactive with follicular cells either in normal or neoplastic glands.

Lymphoma induction in rabbits by an unknown factor derived from an HTLV-II-producing simian (Cynomolgus) leukocyte cell line (Si-IIA) is reported. Thirteen of 17 male Japanese white rabbits (76%) inoculated intravenously with Si-IIA cells developed malignant lymphoma including Hodgkin-like lymphoma between 62 and 167 days after inoculation. Histologically, there was extensive diffuse or nodular infiltration of either large cell type or mixed type lymphoma cells in many organs, frequently involving the spleen, liver, lymph nodes and kidneys, and less frequently the thymus, bone marrow, lungs, heart, skin and gastrointestinal tract. Hodgkin-like lymphoma was also observed in two rabbits. Chromosomal analysis of five cell lines established from tumor-bearing rabbits revealed the male rabbit karyotype. The immunophenotype of these tumor cells was usually T-cell (CD5+ or -, RT1+, RT2+ or -, CD45+, CD4-, RABELA- and MHC class II-DQ+) except for Hodgkin-like lymphoma cells which expressed only CD45. However, integration of the HTLV-II provirus genome could not be demonstrated in the tumor tissues or any of the rabbit cell lines by polymerase chain reaction or Southern blot analysis. Moreover, no lymphoma was induced by inoculation of HTLV-IIC, MOT (other HTLV-II-producing human cell lines) or TALL-1 (control). Two of four rabbits injected with cell-free pellets from Si-IIA cultures died of malignant lymphoma (15-20 days). Five irradiated rabbit cell lines were inoculated but only one (Ra-SLN) induced lymphoma in 1 of 3 rabbits at 27 days. Neither Herpesvirus saimiri nor Herpesvirus ateles (simian oncogenic viruses) was detected in Si-IIA cells by immunofluorescence testing. These data suggest that the high rate of lymphoma induction in rabbits may be caused not by only HTLV-II or well known simian oncogenic viruses, but rather by an unknown passenger agent derived from Si-IIA or HTLV-IIA, with which Si-IIA was established.

The sensitivity and specificity of single cell polymerase chain reaction (PCR) were studied. Its high sensitivity enabled detection of a single-copy gene, such as human T-lymphotropic virus type I genome in paraffin sections. The rate of obtaining positive signals with this method was affected by the number of copies of the gene in the target cell. Specificity was satisfactory if the procedure was properly and carefully followed. Since the single cell PCR is a time-consuming method which requires skill and experience to pick up the target cells accurately, the applicability of this method is limited. It works best when it is used to analyze a single or a few copy genes in histologically identified cells.

Expression of the CD30 antigen in lymphoid neoplasms and reactive lesions were examined immunohistologically using the monoclonal antibody BerH2. CD30 antigen was expressed in 17 of 18 patients with Hodgkin's disease (HD), all of three anaplastic large cell lymphomas (ALCL), 11 of 52 T-cell malignant lymphomas (TML) including ALCL, 13 of 153 B-cell malignant lymphomas (BML) including ALCL, two of three malignant histiocytosis and one of four plasmacytomas. Although a single case of small cell lymphoma was positive, in cases of mixed cellular morphology, neoplastic cells of larger or more pleomorphic nuclei tended to be stained more extensively and intensively. This antigen is expressed in TML significantly more often than in BML (P < 0.05). CD30 antigen was expressed less frequently at clinical stage I than those of stages II to IV. There was no significant relationship between CD30 antigen expression and that of proliferating cell nuclear antigen or CD43 antigen in non-Hodgkin's lymphomas (NHL). The CD30 antigen expression did not affect the prognosis of NHL overall, but in high grade TML, CD30 antigen-positive individuals tended to have a more favorable prognosis than those that were negative. In reactive diseases, some plasma cells, immunoblasts, interdigitating cells, follicular center cells and histiocytes were stained positively, but not always, and with variable intensity. These results suggest that CD30 antigen is expressed in various cell lineages to some extent and may relate to cellular activation in some instances. When making the histopathologic diagnosis of HD or NHL including ALCL, CD30-positive cell should be carefully interpreted.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the bcl-2 protein and bcl-2 mRNA at the individual cell level was semiquantitatively examined in normal quiescent peripheral blood lymphocytes and pokeweed mitogen- or concanavalin A and interleukin-2-induced lymphoblasts in vitro by microscopic fluorometry using immunofluorescence and fluorescein-labeled in situ hybridization. Approximately 90% of normal quiescent T and B lymphocytes expressed bcl-2 protein at a level which was compatible with that of bcl-2 mRNA. On the contrary, most mitogen-induced lymphoblasts showed a posttranscriptional suppression of bcl-2 protein expression. However, bcl-2 protein was not downregulated by the posttranscriptional suppression in all lymphocytes activated in vitro, but approximately 15% of the lymphoblasts still expressed bcl-2 protein at a higher level than nontransformed quiescent small lymphocytes; thus bcl-2 protein expression in lymphoblasts showed a distinct bimodal pattern. Furthermore, it was supposed that lymphoblasts with no detectable bcl-2 protein might fall into apoptosis but the remainder, expressing high levels of bcl-2 protein, could escape apoptosis. Thus, the bcl-2 gene may play an important role as a regulator of apoptosis in the human immune system.

