Human neutrophil-derived histamine-releasing activity (HRA-N) causes the release of serotonin but not arachidonic acid metabolites from rat basophilic leukemia cells.

The effects of neutrophil-derived histamine-releasing activity (HRA-N) on arachidonic acid (AA) metabolism is unknown. Human basophils exposed to HRA-N released 25% of total histamine but no leukotriene C4 (LTC4). To confirm this phenomenon, rat basophilic leukemia (RBL) cells were exposed to HRA-N as well as anti-IgE, or calcium ionophore A23187. RBL cells incubated with A23187 released 44% of available serotonin and 59 and 124 pmol/10(6) cells of prostaglandin D2 (PGD2) and LTC4, respectively. Anti-IgE stimulation resulted in 34% serotonin release and the generation of 34 pmol PGD2 per 10(6) cells and 72 pmol LTC4 per 10(6) cells. In contrast, HRA-N (2 U/ml) induced 20% serotonin release, 4 pmol PGD2 per 10(6) cells, and 0.6 pmol LTC4 per 10(6) cells. Neither increasing the dose nor the incubation time of HRA-N enhanced the generation of AA metabolite. Additionally, the spectrum of AA metabolites generated by RBL cells in response to those agents was examined by reverse-phase high-performance liquid chromatography. RBL cells stimulated with A23187 released PGD2, LTB4, and its isomers, LTC4, and 5-hydroxyeicosatetraenoic acid. In contrast, HRA-N stimulation resulted in only minimal PGD2 generation and no other discernable AA metabolites. Thus, HRA-N causes selective release of serotonin without inducing AA metabolites. These data suggest that HRA-N activates mast cells through a unique pathway.


Chronic idiopathic urticaria: profiles of skin mast cell histamine release during active disease and remission.

Chronic idiopathic urticaria (CIU) is characterized by the increased releasability of histamine by mast cells in normal-appearing skin. In active CIU, this abnormality is consistently present. To determine if this finding subsides when the disease goes into remission phase, we analyzed the histamine secretion in patients with CIU in remission (CIUR) compared with that of patients with CIU and in normal control (NC) subjects, with the skin-chamber technique. The profiles of histamine release in sites challenged with compound 48/80 were significantly different in the groups with CIU and CIUR. Furthermore, patients with CIUR did not differ from NC subjects in terms of histamine releasability under compound 48/80 stimulation (p greater than 0.1). These data suggest that the state of excessive skin mast cell sensitivity is a reversible and transient phenomenon in CIU disease.


Measurement of histamine-releasing factor activity in individual nasal washings: relationship with atopy, basophil response, and membrane-bound IgE.

We collected individual pools of nasal washings (NWs) from 15 allergic and 15 nonallergic subjects to determine histamine-releasing factor (HRF) activity and to ascertain the relationship of these cytokines with atopic status, basophil releasability, and cell membrane-bound IgE. NWs were concentrated, dialyzed, and assayed with basophils from a single donor. Samples from 12 of 15 allergic subjects and from all the nonallergic subjects revealed greater than or equal to 15% histamine release (HR), 33.5% +/- 21.3% (mean +/- SD) and 38.6% +/- 19.6%, respectively (p greater than 0.05). When we assayed the same samples with autologous basophils, the allergic group demonstrated higher HR than the nonallergic group (31.9% +/- 19.7% versus 4.8% +/- 4.3%; p less than 0.001). A standard lot of mononuclear cell-derived HRFs was also screened with basophils from both groups. Means for HR from basophils of allergic and nonallergic subjects were 51.9% +/- 16.7% versus 26.3% +/- 8.2%, respectively (p less than 0.001). Pretreatment of basophils with lactic acid led to abrogation of sensitivity to HRF. Acid-stripped cells incubated with sera from patients with asthma regained their capacity to release histamine. We found that HRF activity can be detected in NWs of most donors, and there is no difference among allergic and nonallergic subjects. Our results suggest that the capacity of these cytokines to induce HR depends on several factors: atopic status, basophil releasability, and membrane-bound IgE.


Re-innervation of submucosal arterioles by myenteric neurones following extrinsic denervation.

We used a combination of selective lesions, immunohistochemistry and video monitoring of arteriolar diameter to determine the source of the changes in vasodilator innervation to guinea pig ileal submucosal arterioles which occur following removal of their extrinsic sympathetic and sensory nerve fibre input. A non-cholinergic neurogenic vasodilation appeared in arterioles in which extrinsic denervation was performed 50-90 days previously. The non-cholinergic innervation did not result from regrowth of extrinsic fibres because the neurogenic response was not altered by combining long-term denervation with capsaicin treatment or re-denervation 7 days prior to examination. However, non-cholinergic neurogenic vasodilations were not observed in arterioles which had been subjected to long-term denervation combined with a myectomy 7 days prior to examination. Immunohistochemical co-localization of SP and CGRP in these vessels confirmed previous findings that a prominent SP perivascular nerve plexus appeared after long-term denervation. Perivascular SP-containing fibres that appeared after long-term denervation were unaffected by capsaicin or re-denervation but were absent from preparations in which long-term denervation and myectomy were performed. These results demonstrate that myenteric neurones are the source of the non-cholinergic innervation which appears after extrinsic denervation and support our previous conclusion that SP is the neurotransmitter responsible for this non-cholinergic vasodilation in submucosal arterioles of the small intestine.


Urinary enzymes excretion in pancreatic diseases. Clinical role and pathophysiological considerations.

The amylase-creatinine clearance ratio was first proposed as a useful tool in the diagnosis of acute pancreatitis, and later it was claimed that trypsin creatinine clearance ratio was a sensitive and accurate test of pancreatic cancer. More recent observations have undermined the role of both clearances in the diagnosis of acute pancreatitis, and their utility in patients with chronic pancreatic diseases has largely been ignored. Three orders of factors, (a) the physicochemical characteristics of the protein, (b) the glomerular filtration rate variations, and (c) renal tubular damage, may have a role in determining the changes in the plasma-urine transfer of enzymes such as amylase and trypsin. Amylase urinary output is related both to variations in amylase serum levels (since this enzyme probably is not intensively reabsorbed by the tubule) and to the presence of renal tubular damage. Trypsin plasma-urine transfer changes depend greatly on the presence of tubular alterations. Elastase 1 and phospholipase A2 urinary outputs can also be predicted on the basis of the presence of tubular damage. Renal tubular alteration in pancreatic diseases may depend on the damaging effect of toxic substances (proteolytic enzymes, for example) released by the inflamed pancreas; the role of liver damage and of extrahepatic jaundice, which are frequent findings in chronic pancreatic diseases, should also be considered. However, toxic compounds such as ethanol, which can alter the pancreas and possibly the kidney, could also have a key role in the genesis of urinary findings in pancreatic diseases.


The intraoperative localization of the obscure bleeding site using fluorescein.

Despite visceral angiography and radiolabeled gastrointestinal scintigraphy, the localization of gastrointestinal bleeding can still be a challenge. Even preoperative localization with visceral angiography is no guarantee of successful intraoperative localization or subsequent pathological confirmation. We report a case of obscure gastrointestinal bleeding of small bowel origin, which was localized intraoperatively by fluorescein injected via a superselectively placed catheter.


Techniques to optimize post-embedding single and double staining for amino acid neurotransmitters.

We report a number of technical refinements for single and double staining with post-embedding electron microscopy for glutamate, aspartate, and gamma-aminobutyric acid. Best results were obtained with 2.5% glutaraldehyde in the fixative and by minimizing the duration of plastic polymerization and the interval between cutting and reacting. Quantitative documentation of the ability of exogenous glutamate, aspartate, and gamma-aminobutyric acid to block their immune staining is provided. Increased intensity of staining with the glutamate and aspartate antisera resulted from preincubation of glutamate antiserum with aspartate and aspartate antiserum with glutamate. To perform double staining with antisera raised in the same species, it was necessary to block antigenicity of the first antiserum; best results were obtained with hot paraformaldehyde fumes. By using a detergent instead of etching, these methods permitted the simultaneous visualization of tracers to identify neuroanatomic pathways.


Direct flow cytometric quantification of alkaline phosphatase activity in rat bone marrow stromal cells.

Cell preparations in cytochemistry are conventionally analyzed with transmitted light after fixation and reaction with agents such as azo-coupling dyes. With cell suspensions stained with fluorescent cytochemical dyes, cells can also be analyzed and sorted by flow cytometry. We have exploited the intense red fluorescence of Fast Red Violet LB generated in cytochemical reactions to perform flow cytometric analyses of alkaline phosphatase (AP) expression in rat bone marrow stromal cells. By modifying staining protocols of single-cell suspensions, we demonstrate that in comparison to staining with Fast Red TR, the method is specific, can distinguish among various levels of enzyme expression within the whole population, and permits enzyme kinetic studies of heterogeneous cell populations. The method was applied to study the effect of the glucocorticoid dexamethasone (Dx) on cell proliferation and AP expression. In low AP-expressing cells, Dx treatment at 10(-8) M increased the [3H]-thymidine labeling index from 3.85% to 5.24% (p less than 0.01). In contrast, high AP-expressing cells were unlabeled by [3H]-thymidine. The staining and analytical methods reported here facilitate the detection, isolation, and quantification of subpopulations of bone marrow stromal cells that express alkaline phosphatase activity. These experiments demonstrate the value of flow cytometry as an adjunct to conventional cytochemical methods.


Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexaammine trichloride.

We tested various cationic dyes chemically related to ruthenium hexaammine trichloride (RHT) [i.e., the RHT-cyclohexanedione complex (RHT-CC), pentaamine ruthenium N-dimethylphenylenediimine trichloride (PRT), tris-(bipyridyl)ruthenium (II) chloride (TRC), tris (bipyridyl) iron (II) chloride (TIC), and cobalt hexaammine trichloride (CHT)] for their effectiveness in precipitating cartilage matrix proteoglycans in situ. Dyes were introduced into media at the onset of processing and were present throughout both aldehyde fixation and osmium tetroxide post-fixation. Contrary to expectation, most of the dye-proteoglycan complexes generated and stable under aldehyde fixation conditions were found to be unstable during post-fixation despite the continuing presence of the dye. A similar phenomenon was also found for the cationic dyes commonly used for precipitation of proteoglycans in cartilage tissue sections (such as Acridine Orange, Alcian Blue, Azure A, Methylene Blue, and Ruthenium Red). Only two dyes, i.e., RHT and the newly tested RHT-CC, formed proteoglycan precipitates sufficiently stable to resist disruption and extraction during osmium tetroxide post-fixation. The latter may be particularly useful in semiquantitative analyses of proteoglycan content in unstained tissue sections owing to its intense brown-black color. For applications in which the osmium tetroxide post-fixation step may be omitted, TRC and PRT may also be valuable for semiquantitative histochemistry by virtue of their stable fluorescence and intense violet color signals, respectively.