BALB/c mice either were injected subcutaneously with 25 micrograms/g or 50 micrograms/g body weight 7,12-dimethyl-benz[a]anthracene (DMBA) at the age of 13 weeks, or received repeated s.c. injections of azathioprine (0.1 mg/g) starting from the age of 6 or 18 weeks, or they received treatments with both agents. Of the 317 mice treated, 69 developed lymphomas by the age of 50 weeks. None of the 31 non-treated mice developed lymphomas during this period. The lymphomas, classified according to Pattengale-Taylor's classification for murine lymphomas/leukemias, were examined immunohistochemically and immunogenetically. The lymphomas induced with DMBA showed a male preponderance and a higher incidence of the B cell type, especially in the lymph node and spleen. Azathioprine induced predominantly T-lymphoblastic lymphomas in the thymus and these were found more frequently in females. Treatment with both DMBA and azathioprine remarkably increased the incidence of B lymphomas, especially follicular center cell lymphomas in the spleen, which are the predominant type of spontaneous lymphomas occurring in older BALB/c mice. Some of the treated mice also developed prelymphomatous lesions.

An HTLV-I-immortalized human T cell line (JP-2), a N-methyl-N'-nitro-N-nitrosoguanidine-treated JP-2 line (JP-2T), and an adult T cell leukemia cell line (ATL-1T) were examined morphologically and phenotypically. All of these cell lines expressed some T cell markers, including CD4, and showed rearrangement of T cell receptor (TCR) genes, but they lacked CD3 and TCR antigens and expressed some myelomonocytic markers (CD68, HL-21, CD15, CD16). JP-2 cells grew in suspension, but JP-2T and ATL-1T cells, which mostly adhered to the surface of culture vessels, showed macrophage-like morphological features and expressed more monocyte/macrophage markers (lysozyme, alpha 1-antitrypsin) and fibronectin. ATL-1T cells transplanted into SCID mice lost the macrophage features. These results suggest that HTLV-I infected T cells can express some macrophage features and that these cells may provide a model that will be useful in elucidating the phenotypic variability of T cell lymphomas.

A 71-year-old male was admitted to an emergency hospital owing to severe dyspnea of sudden onset. Bilateral tension pneumothorax with two giant bullae occupying more than the third of the right lung was disclosed on the chest X-ray. Bilateral chest drainages and cardiopulmonary resuscitation were performed. He could be freed from ventilatory support several days after tracheostomy and was referred to our hospital. The pulmonary function was severely depressed: VC (%) 1.20 l (40%) and FEV1.0 (%) 0.55 l (46%). In spite of oxygen inhalation he could not keep sitting due to dyspnea (Hugh-Jones grade V). Right multiple bullectomy was performed successfully and his postoperative course was uneventful. He was discharged eight weeks later. Seven months after surgery his pulmonary function was remarkably improved: VC (%) 2.05 l (64%) and FEV1.0 (%) 0.94 l (46%). He became to walk almost equally with a healthy man one year later (Hugh-Jones grade II).

Two cases of spindle cell hemangioendothelioma (SCH) are reported. One of the patients was a 16 year old Japanese female, who had been suffering from Ollier's disease (multiple enchondromatosis) since 3 years of age and had developed multiple SCH in the right leg at the age of 11 years. Spindle cell hemangioendothelioma lesions coincided with the site of enchondromatosis and increased in number thereafter. This is the first report of Ollier's disease complicated with multiple SCH. Another patient, a 33 year old Japanese female, who was a carrier of hepatitis B virus (HBV), developed solitary SCH in the lateral aspect of the right ankle where a lipoma was extirpated 10 years previously. Tumor cells of both cases were composed of four cell types: (i) spindle cells; (ii) epithelioid cells; (iii) vacuolated endothelial cells; and (iv) usual endothelial cells. Endothelia in the cavernous area and vacuolated cells reacted to Ulex europaeus agglutin 1 (UEA-I), factor VIII-related antigen and vimentin. Spindle cells and epithelioid cells reacted only to vimentin.

The acute antihypertensive effects of 3-pyridine carboxylic acid 5-[(cyclo-propylamino)carbonyl]-1,4-dihydro-2,6-dimethyl-4-(2-nitroph eny l) octyl ester (NP-252, CAS 132031-81-3) administered orally to conscious spontaneously hypertensive rats (SHRs) and renal hypertensive rats (RHRs) were evaluated using the impedance plethysmographical technique as a modified tail cuff method, and compared with those of nifedipine (NF). In the fitness test of this indirect method, the average value of blood pressure (BP) measured in 7 conscious SHRs was 201 +/- 6.9 mmHg. This value showed good correlation with that (201 +/- 8.8 mmHg) of systolic BP measured by the direct method in the same animals. In the comparative study of antihypertensive activities of the compounds on both models of hypertension using this method, NP-252 and NF dose-dependently lowered BP having a different peak time and restoration after dosing. Therefore, the antihypertensive activities were compared using a 20% effective dose (ED20) for producing hypotension, and the ED20 values of NP-252 and NF were 2.55 and 2.00 mg/kg in SHRs, and 1.25 and 0.67 mg/kg in RHRs, respectively. Moreover, the duration of actions of the compounds were evaluated by the simulated duration time (SDT) which was calculated from the peak time of BP-fall and the pharmacological half life time for the maximum BP-fall and the SDT values of NP-252 and NF were 1.85-4.70 and 0.90-0.75 h in SHRs, and 3.30-12.80 and 0.57-6.90 h in RHRs, respectively. Also, the BP-falls by the compounds were accompanied by an increase in heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of semotiadil on the voltage-dependent Ca current (ICa) were investigated in dispersed smooth muscle cells of the rabbit portal vein. At a holding potential of -100 mV, semotiadil (> or = 0.1 microM; dissolved in dimethylsulphoxide, DMSO) inhibited the ICa in a concentration-dependent manner (IC50 = 2.0 microM, Hill's coefficient = 1.0). At a holding potential of -80 mV or -60 mV, the concentration-inhibition curve observed in the presence of semotiadil was shifted to the left compared with that observed at -100 mV; and semotiadil shifted the voltage-dependent inactivation curve to the left. The curve for the decay of ICa was fitted with two time constants. Semotiadil (< 1 microM) reduced the slow but not the fast time constant. The curve for the recovery from ICa inactivation also consisted of two time constants, and semotiadil (1 microM) prolonged the slow recovery. Semotiadil dissolved in deionized water more potently inhibited ICa than semotiadil dissolved in DMSO. At pH 10.0, semotiadil did not modify the voltage-dependent inactivation curve. However, recovery from the inactivation was much faster at pH 10.0 than at pH 7.3. These results indicate that the voltage-dependent inhibition of ICa by semotiadil may be due to binding of the ionized drug during the inactivated state and also inhibition of the transition from the inactivated to the resting state. Long-lasting inhibition of ICa after removal of semotiadil may be due to tight binding of semotiadil on the channel through a hydrophobic site.