Regulation of rat pulmonary artery endothelial cell transforming growth factor-beta production by IL-1 beta and tumor necrosis factor-alpha.

Recent studies suggest that transforming growth factor-beta (TGF-beta) production is up-regulated at sites of tissue injury, inflammation and repair, or fibrosis. Endothelial cells represent a potentially important in vivo source of TGF-beta; however, the identity of endogenous modulators of TGF-beta production by these cells remains unclear. To address this issue, the effects of the cytokines, IL-1 beta, and TNF-alpha on TGF-beta production by rat pulmonary artery endothelial cells were examined. Conditioned media from cells treated with 0 to 20 ng/ml IL-1 beta and/or TNF-alpha were assayed for TGF-beta activity using a mink lung epithelial cell line. The results show that rat pulmonary artery endothelial cells secreted undetectable amounts of active TGF-beta in the absence of cytokines. However, upon acidification of the conditioned media before assay, a time-dependent increase in TGF-beta activity was noted in media from both untreated and cytokine-treated cells. However, both IL-1 beta and TNF-alpha treatment caused the secretion of significantly greater amounts of TGF-beta activity than control cells, in a dose-dependent manner, with maximal response obtained at cytokine doses of greater than 10 ng/ml. At equivalent doses of cytokine tested, the magnitude of the response was significantly greater with IL-1 beta. These responses were paralleled by increases in steady state mRNA levels for TGF-beta 1. Addition of both cytokines resulted in a synergistic response. Synergism with IL-1 beta was also noted with the fibrogenic agent bleomycin. Kinetic studies indicated that a minimum of 4 h of treatment with either IL-1 beta or TNF-alpha was required for detection of significant increases in either secreted TGF-beta activity or steady state TGF-beta 1 mRNA levels. Thus, endothelial cells could play a role in various TGF-beta-dependent processes in vivo, in situations wherein IL-1 beta and/or TNF-alpha may be present at comparable concentrations.


Ashman's phenomenon--a source of nonsustained wide-complex tachycardia: case report and discussion.

The refractory period of the right bundle branch is increased when the R-R interval between the prior two conducted impulses is long. Thus, an impulse that arrives soon after the second of two impulses separated by a long R-R interval may be aberrantly conducted with a right bundle branch block morphology on electrocardiogram. This aberrant conduction is termed "Ashman's phenomenon" and is often responsible for isolated wide QRS complexes in the presence of underlying atrial fibrillation. This process may also produce runs of wide QRS complexes that must be distinguished from nonsustained ventricular tachycardia. A case of such multibeat Ashman's phenomena is presented, and the characteristics used to identify this phenomenon are discussed. A brief review of several recent studies on the differentiation of sustained ventricular tachycardia from supraventricular tachycardia with aberrancy in the setting of a regular underlying rhythm is given as well.


Granulocyte colony-stimulating factor (G-CSF) gene transduction in murine adenocarcinoma drives neutrophil-mediated tumor inhibition in vivo. Neutrophils discriminate between G-CSF-producing and G-CSF-nonproducing tumor cells.

We have previously demonstrated that the murine colon adenocarcinoma C-26 cell line transduced with the human gene for the granulocyte CSF (G-CSF) loses tumorigenic activity through a mechanism that involved massive targeting of neutrophils at the site of tumor injection. The suppression of tumorigenicity by G-CSF was limited to the G-CSF-producing cells and was not transferred to nonproducing C-26 cells in a mixed tumor transplantation assay. We present direct evidence that neutrophils are involved in this phenomenon. We firstly examined, by electron microscopy (EM), the morphology of tumor infiltrates obtained 2, 5, and 10 days after s.c. injection of a mixture of G-CSF-producing and -nonproducing C-26 cells into syngeneic BALB/c mice. The EM analysis showed at 5, but not at 2 or 10 days, the presence of neutrophils in intimate contact with tumor cells. We then investigated whether neutrophils discriminate between G-CSF-producing and -nonproducing C-26 cells. To this aim, C-26 cells were transduced, via retroviral vector, with the Escherichia coli LacZ gene and mixed tumor transplantation assays were performed by injecting a mixture of G-CSF-producing beta-gal- and G-CSF-nonproducing beta-gal+ C-26 cells at different ratios. Histologic and EM analysis of the tumors growing at the site of injection were carried out. Five days after injection, treatment with x-gal revealed, at the histochemical level, the presence of neutrophils around G-CSF producing beta-gal- cells; cell-cell contacts and fusion of cell membranes were detected by EM only between neutrophils and G-CSF-producing cells. In vitro experiments, performed in Boyden chambers, confirmed that the G-CSF produced by C-26 cells was a chemoattractant for neutrophils. In addition, a colorimetric, cytostatic assay revealed that neutrophils were able to inhibit the growth of G-CSF-producing but not of G-CSF-nonproducing C-26 cells. Thus the tumor take after injection of G-CSF-producing C-26 cells seems to be controlled in situ through two major mechanisms namely neutrophil chemotaxis and neutrophil-mediated tumor inhibition. The results indicate that neutrophils can discriminate between G-CSF-producing and -nonproducing tumor cells and that neutrophils infiltrate the tumor mixture as long as G-CSF-producing cells are present.


A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.


Mechanisms of lipopolysaccharide-induced neutrophil retention. Relative contributions of adhesive and cellular mechanical properties.

Intravascular LPS rapidly induces neutrophil sequestration in pulmonary capillaries by mechanisms that, although currently unknown, must take into account the size difference between the neutrophil and capillary diameter. To determine whether LPS alters neutrophil stiffness, and hence the ability of neutrophils to traverse capillaries, neutrophil passage through pulmonary capillaries was modeled by passage through filters with 6.5-microns pores. LPS increased retention in the pores in a concentration-dependent fashion that required the presence of heat-inactivated platelet-poor plasma, and was evident as early as 10 min after stimulation. The effect of LPS on the structural properties of the neutrophil was then studied. LPS induced f-actin reorganization in neutrophils in the presence of plasma. Disruption of actin organization and assembly with cytochalasin D completely inhibited early LPS-induced retention and attenuated retention at later timepoints, indicating that LPS-stimulated retention depends on filament organization. LPS-induced actin assembly and retention were abrogated by an antibody directed against CD14, a putative LPS receptor. CD18-dependent adherence of neutrophils contributed significantly to retention only at later timepoints with no significant contribution to retention at 20 min as determined by inhibition of adherence with the mAb 60.3. Morphometric assessment of neutrophil accumulation in the lungs of rabbits given 1 microgram LPS showed a marked increase in apparent neutrophil number, which was unaltered by antibodies to CD18, suggesting that mechanisms other than adhesion may account for accumulation in vivo. Direct measurements showed that neutrophil stiffness increased with exposure to LPS in a fashion similar to LPS-induced retention and actin organization. Pretreatment of neutrophils with cytochalasin D attenuated the increased stiffness. These data suggest that reorganization of filamentous-actin induced by LPS leads to cell stiffening and retention in capillary-sized pores. Although the organization of f-actin continues to be important in retention at later time points, adherence of cells also contributes significantly to cell retention. The changes in mechanical properties of the neutrophil may be important in the sequestration of neutrophils in pulmonary capillaries noted in endotoxemia.


Antigen receptor-mediated protein tyrosine kinase activity is regulated by a pertussis toxin-sensitive G protein.

Ligation of the Ag receptor on B cells is associated with a rapid increase in phosphorylation on tyrosine residues of multiple substrates. One of the substrates is the phosphoinositide-specific phospholipase C-gamma 1. Because activation of phospholipase C-gamma 1 seems to be dependent on tyrosine phosphorylation, it is assumed that the two signaling pathways, phosphatidylinositol turnover and tyrosine phosphorylation, might be linked. However, since the Ag receptor does not possess a kinase domain, it remains unclear how these signaling pathways are regulated by the Ag receptor. Previous studies have proposed the existence of a receptor-coupled G protein that regulates inositol phosphate production in B cells. We confirm that phosphoinositide turnover is regulated by a pertussis toxin (PT)-sensitive G protein, most probably by controlling phosphorylation of phospholipase C-gamma 1. We show that treatment of permeabilized B cells with a nonhydrolyzable GTP analogue guanosine 5'-[3-thio]triphosphate induced an increase in tyrosine phosphorylation of multiple substrates that are identical to the proteins phosphorylated after anti-IgM stimulation. Furthermore, binding of the inactive form of G proteins with guanosine 5'-[2-thio]-triphosphate blocked anti-IgM induced tyrosine phosphorylation in permeabilized B cells. The results indicate that an Ag receptor-coupled G protein controls protein tyrosine kinase activity. We show that this G protein is sensitive to PT because tyrosine phosphorylation mediated by the Ag receptor was inhibited by this toxin in a concentration-dependent manner. Similar concentrations of PT also blocked tyrosine phosphorylation on phospholipase C-gamma 1 and generation of inositol phosphates. Preincubation of intact B cells with PT resulted in inhibition of c-fos mRNA expression and DNA synthesis in anti-IgM stimulated B cells, indicating that post-transcriptional events are also controlled by the Ag-receptor coupled G protein. We conclude that Ag receptor-associated protein tyrosine kinase activity is regulated by a G protein. This PT-sensitive G protein also regulates phosphorylation and activation of phospholipase C-gamma 1 as well as later events in B cell activation such as c-fos mRNA expression and proliferation.


Independent regulation of c-myc, B-myb, and c-myb gene expression by inducers and inhibitors of proliferation in human B lymphocytes.