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutyrate (PDBu) and H-7 on Ca(2+)-dependent K+ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (Ioo). Neomycin (30 microM), an inhibitor of phospholipase C, and intracellular applications of heparin (10 micrograms/ml) and guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]; 1 mM) partly but consistently inhibited the generation of Ioo, whereas a higher concentration of heparin (100 micrograms/ml) transiently enhanced then suppressed the generation of Ioo. Inhibition of Ioo generation by heparin was more powerful at the holding potential of +20 mV than at -20 mV. Inositol 1,4,5-trisphosphate (InsP3; 30 microM) continuously generated Ioo at holding potentials more positive than -60 mV. Noradrenaline (10 microM) and caffeine (3-20 mM) transiently augmented, then reduced the generation of Ioo. Heparin (10 micrograms/ml) completely inhibited responses induced by InsP3 and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5'-triphosphate (GTP; 200 microM) or low concentrations of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]; < or = 3 microM) continuously augmented the generation of Ioo. High concentrations of GTP[gamma S] (> or = 10 microM) transiently augmented, then inhibited Ioo. Neither GTP[gamma S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of Ioo when applied in the presence of GDP[beta S] (1 mM), neomycin (30 microM) or heparin (10 micrograms/ml). PDBu (0.1 microM) reduced the generation of Ioo but failed to produce an outward current following application of caffeine (3-5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 microM). In the presence of H-7, GTP[gamma S] continuously enhanced the generation of Ioo. The suppression of the generation of Ioo during application of noradrenaline (10 microM) was reduced by pretreatment with H-7. Thus both InsP3 and protein kinase C contribute to the generation of Ioo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP3 antagonist on the InsP3-induced Ca(2+)-release channel (PIRC). InsP3 opens PIRC and protein kinase C may deplete the stored Ca2+ by either inhibiting the reuptake of Ca2+ or by enhancement of the releasing actions of InsP3.

The cardiovascular effects of 3-pyridine carboxylic acid 5-[(cyclopropylamino)carbonyl]-1,4-dihydro-2,6-dimethyl-4-(2-nitro phenyl) oxtyl ester (NP-252, CAS 132031-81-3) were examined in anesthetized closed- or open-chest dogs and Langendorff perfused rabbit hearts, and compared with those of nifedipine (NF) and nicardipine (NC). In anesthetized closed- and open-chest dogs, NP-252 (i.v.) selectively increased vertebral and coronary blood flow with a fall in mean blood pressure (MBP). These effects were nearly equipotent with those of NF and NC, but more durable. A similar effect was obtained by intraduodenal administration of NP-252. Also, NP-252 (i.v.) decreased MBP, total peripheral resistance (TPR), left ventricular pressure and dLVP/dtmax while cardiac output (CO) and stroke volume (SV) increased without apparent changes in heart rate and myocardial contractility index. Decreases in MBP and TPR by NP-252 were equipotent with NF and NC, whereas increases in CO and SV were more potent than those of the two drugs. These actions of NP-252 were longer than those of the reference agents. Further, in perfused rabbit hearts, NO-252 increased coronary perfusion flow (CPF), accompanying a slight negative inotropy. Its effect in CPF was equipotent with NF and 2 times less than that of NC, however the duration was much longer than those of the reference drugs. On the other hand, the negative inotropic effect of NP-252 was less than NF, but greater than that of NC. In the preparations controlled by pacing, NP-252 and NF lengthened atrio-His bundle conduction time, without affecting His bundle-ventricular conduction time. Lengthening effect of NP-252 was about 4 times less than that of NF. These results suggest that NP-252 acts more selectively on vascular smooth muscle than cardiac muscle, and has almost equipotent but markedly longer vasodilating actions as compared to NF and NC. It may be expected that NP-252 is a new generation of tissue specific and long acting Ca2+ antagonist.

Eleven cases of Hodgkin's disease (HD) were examined for the presence of the Epstein-Barr virus (EBV) genome, using the polymerase chain reaction (PCR) to detect EBV DNA in whole paraffin-embedded tissue specimens and in single cells picked out from the specimens with a micromanipulator. The EBV genome was detected in 5 of the 11 cases by conventional PCR. Single cell PCR demonstrated the EBV genome in Reed-Sternberg cells from all the EBV-positive cases, but not from any of the EBV-negative cases. Background lymphocytes and lysozyme-positive histiocytes from EBV-positive cases did not contain the EBV genome. These results indicate an etiological association of EBV with some cases of HD.

A 79-year-old woman had cutaneous T cell lymphoma. After a long clinical course, numerous tumors and an enormous increase in peripheral granular lymphocytes without lymphadenopathy developed suddenly. Surface markers and DNA analysis of the tumor cells from her skin and peripheral blood and electron microscopic examination suggested that granular lymphocytic leukemia had developed in the terminal stage of cutaneous T cell lymphoma. To our knowledge this association has not been reported previously.

Infectivity of human T-lymphotropic virus type I (HTLV-I) to human nervous tissue cells was explored using co-cultivation with X-irradiated, HTLV-I-producing MT2 cells. Examined cells included normal cerebellar cells, brain tumor cells (astrocytoma, medulloblastoma, meningioma, hemangioblastoma, and schwannoma), and various cell lines (astrocytoma, ependymoma, oligodendroglioma, medulloblastoma, and neuroblastoma). Successful HTLV-I infection was confirmed immunohistochemically using monoclonal antibodies to HTLV-I p19, p24, and pX product. All cell lines and primary cultures from normal cerebellar tissues and brain tumors could be infected with HTLV-I. Double immunostaining showed that glial fibrillary acidic protein-, S-100 protein- or vimentin-positive cells were susceptible to infection. Neurofilament- or neuron-specific enolase-positive cells in medulloblastoma could also be infected. Reverse-transcriptase assay revealed the productive infection in U251-MG (astrocytoma) and KG-IC (oligodendroglioma) lines. Co-cultivated U251-MG cells formed syncytial polykaryons after serial passages, and polymerase chain reaction assay detected HTLV-I genome in U251-MG syncytial polykaryons and p19+ mononuclear cells. HTLV-I viral RNA was also detected in infected U251-MG cells by in situ hybridization. These data show that HTLV-I may have a wide spectrum of infectivity in human nervous tissues.

For the diagnosis of allergic rhinitis we measured the specific IgE and the total serum IgE in the blood. Although we had measured them by the conventional Phadebas RAST and the Pharmacia IgE RIA before, we now adopted the new Pharmacia CAP System (CAP RAST FEIA and CAP IgE FEIA). To compare these methods, we measured the specific IgE to the cedar pollen and total serum IgE in the same blood of 224 hospital staff at the regular physical examination. As a result the new Pharmacia CAP System proved to be simpler, safer, faster, easier, more specific, and more sensitive than the Phadebas RAST and the Pharmacia IgE RIA. And also these methods correlated well with each other.

The effects of E4080 [(E)-N-[3-[N'-(2-(3,5-dimethoxyphenyl)ethyl)-N'-methyl)amino)propyl]-4- (4-(1H-imidazol-1-yl)phenyl)-3-butenamide dihydrochloride dihydrate] on ionic currents recorded from the rabbit portal vein were investigated by using the patch-clamp technique. A depolarization of the membrane produced an inward Ca current (ICa), a transient outward current (ITO), a sustained outward current (ISO) and an oscillatory outward current (IOO), whereas a hyperpolarization of the membrane produced a hyperpolarization-activated current (Ih). When ICa was evoked by a depolarizing pulse to 0 mV from the holding potential of -80 mV, 1 microM E4080 increased and higher concentrations (10 microM) inhibited ICa, whereas, at the holding potential of -60 mV, E4080 (greater than 1 microM) consistently inhibited ICa, in a concentration-dependent manner. E4080 inhibited ITO, ISO (greater than or equal to 1 microM) and Ih (greater than or equal to 0.1 microM) concentration dependently. Inasmuch as 1 microM E4080 did not inhibit ICa at the holding potential of -80 mV, the inhibition of ITO induced by 1 microM E4080 was not related to the inhibition of ICa. When a continuous depolarization (0 mV) was applied to the cell, E4080 (1 microM) produced a maintained outward current (14.0 +/- 15.3 pA), which was inhibited by glibenclamide. I infinity was inhibited by E4080 (greater than or equal to 0.1 microM) and application of 1 microM glibenclamide partly restored I infinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Annular erythema developed in 22 patients with Sjögren syndrome. Clinically, the annular erythema was subdivided into three forms: Sweet disease-like annular erythema with an elevated border (14 cases); subacute cutaneous lupus erythematosus (SCLE)-like marginally scaled erythema (5 cases); and papular erythema (3 cases). Histopathologically, features commonly seen in annular erythema are deep perivascular and/or periappendageal infiltration of the lymphocytes with an admixture of neutrophils or plasma cells and less frequent epidermal changes suggestive of cutaneous lupus erythematosus. Immunoglobulin or complement deposition along the dermoepidermal junction of lesional skin was observed in 8 of 18 cases, and most of the dermal infiltrates consisted of CD4(+), 4B4(+) cells. The appearance of anti-SS-A(Ro) (100%) and anti-SS-B(La) (77%) was significantly higher in patients with annular erythema. These results suggest that patients with Sjögren syndrome might have a distinct annular erythematous lesion that is both clinically and histopathologically different from SCLE, although close immunologic abnormalities exist in these two diseases.