Although a detailed picture is emerging about the nature of the second messengers involved in B cell activation and proliferation, little is yet known about the intracellular events taking place further downstream. The c-myb proto-oncogene, the structurally related B-myb gene, and c-myc probably code for transcription factors, have been demonstrated to be necessary for the proliferation of hemopoietic cells, and their expression is indeed induced after mitogenic stimulation of T and B lymphocytes. They are therefore likely to be key elements in the regulation of gene expression during proliferation. We have set out to study the regulation of the expression of these two myb genes and of that of c-myc in relation to entry into the different phases of the cell cycle during mitogenic stimulation of resting human B lymphocytes. Resting tonsillar B cells stimulated with the anti-CD20 antibody 1F5 alone are induced to enter the G1 but not the S phase of the cell cycle, whereas co-stimulation with the anti-CD40 antibody G28.5 further drives them to enter the S phase and proliferate. The G28.5 antibody alone has been reported to partially activate and increase the alertness of resting B cells without inducing them to enter G1. In this report we show that increasing the strength of the activating signal leads to progressive induction of the proliferation-related genes studied. Thus the G28.5 antibody alone induces c-myc mRNA only in resting B cells, 1F5 induces both c-myc and B-myb, and the full mitogenic signal given by both antibodies together is accompanied by increased expression of all three--c-myc, B-myb, and c-myb genes. In addition, using a semi-quantitative polymerase chain reaction method, we show that different inhibitors of B cell proliferation, namely, cyclosporin A, an anti-CD19 antibody (HD37), and transforming growth factor beta 1 (TGF-beta 1), inhibit differentially the induction of these same genes after mitogenic stimulation of B cells. Whereas cyclosporin A inhibits induction of all three genes, TGF-beta 1 specifically blocks B-myb induction and CD19 has little effect on either of the genes tested. We conclude that c-myb, B-myb, and c-myc are regulated independently from one another, that induction of c-myc and B-myb together is not sufficient to trigger B cell proliferation, and we suggest that expression of all three is a prerequisite for proliferation to occur.


Immunogenetics of collagen-induced arthritis in rats. Both MHC and non-MHC gene products determine the epitope specificity of immune response to bovine and chick type II collagens.

Seven inbred, RT1-congenic rat strains were immunized with native bovine (BII), porcine (PII), or chick (CII) type II collagen and observed for onset, incidence, and severity of arthritis. Clinical results were compared with IgG reactive with native rat type II collagen (RII) and the purified, renatured cyanogen-bromide peptides of BII, CII, or RII. Immunodominant responses to CB11, CB9,7, and CB12 of RII were identified. Secondary responses to CB8 and CB10 also occurred. Reproducible patterns of peptide reactivity were defined in each strain and reflected both RT1 and non-RT1 genotypes plus the species of immunizing collagen. BN non-RT1 gene products moderated clinical arthritis but increased the levels of reactivity to CB11 in three strains carrying RT1l,n,av1 haplotypes. WF (RT1u) rats were susceptible to collagen-induced arthritis (CIA) and developed very high levels of autoantibodies with dominant responses to rat CB11 after CII injections and to rat CB11 and CB9,7 after BII injections. DA (RT1av1) rats developed the most severe arthritis but had only moderate (total) levels of anti-RII IgG: a broad response to CB11, CB10, and CB9,7 after CII injections but predominantly to CB12 and CB9,7 after BII injections. Three RT1n strains--DA.1N(BN), WF.1N(MAXX), and BN--were resistant to BII-induced CIA but developed mild arthritis after immunization with CII. After BII: BN IgG reacted with CB9-7, CB11, and CB12; DA.1N and WF.1N IgG reacted with CB9,7 and CB12. After CII: BN IgG reacted broadly with CB11, CB9-7, CB12, and CB8; WF.1N IgG reacted to CB9-7, CB11, CB8, and CB12; DA.1N IgG reacted with CB8, CB11, and CB9-7. Thus, selective induction of CIA in BN, WF.1N, and DA.1N rats by CII correlated with serum IgG reactivity to rat CB11, but overall strain results identified no single cyanogen-bromide peptide as expressing the sole "arthritogenic" epitope in CIA.


Comparison of the T cell receptors on insulin-specific hybridomas from insulin transgenic and nontransgenic mice. Loss of a subpopulation of self-reactive clones.

Transgenic mice expressing the human insulin gene do not produce insulin-specific antibody after injection of human insulin. Nevertheless, they have some peripheral T cells that proliferate to human insulin in vitro. To investigate the nature of these T cells, human insulin-specific T cell hybridomas were produced from transgenic and nontransgenic mice. Transgenic hybridomas required more insulin to achieve maximum responses and they produced lower levels of lymphokines than nontransgenic hybridomas. The majority of nontransgenic hybridomas recognized only human and pork insulin whereas transgenic hybridomas recognized beef, sheep, and/or horse insulin in addition to human and pork insulin. The TCR expressed by transgenic and nontransgenic hybridomas were determined by Northern analysis. Both types of hybridomas used several different V alpha and V beta gene families and no favored association between V alpha and V beta gene usage was detected in either type. V beta 1 was used by 7 of 16 nontransgenic hybridomas but only by 1 of 16 transgenic hybridomas. V beta 6 receptors were predominantly expressed by the transgenic hybridomas and all V beta 6-bearing hybridomas recognized beef as well as human insulin. The differences in Ag reactivity and TCR gene usage suggest that V beta 1-bearing human insulin-reactive T cells were clonally deleted or inactivated in the transgenic animal. Other clones, representing a minor subpopulation in nontransgenic mice, were recovered from transgenic mice.


Signals delivered via MHC class II molecules synergize with signals delivered via TCR/CD3 to cause proliferation and cytokine gene expression in T cells.

We examined the role of MHC class II molecules in transducing signals to activated human T cells. Cross-linking of MHC class II molecules synergized with submitogenic amounts of anti-CD3 mAb in causing proliferation and secretion of the cytokines IL-2, IL-3, IFN-gamma, and TNF-alpha by MHC class II-alloreactive T cell lines. Signaling via MHC class II molecules in T cells resulted in activation of tyrosine kinases, in generation of inositol phosphates, and in Ca2+ mobilization that was abrogated by the tyrosine kinase inhibitor herbimycin A. Thus, like signaling via TCR/CD3, signaling via MHC class II molecules involved tyrosine kinase-dependent activation of phospholipase C, resulting in phosphoinositol turnover and Ca2+ flux. However the signaling pathways coupled to MHC class II molecules and to TCR/CD3 differed, because engagement of the transmembrane phosphatase CD45 inhibited Ca2+ fluxes triggered via TCR/CD3 but not Ca2+ fluxes triggered via MHC class II molecules.


Expression of transforming growth factor type beta on human epidermal dendritic cells.

Our study demonstrates that epidermal Langerhans cells (LC) of healthy human skin produce Transforming growth factor-beta (TGF-beta). This multipotent mediator might keep the immunocompetent LC in an "immature" stage, in which they are able to capture and process antigen. Development to potent antigen-presenting cells would become possible after migration from the epidermal network to the draining lymph node to stimulate (auto-)reactive T-cell clones.


Selective involvement of keratins K1 and K10 in the cytoskeletal abnormality of epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma).

Aggregation of tonofilaments within epidermal keratinocytes is a characteristic histologic feature of epidermolytic hyperkeratosis including the generalized form known as bullous congenital ichthyosiform erythroderma. The histologic distribution and the keratin composition of the altered tonofilaments were investigated to determine whether the aggregation was specific to any particular keratin(s). Skin samples from seven patients and one mid-trimester fetus with generalized epidermolytic hyperkeratosis, and from one patient with a localized or "nevoid" form of epidermolytic hyperkeratosis, were analyzed by using various microscopical and immunocytochemical methods. A conjunctival sample and cultured epidermal keratinocytes from one patient with generalized epidermolytic hyperkeratosis were also examined by electron microscopy and immunocytochemistry. Ultrastructurally, tonofilament aggregates were distributed within the suprabasal stratified epithelial cell layers of the epidermis, of the infundibular part of outer root sheaths, and of the sebaceous ducts and sweat ducts, selectively following the known distribution pattern of keratins K1 and K10. The abnormal tonofilaments were not found in any other cutaneous epithelia, in conjunctival epithelium, or in cultured keratinocytes, where K1 and K10 are absent or only minimally expressed. Immunoelectron microscopy showed that among the keratins detected in suprabasal epidermolytic hyperkeratosis epidermis (K1/K5/K10/K14/K16), the aggregated tonofilaments predominantly expressed K1 and K10 rather than other keratins. These results suggest that the keratin filament abnormality in epidermolytic hyperkeratosis principally involves K1 and K10 and raise the question whether epidermolytic hyperkeratosis might be primarily a disorder of one or both of these keratins.


Simple assays of retinoid activity as potential screens for compounds that may be useful in treatment of psoriasis.

Human epidermal cell cultures were used to study the effects of retinoids on keratinocyte differentiation. Keratin profiles were studied by quantitative gel electrophoresis of culture extracts, whereas the extent of envelope formation was assessed in an enzyme-linked immunosorbent assay (ELISA) using an antibody that specifically recognizes keratinocyte envelopes. Exposure of cultures to a variety of different retinoids produced both dose-dependent decreases in keratin 16 with consequent increases in the keratin 14: keratin 16 ratio, and a decrease in envelope formation. The order of activity in both assays was similar: arotinoid ethyl ester (Ro 13-6298) greater than or equal to arotinoid acid (Ro 13-7410) much greater than all trans retinoic acid (Ro 1-5488) greater than acitretin (Ro 10-1670) greater than or equal to etretinate (Ro 10-9359), the only difference being that acitretin was slightly more active than etretinate in the keratin assay whereas these retinoids were equi-active in the envelope assay. Analysis of the lesional keratins of psoriasis patients showed that etretinate caused a reduction in keratin 16 and an increase in the keratin 14:keratin 16 ratio, although the magnitude of these changes and their correlation with clinical improvement was variable. As the in vitro assays reported here are simple and quick, they allow rapid screening of compounds for retinoid-like activity.


Specificity of antibodies and tuberculin response after occupational exposure to tuberculosis.

Specific antibody levels and delayed-type hypersensitivity skin responses to antigens of Mycobacterium tuberculosis in 39 hospital staff who were heavily exposed to tuberculosis (TB) were compared with those in 36 factory employees from Indonesia. Antibody levels to the TB68 epitope of the 14-kDa antigen were significantly greater, while titers to the TB23 (19-kDa) and TB72 (38-kDa) epitopes and lipoarabinomannan (LAM) were lower in exposed than in nonexposed subjects (all P less than .02). The intensity of tuberculin responses correlated positively with anti-LAM and negatively with anti-19-kDa antibody levels. Possible reasons for the selective humoral response of chronically exposed healthy subjects to the 14-kDa antigen, but not to other antigens immunogenic in patients with tuberculosis, are discussed.


Analysis of the Lewisx epitope in human pancreas and pancreatic adenocarcinomas.

The Lewisx epitope (Gal beta 1-4[Fuc alpha 1-3]GlcNAc-R) can be detected in most ductal pancreatic carcinomas. However, few data pertain to its expression in normal exocrine pancreas and its biochemistry. We compared the expression of the Lewisx epitope in normal pancreas with its expression in pancreatic adenocarcinomas and tumor cell lines and to further characterize the nature of the antigens bearing this epitope with immunochemical methods. On frozen tissue sections of normal pancreas, the Lewisx epitope was detected focally in acinar cell granules; sialosyl-Lewisx was detected in centroacinar cells and the cytoplasm of intralobular ducts. Sixteen of 19 pancreatic carcinomas expressed the Lewisx antigen on tissue sections; however, there was no correlation with prognostic parameters, such as tumor grade or stage. Eighteen of 19 tumor specimens were positive for sialosyl-Lewisx. Analysis of pancreatic carcinoma cell lines (CAPAN-1, CAPAN-2, and DAN-G) revealed intracytoplasmic expression of sialosyl-Lewisx epitopes in these cells, and cell surface reactivity was detected on flow cytometry for sialosyl-Lewisx. Lectin-affinity chromatography of cytoplasmac preparations showed that Lewisx-positive antigens of normal human pancreas bind to SBA and UEA-I lectins but have little or no affinity to WGA, GS-I, or DBA lectins, indicating the presence of Fuc and Gal oligosaccharide residues. It was concluded that the Lewisx and sialosyl-Lewisx antigens are nonpolymorphic carbohydrate determinants that are present in secretory granules of acinar cells, ductules, and pancreatic secretions. This makes the Lewisx antigen suitable for the analysis of the secretory process in the exocrine pancreas and the study of secretory differentiation antigens in ductal pancreatic carcinoma.


[Combination chemotherapy with 254-S, ifosfamide and peplomycin for advanced or recurrent cervical cancer].

From favorable results with 254-S, a new cisplatin analogue, single administration, we have conducted a clinical study to investigate the efficacy of combination of 254-S, ifosfamide and peplomycin, each of which has a different dose limiting factor. A total of 45 patients, including 22 patients with stage III and IV cervical cancer and 23 cases with recurrent cervical cancer, were treated with at least two courses of 254-S (100mg/m2, iv. Day 1), ifosfamide (1,500mg/body, iv. Day 1-5) and peplomycin (5mg/body, im. Day 1-6), and tumor response was evaluated clinically and by CT scanning. The response rate obtained in patients with advanced disease was 81.8% (PR = 17, CR = 1) and that in cases with recurrence was 60.9% (PR = 12, CR = 2). Myelosuppression was the dose limiting factor. In the 121 courses, grade 3 and 4 of leucopenia and thrombocytopenia were observed with an incidence of 44% and 32%, respectively and DIC occurred in 3 cases with poor PS though they recovered after reducing the 254-S dose to 80 mg/m2. The other toxicities were mild except for alopecia. Anaphylaxia was observed in a case at the second administration though the patient recovered in 15 minutes. There was no death. As to prognosis, a significant prolongation of survival period was observed in recurrent cases and 4 cases are alive (NED) after one and a half year. In the advanced cases, until now 3 cases of stage IV have died from the disease. We have concluded that this regimen is effective as a neoadjuvant chemotherapy for advanced cervical cancer and useful for the treatment of recurrent cervical cancer.


Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.

Retinas from the scallop Pecten irradians were enzymatically dispersed, yielding a large number of isolated photoreceptors suitable for tight-seal recording. Whole-cell voltage clamp measurements demonstrated that the phototransducing machinery remained intact: quantum bumps could be elicited by dim illumination, while brighter flashes produced larger, smooth photocurrents. Single-channel currents specifically activated by light were recorded in cell-attached patches, and were almost exclusively confined to the rhabdomeric region. Their density is sufficiently high to account for the macroscopic photoresponse. Channel activation is graded with stimulus intensity in a range comparable to that of the whole-cell response, and can be recorded with illumination sufficiently dim to evoke only quantum bumps. Light-dependent channel openings are very brief, on average 1 ms or less at 20-22 degrees C, apparently not because of blockage by extracellular divalent cations. The mean open time does not change substantially with stimulus intensity. In particular, since dwell times are in the millisecond range even with the dimmest lights, the channel closing rate does not appear to be the rate-limiting step for the decay kinetics of discrete waves. The latency of the first opening after light onset is inversely related to light intensity, and the envelope of channel activity resembles the time course of the whole-cell photocurrent. Unitary currents are inward at resting potential, and have a reversal voltage similar to that of the macroscopic light response. Voltage modulates the activity of light-sensitive channels by increasing the opening rate and also by lengthening the mean open times as the patch is depolarized. The unitary conductance of the predominant class of events is approximately 48 pS, but at least one additional category of smaller-amplitude openings was observed. The relative incidence of large and small events does not appear to be related in a simple way to the state of adaptation of the cell.


Diverse K+ channels in primary human T lymphocytes.

We used patch clamp techniques to identify and characterize a variety of K+ channels in primary human peripheral T lymphocytes. The most common channel observed in cell-attached configuration was voltage gated and inactivating. In ensemble averages, the kinetics of its activation and inactivation were similar to those of the whole-cell, voltage-gated K+ current described previously (Cahalan, M. D., K. G. Chandy, T. E. DeCoursey, and S. Gupta. 1985. J. Physiol. [Lond.]. 358:197-237; Deutsch, C., D. Krause, and S. C. Lee. 1986. J. Physiol. [Lond.]. 372:405-423), suggesting that this channel underlies the major portion of the outward current in lymphocytes. A small fraction of the time, this or another very similar channel was observed to inactivate significantly more slowly. Another channel type observed in cell-attached recording was seen less frequently and was transient in its appearance. This channel has a unitary conductance of approximately 10 pS, similar to the voltage-gated channel, but its voltage-independent gating, lack of inactivation, and different kinetic parameters showed it to be distinct. In whole-cell recording there is often a significant plateau current during sustained depolarization. Experiments using whole-cell and excised outside-out configurations indicate that at least part of this residual current is carried by K+ and, as opposed to the predominant voltage-gated current, is charybdotoxin insensitive. These findings are consistent with evidence that implicates charybdotoxin-sensitive and -insensitive components in T lymphocyte proliferation and volume regulation.


Influenza virus infection elicits class II major histocompatibility complex-restricted T cells specific for an epitope identified in the NS1 non-structural protein.

A T cell epitope of the influenza virus NS1 molecule was identified and shown to be a determinant used in class II major histocompatibility complex-restricted T cell responses to infectious virus. An I-Ed-restricted BALB/c mouse T hybridoma clone recognizing influenza virus A/Puerto Rico/8/34 (PR8; subtype H1N1) but not A/Udorn/72 (subtype H3N2) secreted lymphokines in response to purified recombinant NS1 or fusion proteins containing amino acids 1 to 81 or 1 to 42 of NS1. As expected for recognition of a non-virion protein, the clone failed to respond to u.v.-inactivated virus. The antigenic determinant was localized by synthetic peptides to amino acids 13 to 32 of NS1, explaining the lack of recognition of A/Udorn/72 virus which has an alanine to valine substitution at position 23 within the determinant. A single intranasal dose of infectious PR8 virus was found to elicit T cells that responded to peptide NS1 13-32, suggesting that this determinant is a significant target of T cells in normal infections. To stimulate helper T cell responses similar to those achieved with infectious virus, influenza virus vaccines may therefore have to include NS1 in addition to virion components.


Distribution of epitopes within the amino acid sequence of the Epstein-Barr virus major envelope glycoprotein, gp340, recognized by hyperimmune rabbit sera.

Epstein-Barr virus (EBV) is a major human pathogen for which the development of an effective vaccine remains an important goal. Rabbits were immunized with one of a set of 10 fusion proteins representing protein fragments from the EBV receptor-binding ligand and candidate subunit vaccine gp340. Sera from recipients of fragments from the amino-terminal half of the polypeptide chain bound gp340 in Western blot assays and ELISA but were not virus-neutralizing. The fine epitope specificity of these sera, and of EBV-neutralizing rabbit sera raised against whole EBV and gp340-containing immune-stimulating complexes, were assessed in a peptide ELISA. All but two of these sera bound peptides located between positions 236 and 327 in the 907 amino acids of the gp340 polypeptide chain. Among these it was possible to identify regions containing candidate virus-neutralizing B cell epitopes. The use of a gp340 fusion protein affinity column to isolate antibodies from EBV-neutralizing rabbit sera specific for this region suggests the presence of both continuous and discontinuous B cell epitopes with potential roles in EBV neutralization.


Antipeptide antisera define neutralizing epitopes on the adenovirus hexon.

The adenovirus (Ad) hexon contains both group-and type-specific antigenic determinants. To identify the latter, peptides were synthesized corresponding to residues 281 to 292 from loop 1 and 441 to 455 from loop 2 of the Ad2 hexon. These sequences display type-specific variation and have been shown by X-ray crystallography to be present on the surface of the virion. Antisera raised against the peptides bound both peptide and the native hexon in ELISA, and blocked virus infectivity, as determined by immunofluorescence or neutralization assays. The loop 1 peptide was shown to inhibit binding of the corresponding antiserum to the native hexon in ELISA and to abolish its neutralizing activity. Neither the loop 1- nor loop 2-specific antiserum neutralized the infectivity of Ad4 or Ad40. Neutralization did not appear to result from aggregation of virus particles and thus their inability to attach to the cell, because virions treated with immune serum were internalized to the same extent as those treated with preimmune serum. Examination of the immune response elicited by Ad2 infection revealed that antibodies directed against the L1 and L2 epitopes were also present in human serum. Thus, the variable regions exposed on the surface of the Ad2 hexon represent type-specific neutralizing antigenic determinants.


Diversity of the antibody responses produced in ponies and mice against the equine influenza A virus H7 haemagglutinin.

A large panel of mouse monoclonal antibodies was produced and tested against field isolates of the equine H7N7 influenza A virus subtype. Only a limited degree of H7 haemagglutinin variation was detected. At least four antigenic sites were identified by selecting variant viruses in eggs. The limited variation in the field did not correlate with the frequency of variant viruses detected in eggs; this frequency was similar to those reported for other influenza viruses. We sought to determine whether the limited amount of variation could be correlated with an epitope-restricted antibody response in vaccinated horses. To this end, limiting dilution cultures were established with peripheral blood leukocytes from vaccinated ponies and the antibodies released into culture supernatants were assayed for binding to variant H7 viruses in ELISA. Three neutralizable antigenic sites mapped by mouse antibodies were also recognized by antibodies in pony limiting dilution culture supernatants, indicating that the equine antibody response against the influenza virus H7 haemagglutinin is diverse, and should be effective in selecting variant viruses.


Complete replication of a satellite RNA in vitro by a purified RNA-dependent RNA polymerase.