The efficacy of ofloxacin for the treatment of lower respiratory tract infection was evaluated in aged patients with chronic lung disease. Results are the following: improvement of leukocytosis and arterial oxygen tension was observed; peripheral blood cell analysis showed a decrease in natural killer cell counts and in helper/suppressor T cell ratio; superoxide production by peripheral blood white cells was decreased after treatment; interleukin-2 production was rather increased. We concluded that improvement in the immunological parameters indicated the efficacy of ofloxacin for lower respiratory tract infection in aged patients with chronic lung disease.

In four patients with primary Sjögren's syndrome a distinct annular erythema developed, which was characterized by a wide, elevated border (which can be likened to a doughnut ring) and central pallor. Histologically, there was a coat sleeve-like infiltration of lymphocytes around blood vessels and nuclear debris in the connective tissue. Vasculitis or epidermal changes suggestive of lupus erythematosus were not observed, and there was no immunoglobulin deposition along the basement membrane zone. This annular erythema may be a cutaneous manifestation of Sjögren's syndrome.

In this study, we demonstrated that the patients with PSS who develop proteinuria during the course are much more prone to develop lung fibrosis, telangiectasia, serositis and esophageal hypomotility. They also exhibited positive LE cell phenomenon, lupus band test, thrombocytopenia and leukopenia. The mortality rate is significantly higher in proteinuria positive patients and abnormal clinical data described above seem to be the risk factors for the development of renal involvement in PSS.

General pharmacological properties of guanabenz (GUB), a new anti-hypertensive agent, were studied in comparison with those of clonidine (CLD) and guanethidine (GUD). Intravenous or peroral administration of GUB caused a contraction of the nictitating membrane in cats and mydriasis in mice, while it produced an inhibitions of the gastrointestinal motility in dogs; the motility of isolated rabbit ileum; and chacol transport, salivation and gastric acid secretion in rats. GUB had no or slight inhibitory actions on contractile responses induced by peripheral sympathetic or parasympathetic nerve stimulation in various organs; however, it had antagonistic actions against the norepinephrine-induced contraction of isolated guinea-pig vas deferens. The contractile responses to epinephrine and tyramine in the nictitating membrane and to sympathetic nerve stimulation in isolated guinea-pig vas deferens were potentiated by GUB. GUB specifically antagonized the serotonin-induced contraction of the isolated rat fundus strip and nonspecifically inhibited acetylcholine, histamine or Ba2+-induced contractions of isolated guinea-pig ileum at higher concentrations. GUB exhibited local anesthetic actions and diuretic effects, but had no particular actions on neuromuscular transmission, isolated rat uterus, guinea-pig tracheal muscle and the hematic system. These effects of GUB were found to be almost identical with but less potent than those of CLD. The effects of GUD were basically different from GUB.

Co-cultivation of thymus and spleen cells of Fisher and Lewis rats with lethally irradiated MT-2 cells harboring human T-cell leukemia virus type I (HTLV-I) resulted in the establishment of lymphoid cell lines, FIRT-1, FIRS-1, LERT-1, and LERS-1, respectively. Cells of these cell lines had rat T-cell characters as demonstrated by the positive reaction to monoclonal antibodies (MAbs) to rat T cell antigens (Thy 1 and pan T). They lacked surface immunoglobulins and strongly expressed rat interleukin-2 receptor antigen (Tac) and Ia antigen. Karyotypic analysis revealed that they had the normal rat karyotype in early cultures, but showed marked aneuploidy after long cultivation. None of them expressed HTLV gag proteins (p19 and p24) or virus particles, but they contained HTLV-I proviral DNA monoclonally and weakly expressed pX gene products (p40x). They were not transplantable into syngeneic newborn rats.