The 334 nucleotide R satellite RNA was used as a template for purified RNA-dependent RNA polymerase (RdRp) from cucumber mosaic virus-infected tobacco plants. The products of the reaction were dsRNA and positive-strand RNA of the same size as the R satellite RNA. Similar products were obtained when T7 RNA polymerase positive-strand transcripts of a cDNA clone of the satellite RNA, designed to have the same 5' and 3' ends as the satellite RNA, were used as templates. The formation of the positive strands demonstrates complete replication of the satellite RNA. A positive-strand transcript with 65 and 255 additional nucleotides at the 5' and 3' ends of the satellite RNA respectively was also utilized as a template by the RdRp, but only dsRNA was formed. However, no products could be detected when the RdRp was programmed with transcripts corresponding to the negative-strand satellite RNA, either with no additional terminal nucleotides or with 24 and 310 additional nucleotides at the 5' and 3' ends respectively.


Characterization of inner ear sensory hair cells after rapid-freezing and freeze-substitution.

In order to determine the ultrastructural organization of normal cells and to understand better the anatomical substrates for outer hair cell motility, cryofixation was performed on the sensory epithelium of the inner ear of guinea pigs prior to substitution of frozen water with organic solvents containing chemical fixatives. In this way cells would not be altered by the direct application of the chemicals commonly used for preservation, which are also known to cause fixation-induced shape changes in outer hair cells. Following rapid freezing and freeze-substitution, preservation of cells within the isolated sensory epithelium containing the organ of Corti was similar to that seen in conventionally fixed cells. However, in rapidly frozen and freeze-substituted outer hair cells the cytoplasm and the cellular membranes differed from those seen in conventionally fixed preparations. The cytoplasmic matrix was densely packed with filaments and stained homogeneously, suggesting better preservation of the cytoskeleton and less extraction of the soluble ground substance. Cell membranes were smooth, indicating that fixation-induced shape changes and shrinkage had been avoided. The subsurface cisternal system of intracellular membranes lining the lateral wall of the outer hair cells was composed of continuous, tightly packed, parallel rows of membranous lamellae. Thus rapid-freezing and freeze-substitution are important techniques by which structural alterations correlated with outer hair cell motility can be separated from fixation-induced cell shape changes.


Phase I study of taxol and granulocyte colony-stimulating factor in patients with refractory ovarian cancer.

To increase the taxol dose beyond the current standard dose intensity of 175 mg/m2 per 21 days in patients with refractory ovarian cancer. Fifteen patients who had platinum-refractory or recurrent advanced-stage ovarian cancer were treated with taxol in a phase I trial and were given granulocyte-colony stimulating factor (G-CSF). Taxol was administered at doses of 170, 200, 250, and 300 mg/m2 every 3 weeks. G-CSF was given as a daily subcutaneous injection that started 24 hours after the completion of the taxol infusion. Four patients required either taxol dose reduction or delay. The dose-limiting toxicity (DLT) was peripheral neuropathy, and it occurred at 300 mg/m2. This toxicity was manifested clinically as a stocking-and-glove sensory disturbance that primarily affected proprioception, and was associated with objective changes on nerve conduction studies in affected individuals. Mucositis was rarely observed. Substantial myelosuppression was observed, but was not dose-limiting. Five of 14 assessable patients experienced an objective response to therapy, with another five individuals who experienced a 30% to 45% reduction in tumor mass. Taxol can be safely administered in doses up to 250 mg/m2 with G-CSF support, which may make it possible to study taxol dose intensification.


Neurofilament proteins and the mesolimbic dopamine system: common regulation by chronic morphine and chronic cocaine in the rat ventral tegmental area.

The ventral tegmental area (VTA) and its dopaminergic projections appear to mediate some of the rewarding properties of opiates, cocaine, and other drugs of abuse. In a previous study, we demonstrated that chronic morphine and cocaine exert common actions on tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in this dopaminergic brain reward region (Beitner-Johnson and Nestler, 1991). In the present study, we investigated the effects of chronic morphine and cocaine on other phosphoproteins in the VTA by back phosphorylation and two-dimensional electrophoretic analysis. It was found that a number of phosphoproteins, in addition to tyrosine hydroxylase, were regulated similarly by the two drug treatments in this brain region. Several of these morphine- and cocaine-regulated phosphoproteins were identified as neurofilament (NF) proteins. Chronic, but not acute, administration of either morphine or cocaine was found to decrease levels of the three NF proteins, NF-200 (NF-H), NF-160 (NF-M), and NF-68 (NF-L), by between 15% and 50% in the VTA by back phosphorylation, immunolabeling, and Coomassie blue staining. Such regulation of NF proteins was selective, in that no detectable changes were observed in the levels of eight other major cytoskeletal or cytoskeletal-associated proteins analyzed. Furthermore, NF levels were not altered by chronic treatment with either imipramine or haloperidol, two psychotropic drugs without reinforcing properties, or by chronic stress. Morphine and cocaine regulation of NFs showed regional specificity, as NF levels were not altered in the substantia nigra, or other parts of the brain or spinal cord, by these drug treatments. NFs are thought to function as determinants of neuronal morphology and to be associated with axonal transport. Thus, decreased NF levels in the VTA in response to chronic morphine and chronic cocaine could lead to drug-induced alterations in the structural and functional properties of this brain region, which may represent, in turn, part of a common biochemical basis of morphine and cocaine addiction and craving.


SAG: a Schwann cell membrane glycoprotein.

We report on characterization of a 170,000 Da glycoprotein found exclusively in the PNS. We refer to this protein as the Schwann cell membrane glycoprotein (SAG). SAG contains the HNK-1 carbohydrate, which is considered by some to be a marker of adhesion molecules. Its N-terminal sequence is not similar to previously known polypeptide sequences. SAG is found exclusively in the PNS, is present in rat sciatic nerve prior to myelination, and is in both myelinating and nonmyelinating Schwann cells. Tumors of Schwann cell lineage express SAG where axons are present (neurofibromas) but do not in the absence of axons (schwannomas). Schwannoma cells in culture do not express SAG even when exposed to forskolin, an activator of adenylate cyclase. However, schwannoma cells grown in the presence of a neuronal cell line (PC12) express SAG.


Effects of 1-[(2-thiazolin-2-yl)amino]acetyl-4-(1,3-dithiol 2-ylidene)-2,3,4,5-tetrahydro-1H-1-benzazepin-3,5-dione hydrochloride (KF-14363) on liver regeneration and function of hepatic mitochondria in partially hepatectomized rats.

Effects of 1-[(2-Thiazolin-2-yl)amino]acetyl-4-(1,3-dithiol 2-ylidene)-2,3,4,5-tetrahydro-1H-1-benzazepin-3,5-dione hydrochloride (KF-14363), a hepatoprotective compound, on liver regeneration, liver nucleic acid levels, mitochondrial respiration activity and hepatic energy metabolism after partial hepatectomy (OPE) in rats were studied. The liver regeneration rate was significantly increased in rats administered 100 mg/kg of KF-14363 for 6 d as compared with the control group administered vehicle. The serum glucose level in rats administered 100 mg/kg of KF-14363 measured 3 h after OPE (OPE 3 h) was significantly higher than that in rats administered vehicle (group A) and the serum insulin level in the KF-14363 group was more than double that of group A. KF-14363 also significantly increased liver nucleic acid levels in OPE rats. In mitochondrial function experiments, KF-14363 (100 mg/kg) was orally administered 2 h after OPE. Three hours after OPE (1 h after KF-14363 administration), state 3 respiration was significantly higher in the OPE 3 h + KF-14363 group than in the OPE 3 h + water group. The adenosine diphosphate/oxygen (ADP/O) ratio was also significantly higher in the OPE 22 h (22 h after OPE) + KF-14363 group than in the OPE 22 h + water group. On hepatic energy metabolism, KF-14363 increased adenosine diphosphate and triphosphate levels. Total adenine nucleotide level in the OPE 3 h + KF-14363 group was significantly higher than that in the OPE 3 h + water group. Adenylate energy charge (AEC) was slightly decreased in the OPE 3 h + water group and significantly decreased in the OPE 22 h + water group than in the corresponding sham group.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction mode of dicumarol and its derivatives with human serum albumin, alpha 1-acid glycoprotein and asialo alpha 1-acid glycoprotein.

The interaction of dicumarol and its seven other derivatives with human serum albumin (HSA), alpha 1-acid glycoprotein (AGP) and desialylatedAGP (asialoAGP) has been investigated by circular dichroism (CD) and fluorescence. The binding parameters of dicumarol and its derivatives obtained from fluorescence almost agreed with those obtained from CD. The binding data indicated that the total binding affinities (nK) to HSA were higher than the binding affinities to AGP and asialoAGP. Hydrophobic interaction was the driving force for the binding to all the three proteins and nK values in the binding process were found to be increased with the increase of hydrophobicity of the compound. This was evidenced by the attempts taken to correlate binding affinities with partition coefficients. Both electrostatic and van der Waals interactions were not found to play any significant role in the binding of these compounds to any of these three proteins. However, in case of the AGP and asialoAGP binding, apart from the hydrophobic interaction, some other forces may be involved as evidenced from the experimental data. Binding was exothermic, entropy driven and spontaneous. The change of enthalpy (delta H degree) was compensated for by the change in entropy (delta S degree). Relative contribution of hydrophobic interactions in the binding of these compounds to HSA was higher than to AGP or asialoAGP. Sialic acid was not found to impart any significant role in the binding of these compounds to AGP.


Inhibition of interleukin-1-alpha-induced nitric oxide synthase in vascular smooth muscle and full reversal of interleukin-1-alpha-induced hypotension by N omega-amino-L-arginine.

Interleukin-1-alpha (IL-1) is a cytokine with potentially therapeutic immunoproliferative and tumoricidal activities. Preliminary clinical studies suggest that use of IL-1 may be restricted by dose-limiting hypotension. The purpose of this study was to investigate the role of nitric oxide (NO.) as a possible mediator of this hypotension. Cytokine-treated rat aortic smooth muscle cells were assayed for nitrite production, a stable breakdown product of nitric oxide. Nitric oxide synthase from smooth muscle cells was partially characterized in cytosol preparations using a novel Fe(2+)-myoglobin method to test for nitric oxide production. To determine the role of NO. on the immunorestorative and antineoplastic activity of IL-1, N omega-amino-L-arginine (NAA) or N omega-monomethyl-L-arginine (NMA), inhibitors of nitric oxide synthase, were added to either cultures of IL-1-dependent T cells or A375 melanoma cells exposed to IL-1. To investigate the effects of NAA in vivo, pentobarbital anesthetized dogs, which were made hypotensive by administration of IL-1, received a single intravenous bolus dose of NAA. The effects of NAA were then reversed by the administration of L-arginine. Our results show that cultured IL-1-activated rat aortic smooth muscle cells synthesize nitric oxide, a potent vasodilator. Induction of nitric oxide synthase is augmented by interferon-gamma and blocked by IL-1 receptor antagonist and by inhibitors of RNA or protein synthesis. Nitric oxide synthesis by IL-1-activated smooth muscle cells is inhibited by NAA, NMA, and N omega-nitro-L-arginine (NNA) with ED50 (i.e., effective dose for 50% inhibition) values of 20, 60, and 1000 microM, respectively; this rank order of inhibition is characteristic of an agonist-unregulated, inducible isoform of nitric oxide synthase. In smooth muscle cells, inhibition of NO. synthesis by NAA is reversed by excess L-arginine. Consistent with the induction of unregulated NO. synthesis in vascular smooth muscle in vivo, administration of IL-1 (50 micrograms/kg) to dogs caused a 33.5% decrease in systemic vascular resistance and a 28% decrease in blood pressure within 3 hours. Subsequent administration of NAA (20 mg/kg) rapidly and completely reversed the hypotension and increased systemic vascular resistance; these effects of NAA were reversed by L-arginine. Neither the immunoproliferative nor the tumoricidal activity of IL-1 was diminished by NAA. Our results indicate that (a) vascular smooth muscle is a likely source as well as a target of IL-1-induced NO. synthesis, causing vasodilatation and hypotension, (b) nitric oxide synthase inhibitors can fully reverse this hypotension, and (c) the therapeutically useful properties of IL-1 are not diminished by nitric oxide synthase inhibitors. Administration of inhibitors of nitric oxide synthase can reverse the pathological cardiovascular effects of IL-1 at concentrations that do not interfere with the potentially useful immunoproliferative or tumoricidal effects of this cytokine. In the context of the current clinical trials of IL-1, this finding would represent a very significant advantage.


Leucovorin enhances cytotoxicity of trimetrexate/fluorouracil, but not methotrexate/fluorouracil, in CCRF-CEM cells.

Increased response rates in studies of patients with colon cancer have indicated that the cytotoxic effects of fluorouracil (5-FU) are potentiated by leucovorin (LV) and by methotrexate (MTX). However, preliminary studies using a sequential combination of MTX, LV, and 5-FU showed no additional potentiation. We hypothesized that the lack of additional cell kill with this combination could be due to competition of LV with MTX for cellular uptake and reduced folate polyglutamylation. We have tested this possibility by comparing the cytotoxicity of drug combinations containing MTX with that of drug combinations containing trimetrexate (TMTX), an antifolate that does not compete with LV for uptake or polyglutamylation. Human lymphocytic leukemia CCRF-CEM cells were exposed to MTX or TMTX for 24 hours and to 5-FU during the last 4 hours of antifolate exposure. LV was administered 30 minutes before 5-FU. After 20 hours of exposure to TMTX or MTX, intracellular levels of phosphoribosyl pyrophosphate were elevated to a similar degree, and these levels did not decrease after a 30-minute exposure to LV. No additional cell kill was observed when LV was added to the MTX/5-FU combination, but cytotoxicity was enhanced when LV was added to the TMTX/5-FU combination. This study supports the hypothesis that the lack of additional cell kill when high-dose LV is added to the MTX/5-FU combination may be due to competition of MTX with LV for cellular uptake and/or competition of MTX or its polyglutamates with polyglutamylation of reduced folates. Inasmuch as TMTX does not compete with LV and reduced folates for uptake and polyglutamylation, the synergy obtained with the combination of TMTX plus 5-FU and high-dose LV further supports this hypothesis.


[Intestinal diseases and pathogenic Escherichia coli in early childhood--diagnostic methods and their value].

In the 50th and 60th enteropathogenic Escherichia coli (EPEC) played an important role for epidemic diarrhoe in children hospitals and kindergarten. The diagnose based on the 0-antigen determination and integration in EPEC-correlated 0-antigen groups. We could demonstrate, that 0-antigen identic Escherichia coli strains isolated in a short period and a small district are extremely different in plasmid profiles. A clonal coherence was not found. 0-antigens are marker of Escherichia coli virulence and today EPEC-infections are only sporadic in middle Europe. That's why 0-antigen determination is of questionable importance for the differentiation of enteropathogenicity. On the other side the role and frequency of further intestinal pathogenic Escherichia coli in early childhood is still unknown.


Stuttering and speech naturalness: audio and audiovisual judgments.

Unsophisticated raters, using 9-point interval scales, judged speech naturalness and stuttering severity of recorded stutterer and nonstutterer speech samples. Raters judged separately the audio-only and audiovisual presentations of each sample. For speech naturalness judgments of stutterer samples, raters invariably judged the audiovisual presentation more unnatural than the audio presentation of the same sample; but for the nonstutterer samples, there was no difference between audio and audiovisual naturalness ratings. Stuttering severity ratings did not differ significantly between audio and audiovisual presentations of the same samples. Rater reliability, interrater agreement, and intrarater agreement for speech naturalness judgments were assessed.


Effects of intravenous epinine administration on left ventricular systolic performance, coronary hemodynamics, and circulating catecholamines in patients with heart failure.

Twenty-six patients with mild to moderate heart failure were studied to determine the effects of epinine infusion (at a rate producing plasma levels similar to those measured after oral administration of 100 mg of the prodrug ibopamine) on left ventricular (LV) function (14 patients), and coronary flow and circulating catecholamines (12 patients). The only significant hemodynamic change at an infusion rate of 0.5 microgram/kg/min was a 9% (p less than 0.05) decrease in systemic vascular resistance (SVR). At an infusion rate of 1 microgram/kg/min (mean free epinine plasma levels 14.3 +/- 3.7 ng/ml), heart rate (HR), dP/dtmax (1,405 +/- 255 to 1,490 +/- 320 mm Hg, NS), (dP/dt)/DP40, and the relaxation rate remained unchanged, but the ejection fraction (EF) increased from 32 to 38% (p less than 0.001), cardiac output (CO) increased, and SVR decreased by 22% (p less than 0.05). In a separate group of 12 patients, epinine infusion at rates of 0.5-1 microgram/kg/min produced no significant changes in coronary blood flow or myocardial oxygen uptake. At these infusion rates, arterial norepinephrine (NE) and dopamine (DA) levels decreased slightly and arterial and coronary sinus epinephrine increased. In conclusion, epinine, at concentrations similar to those achieved during therapeutic use of ibopamine, had negligible effect on myocardial contractility and relaxation rate in heart failure patients. Cardiac pump function was improved by a decrease in SVR rather than by inotropic stimulation. The data also suggest that these low concentrations of epinine may modulate the sympathetic nervous system, but further studies are needed to determine whether this effect could have clinical significance.


Renal hemodynamic effects of nonhypotensive doses of angiotensin-converting enzyme inhibitors in hypertension and heart failure rats.

Both circulating and local renin-angiotensin systems (RAS) may contribute to cardiovascular homeostasis under normal and pathophysiologic conditions. They may also play a role in the effects of angiotensin-converting enzyme (ACE) inhibitors. In the present study, we compared systemic and regional hemodynamic effects of nonhypotensive doses of captopril and enalaprilate in normal rats, spontaneously hypertensive rats (SHR), and rats with heart failure due to myocardial infarction (MI). Enalaprilate (0.1 mg/kg) or captopril (3 mg/kg) was injected intravenously (i.v.) in conscious rats equipped with miniature Doppler flow probes on renal and mesenteric artery and abdominal aorta or an electromagnetic flow probe on the ascending aorta to measure cardiac output (CO). This resulted in a shift of the angiotensin-I (ANG I) dose-pressor curve (ED50 of ANG I after saline 0.21 +/- 0.33 micrograms, enalaprilate 1.45 +/- 0.26 micrograms, captopril 2.38 +/- 0.73 micrograms; mean +/- SEM; n = 6-12). In the systemic hemodynamic groups, no significant changes in mean arterial pressure (MAP), CO, or total peripheral resistance (TPR) were observed. In the regional hemodynamic groups, enalaprilate caused a slight (-8 +/- 1 mm Hg) reduction in MAP in normal rats. Resistance in the hindquarters was not affected by ACE inhibitors, whereas only enalaprilate reduced mesenteric resistance in MI rats. In contrast, renal resistance was reduced and renal blood flow (RBF) increased after captopril in normal and MI rats and after enalaprilate in MI rats. Effects were greatest in MI rats (RBF: saline -0.05 +/- 1.9%, enalaprilate 10.3 +/- 2.4%, captopril 10.1 +/- 2.0%).(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of the effect of renin inhibition and angiotensin-converting enzyme inhibition in ovine heart failure.

The acute hemodynamic and hormonal effects of incremental doses of a specific ovine renin inhibitor (RI: EMD 52 297) and captopril were compared in an ovine model of heart failure. Both RI and captopril inhibited the renin-angiotensin II (ANG II) system, although the decrease in plasma aldosterone (ALDO) was significant only during captopril infusion. Both agents exhibited strong vasodilator properties with similar decreases in mean arterial pressure (MAP, maximum decrease: RI = -20.5 +/- 2.2 mm Hg, p less than 0.001; captopril = -19.8 +/- 1.7 mm Hg, p less than 0.001) and left atrial pressure (LAP, maximum, decrease: RI = -6.8 +/- 1.5 mm Hg, p less than 0.01; captopril = -6.9 +/- 0.4 mm Hg, p less than 0.01) along with a slight increase in cardiac output (CO, maximum increase: RI = 0.54 +/- 0.11 L/min; captopril = 0.79 +/- 0.26 L/min). The slope of the response between MAP and LAP was similar in all animals, indicating that the agents have a similar effect on cardiac preload and afterload. The similar hemodynamic actions of RI and captopril in this model of congestive heart failure suggest that beneficial effects are due to inhibition of ANG II. Thus, orally active renin inhibitors may offer a useful therapeutic alternative when side effects preclude use of angiotensin-converting enzyme (ACE) inhibitors.


Regional hemodynamic effects and clearance of endothelin-1 in humans: renal and peripheral tissues may contribute to the overall disposal of the peptide.

We investigated the regional balance of endothelin-1 across the renal and leg vascular bed as well as the hemodynamic effects of exogenously administered endothelin-1 in six healthy men. Net regional endothelin-1 balance was calculated from the respective arteriovenous differences in plasma concentrations and the corresponding plasma flow, the latter being determined by para-aminohippurate or indocyanine-green dye using appropriate catheter techniques. During constant intravenous (i.v.) infusion of endothelin-1 (0.4 pmol/kg/min), a slight increase in diastolic blood pressure (p less than 0.05) and a decrease in heart rate (p less than 0.01) were observed. In contrast, no significant changes in leg hemodynamics were noted. Renal plasma flow decreased by approximately 30%, and renal vascular resistance increased by 50% as compared with the control (placebo) period (p less than 0.01). Renin plasma concentrations did not change substantially during endothelin-1 infusion. During the control (placebo) period, arterial endothelin-1 plasma concentrations averaged 2.1 +/- 1.0 pM. An equilibrated peptide balance across the leg and a slight renal uptake of endothelin-1 was observed. After endothelin-1 infusion, arterial plasma concentrations of the peptide increased to 4.9 +/- 1.3 pM (p less than 0.01), and a net overall renal and limb uptake of endothelin-1 accounted for approximately 9 and 6% of the infused endothelin-1 amount, respectively. Results showed that at systemic endothelin-1 plasma concentrations, comparable to those which occur in a variety of pathologic conditions such as hypertension or cardiogenic shock, besides pulmonary clearance, renal and limb uptake of the peptide may also contribute to the short half-life (t1/2) of endothelin-1 in humans.


Cicletanine improves myocardial function deteriorated by ischemia/reperfusion in isolated working rat hearts.

The effect of cicletanine, a novel furopyridine antihypertensive drug was compared with that of nitrendipine, a dihydropyridine slow calcium channel blocker, on cardiac function and reperfusion-induced ventricular arrhythmias in isolated working rat hearts subjected to 10-min ischemia induced by ligation of the left main coronary artery followed by 10-min reperfusion. Before ischemia, cicletanine and nitrendipine, perfused at concentrations of 3 x 10(-5), 6 x 10(-5), 10(-4), and 2 x 10(-4) or 10(-8) M, respectively, did not influence heart rate (HR), LV developed pressure (LVDP), its first derivative (LVdP/dtmax), and LV end-diastolic pressure (LVEDP), whereas aortic flow (AF) was decreased by 2 x 10(-4) M cicletanine only. Coronary flow (CF) remained unchanged by various cicletanine concentrations but was slightly increased by nitrendipine. In the concentration range of 3 x 10(-5)-10(-4) M, cicletanine improved AF either in ischemia or during reperfusion, whereas 2 x 10(-4) M had no such effect. Nitrendipine slightly attenuated ischemia/reperfusion-induced decrease in AF. Cicletanine and nitrendipine enhanced LVDP during ischemia. Ischemia-induced deterioration of LVdP/dtmax was reduced by cicletanine, during reperfusion, but this parameter was reduced by nitrendipine and the highest cicletanine concentration. Cicletanine decreased LVEDP significantly during ischemia and reperfusion, but nitrendipine had no such effect. All cicletanine concentrations reduced the incidence of irreversible ventricular fibrillation (VF) during reperfusion, an effect roughly concentration dependent in the range of 3 x 10(-5)-10(-4) M, whereas nitrendipine had no influence on arrhythmias.


Residual platelet function under acetylsalicylic acid and the risk of restenosis after coronary angioplasty.

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) was shown not to be preventable by antiplatelet therapy; residual platelet function under treatment with platelet inhibitors could be one cause of this. Therefore, in a prospective investigation, residual platelet function was assessed in 98 patients treated with acetylsalicylic acid (ASA) on three occasions during the first 3 months after successful PTCA. Control cardiac catheterization was obtained in 75 of these patients (77%) with 82 dilated stenoses 173 +/- 117 days after PTCA. Restenosis, defined as diameter stenosis greater than or equal to 50% at control angiography, occurred in 41% of the dilated vessels, and 43% of patients experienced restenosis in at least one vessel. The in vitro platelet aggregatory response to either ADP (0.5, 1, and 10 microM) or collagen (1 and 5 mg/L) as aggregating agents did not differ between patients with and without restenosis. In addition, neither the collagen-stimulated in vitro synthesis of thromboxane nor the basal or prostaglandin E1-stimulated concentrations of cyclic AMP in platelet-rich plasma (PRP) was different in the two groups. There was no significant correlation between any of the parameters of platelet function assessed and the change in coronary luminal diameter observed between immediately after PTCA and control coronary angiography. The mean dose of ASA ingested during follow-up was also not a determinant of the occurrence of restenosis. Thus, residual platelet function under treatment with ASA as measured in vitro in this study, did not influence the occurrence of restenosis after successful PTCA.


Effects of intracoronary administration of endothelin in anesthetized dogs: comparison with Bay k 8644 and U 46619.

The effects of synthetic endothelin on the coronary circulation were studied in pentobarbital-anesthetized dogs and compared with those of Bay k 8644, a dihydropyridine calcium channel agonist, and U 46619, a thromboxane analogue. Intracoronary bolus administration of endothelin reduced coronary blood flow and increased coronary arterial resistance. Similarly, intracoronary bolus administration of equipotent doses of Bay k 8644 or U 46619 significantly reduced coronary blood flow and increased coronary arterial resistance. The coronary vasoconstrictor effects of endothelin were long-lasting as compared with the transient actions of Bay k 8644 and U 46619. Intracoronary bolus injection of endothelin also reduced left ventricular (LV) dP/dt arterial pressure (MAP), and cardiac output (CO). In contrast, Bay k 8644 increased LVdP/dt but did not alter CO or MAP. Intracoronary bolus injection of U 46619 did not affect MAP, CO, or LVdP/dt. In a separate group of animals, intracoronary infusion of nitrendipine significantly increased coronary blood flow and reduced coronary arterial resistance. Other cardiovascular parameters measured were not significantly altered. In the presence of nitrendipine, the effects of intracoronary administration of endothelin and U 46619 on coronary blood flow, coronary arterial resistance, and LVdP/dt were only partially antagonized. On the other hand, the effects of Bay k 8644 were completely prevented in the presence of nitrendipine. These studies show that at doses which reduce coronary blood flow to the same extent, only endothelin produces myocardial depression in anesthetized dogs. The cardiovascular actions of endothelin were only partially antagonized by nitrendipine, suggesting that mechanisms other than calcium influx through voltage-operated channels are involved.


Isoproterenol increases defibrillation energy requirements in dogs.

The effect of intravenous isoproterenol on the energy requirements for successful defibrillation (DF) was examined in anesthetized dogs following cholinergic blockade with atropine (n = 5) and following treatment with d- and d,l-sotalol (n = 16). Defibrillation shocks were administered through left and right ventricular epicardial patch electrodes and the energy requirements for DF were studied using two different methods. Multiple shocks of varying energies were delivered and the energies required for 50% success (E50) and 80% success (E80) in DF were estimated using logistic regression. Atropine (0.04 mg/kg) increased E50 by 32 +/- 30% (p = 0.046) and E80 by 39 +/- 38% (p = ns). Subsequent administration of isoproterenol (10 micrograms/ml), increasing the heart rate by 52 +/- 35% (p = 0.025), resulted in a further 108 +/- 21% (p = 0.015) and 88 +/- 55% (p = 0.02) rise in E50 and E80 values, respectively. In a second set of experiments, d- (n = 9) and d,l-sotalol (n = 7) (4 mg/kg bolus followed by 0.025 mg/kg/min maintenance infusion) were administered and baseline curves relating delivered energy to % success in DF were calculated. Isoproterenol (10 +/- 4 micrograms/min) was given to increase the heart rate by 54 +/- 32% (p less than 0.025), and resulted in decreases in % success at each of two energy levels, falling in the midrange of the dose-response curve. Following d,l-sotalol, % successful shocks fell from 60 +/- 15 to 42 +/- 28% (p less than 0.05); following d-sotalol, the % success fell from 66 +/- 13 to 38 +/- 36% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Salicylate reduces ventricular dysfunction and arrhythmias during reperfusion in isolated rat hearts.

Recent studies have shown the ability of salicylic acid (SA) to trap hydroxyl radicals (OH.) generated during reperfusion in ischemic myocardium. Since OH. is implicated in the pathogenesis of reperfusion injury, we examined the effect of SA on reperfusion-induced arrhythmias and postischemic ventricular dysfunction. Isolated rat hearts perfused by the Langendorff technique were preperfused with Krebs-Henseleit buffer containing SA for 10 min. Hearts were then made ischemic for 30 min, followed by 30 min of reperfusion. In a separate group, SA was administered only at the onset of reperfusion. The left ventricular contractile functions, left ventricular developed pressure (LVDP) and its first derivative (LV dP/dt), coronary flow (CF), and creatine kinase (CK) release were determined before and after ischemia. Epicardial electrocardiogram (ECG) was also recorded to analyze the incidence of ventricular tachycardia (VT) and ventricular fibrillation (VF). SA improved LVDP, LV dp/dt, and CF recovery and reduced CK release compared to the control group. The incidence of VT and VF during reperfusion was also significantly reduced by SA. Analysis of tissue thiobarbituric acid-reactive products indicates that SA decreased oxidative stress during reperfusion. In conclusion, these results suggest that SA reduces myocardial reperfusion injury and attenuates ventricular arrhythmias by trapping OH. radicals upon reperfusion in isolated rat hearts.


Class III antiarrhythmic action and inotropy: effects of dofetilide in acute ischemic heart failure in dogs.

We studied the hemodynamic and metabolic effects of the novel class III antiarrhythmic agent dofetilide (UK-68,798) in acute ischemic heart failure. In pentobarbital-anesthetized dogs, heart failure was induced by microembolization of the area supplied by the main left coronary artery until a stable left ventricular (LV) end-diastolic pressure of 27 +/- 2 mm Hg was achieved. Embolization depressed LV systolic pressure, LV dP/dtmax, LV dP/dtmin, and cardiac output. None of these parameters were changed following i.v. infusion of dofetilide 5, 10, or 25 micrograms/kg, during spontaneous and paced cycle length of 300 ms (n = 9). Heart rate decreased by 12 +/- 8, 19 +/- 7, and 21 +/- 7 beats/min (p less than 0.05), while QT time increased by 23 +/- 7, 33 +/- 9, and 40 +/- 10 ms (p less than 0.05) after 5, 10, and 25 micrograms/kg, respectively. Ventricular effective refractory period increased from 128 +/- 10 to 153 +/- 11 ms after 25 micrograms/kg (n = 4). Arterial concentration and net myocardial uptake of glucose, lactate, and free fatty acids were not significantly influenced by dofetilide. In conclusion, dofetilide, at doses that prolonged repolarization, was devoid of cardiodepressive effects in acute ischemic heart failure.


Spare receptors for beta-adrenoceptor-mediated positive inotropic effects of catecholamines in the human heart.

We studied whether the human heart has spare receptors for beta-adrenoceptor-mediated positive inotropic effects. Thus, we assessed in right atria and left papillary muscles of patients with different degrees of heart failure under identical experimental conditions affinity (pKI values from (-)-[125I]iodocyanopindolol binding) and potency (pD2 values from contractile responses) for isoprenaline, adrenaline, and noradrenaline in comparison with rat heart. Plots of beta-adrenoceptor occupancy versus responses constructed from these data revealed that rat left atria and papillary muscles had a large receptor reserve for all three beta-adrenoceptor agonists: 50% of maximal response was produced with only 1-3% of beta-adrenoceptor occupancy. In human heart, however, receptor reserve was considerably lower: 50% of maximal response required 8-10% (in right atria) and 20-25% (in left papillary muscles) occupation of beta-adrenoceptors. Receptor reserve declined further with an increasing degree of heart failure (and decreasing beta-adrenoceptor number): in end-stage heart failure (New York Heart Association class IV) both in right atria and left papillary muscles a 1:1 ratio between beta-adrenoceptor occupancy and responses was observed. These data show that the human heart has only a small receptor reserve for beta-adrenoceptor agonists. This may explain why a decrease in beta-adrenoceptor number leads to a decrease in beta-adrenoceptor function early in the development of heart failure.


Aortic baroreceptors exert a tonically active restraining influence on centrally mediated depressor responses.

This study tested the hypothesis that aortic baroreceptors exert a central restraining influence on centrally mediated depressor responses and that this mechanism is tonically active and is independent of their modulation of basal arterial pressure. The effects of short-term (48-72 h) aortic baroreceptor deafferentation (ABD) on the acute hemodynamic (peripherally mediated pressor and centrally mediated depressor) effects of clonidine were investigated in conscious and anesthetized normotensive rats. ABD caused an immediate increase in basal arterial pressure and heart rate and a significant attenuation of the baroreceptor reflex control of heart rate. Since only arterial pressure subsided to control levels by 48-72 h, the data suggest that central reorganizational changes were potent enough to overcome the tonically active restraining influence of aortic baroreceptors on basal arterial pressure but not heart rate. Clonidine produced a similar pressor effect in conscious ABD and sham rats but its depressor effect was significantly greater in ABD rats whose baroreceptor reflex control of heart rate was significantly attenuated. Intracisternal (i.c.) administration of 0.1 microgram of clonidine, a dose that had no effect when administered i.v., produced a near-maximal depressor effect in conscious ABD rats vs. no effect in sham rats (-16.7 +/- 4.1 vs. -0.3 +/- 2 mm Hg; p less than 0.001). The depressor effect of clonidine was also enhanced in chloralose-anesthetized rats and coincided with an anesthesia-induced attenuation of the baroreceptor reflex control of heart rate. It is concluded that aortic baroreceptors exert a potent restraining influence on the centrally mediated depressor effect of clonidine. Since ABD had no significant effect on the depressor response to nitroprusside, but enhanced the depressor response to ganglion blockade by hexamethonium, the data suggest that a higher peripheral sympathetic neural activity existed in ABD rats. The reorganizational changes that occurred within 48-72 h after ABD were potent enough to overcome the tonically active restraining influence of aortic baroreceptors on basal arterial pressure but not on the centrally mediated depressor responses. Thus, the buffering influence of aortic baroreceptors and their central projections on centrally mediated depressor responses seems to be tonically involved in blood pressure control.


Effect of lovastatin on suppression and regression of atherosclerosis in lipid-fed rabbits.

The present study was designed to evaluate the effects of lovastatin on suppression and regression of atherosclerosis in the lipid-fed rabbit. Fifty-seven New Zealand rabbits in six groups were fed a 0.3% cholesterol diet for 10 weeks. In the progression phase of the study, group C10 served as a control and received 1 ml of DMSO daily by gavage. Two other groups, L10 and H10, received low (L)-dose (10 mg/day) or high (H)-dose (20 mg/day) lovastatin dissolved in 1 ml of DMSO for 10 weeks. In the regression phase of the study, three groups of rabbits received the high lipid diet for 10 weeks and were then shifted to a normal diet for the second 10 weeks. During the second 10 weeks, the control group C20 received 1 ml of DMSO daily, and groups L20 and H20 received 10 and 20 mg/day of lovastatin by gavage, respectively. In the progression phase of the study, lovastatin significantly attenuated the percent of aortic lesions in groups L10 (8 +/- 7%) and H10 (9 +/- 14%) vs. the control group C10 (31 +/- 17%; p less than 0.01). There was a similar reduction in pulmonary lesions in groups L10 (10 +/- 6%) and H10 (4 +/- 5%) compared to the control group C10 (30 +/- 16%; p less than 0.01). There was also a reduction in plaque thickness in both the aorta and pulmonary artery, and hence an even greater reduction in estimated plaque volume. In the regression phase of the study, lovastatin also significantly reduced the percent of aortic lesions (groups L20 and H20 vs. C20: 27 +/- 18 and 22 +/- 7% vs. 40 +/- 17%; p less than 0.05) and pulmonary lesions (21 +/- 10 and 17 +/- 6% vs. 26 +/- 9%; p greater than 0.05 and p less than 0.05, respectively); the average maximum plaque thickness of aorta (0.24 and 0.26 mm vs. 0.49 mm; p less than 0.01); and the standardized plaque volume per unit area of aorta (4.5 and 3.4 vs. 12.1 mm-%; p less than 0.05 and p less than 0.01, respectively). However, there was no significant difference in the percent of aortic lesions between groups L20 and H20 and group C10 (27 +/- 18 and 22 +/- 7 vs. 31 +/- 17%; p greater than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)


Effects of ibutilide on spontaneous and induced ventricular arrhythmias in 24-hour canine myocardial infarction: a comparative study with sotalol and encainide.

The electrophysiologic and antiarrhythmic effects of ibutilide, sotalol, and encainide were compared in dogs 24 h after myocardial infarction. Ibutilide (0.03 to 0.3 mg/kg i.v.) prevented the induction of ventricular arrhythmias in 100% of the dogs that had demonstrated inducible ventricular arrhythmias prior to treatment. This antiarrhythmic action was associated with significant increases in ventricular refractoriness and monophasic action potential duration. Sotalol (1.0 to 10.0 mg/kg i.v.) increased the ventricular refractory period and monophasic action potential duration and prevented the induction of ventricular arrhythmias in 75% of the dogs that demonstrated inducible ventricular tachyarrhythmias at baseline. Although 10 mg/kg of sotalol was required to prevent the initiation of ventricular tachycardia, this dose produced marked cardiovascular depression and hypotension in 50% of the dogs tested. Neither ibutilide nor sotalol significantly decreased the incidence of spontaneous ventricular arrhythmias. The class IC agent encainide (0.3 to 3.0 mg/kg i.v.) was successful in preventing the induction of ventricular arrhythmias in only 20% of the dogs tested. However, in contrast to ibutilide and sotalol, encainide significantly reduced spontaneous arrhythmias. Atrial and ventricular refractoriness were significantly increased only after the highest dose of encainide tested (3.0 mg/kg). Over the dose ranges studied, the relative efficacy for prevention of pacing-induced ventricular arrhythmias was ibutilide greater than sotalol much greater than encainide. For suppression of spontaneous ventricular arrhythmias, the relative efficacy was encainide much greater than ibutilide = sotalol.(ABSTRACT TRUNCATED AT 250 WORDS)


The sinus node inhibitor UL-FS 49 lacks significant inotropic effect.

UL-FS 49 is a sinus node inhibitor that has been reported to reduce heart rate and may be useful in improving myocardial oxygen supply vs. demand. However, previous studies performed in a variety of preparations have produced mixed results regarding the independent inotropic effect of UL-FS 49. To determine whether UL-FS 49 has an inotropic effect, we measured both steady-state hemodynamic responses and transient hemodynamic responses to random preload and afterload changes, both with and without UL-FS 49. We found that under steady-state conditions, the effect of UL-FS 49 is so small that it would be of doubtful physiologic significance: a 3% increase in stroke volume (p = 0.007) and 7% increase in peak positive dP/dt (p = 0.051), in the presence of no statistically significant differences in end-diastolic pressure, end-diastolic volume, peak systolic pressure, end-systolic pressure, or heart rate. The more powerful multiple linear regression modeling of hemodynamic transient sequences resulting from random preload and afterload changes showed that UL-FS 49 is without a statistically significant direct effect on left ventricular function. We conclude that UL-FS 49 has no physiologically important direct effect on left ventricular pump function.


Effect of isradipine on cardiopulmonary baroreflex function, regional blood flow, and vascular responsiveness in hypertensive patients.

Calcium channel antagonists, when used to treat hypertension, may modulate baroreflex function and vascular responsiveness to endogenous vasoconstrictors. We studied regional blood flow, cardiopulmonary baroreflex function, and pressor responses in nine hypertensive patients (mean age of 44 +/- 7 years), eight males and one female, treated with isradipine (ISR), a dihydropyridine calcium channel antagonist, in a placebo-controlled, crossover trial. Each patient underwent determination of blood pressure and forearm, splanchnic, and renal blood flows (by strain gauge plethysmography and indocyanine green and p-aminohippurate clearances, respectively) at baseline and during cardiopulmonary unloading by lower body negative pressure (LBNP) at -10 and -20 mm Hg. ISR decreased the mean arterial pressure from 105 +/- 2 to 93 +/- 2 mm Hg (p less than 0.01). ISR did not change supine forearm or splanchnic vascular resistances, but renal vascular resistance fell 30% during treatment (from 0.12 +/- 0.02 to 0.09 +/- 0.01 mm Hg min/ml, p less than 0.05). Cardiopulmonary baroreceptor unloading by LBNP elicited comparable effects on forearm, splanchnic, and renal vascular resistance before and during ISR treatment. Baroreceptor unloading during placebo did not change plasma NE or PRA; during ISR, LBNP elicited a progressive rise in these hormones. The pressor response to NE was potentiated during ISR treatment (p less than 0.05); in contrast, the pressor response to angiotensin II infusion was blunted by calcium blockade (p less than 0.05). The present study, therefore, demonstrates that calcium channel blockade with ISR preserves, and may even augment, cardiopulmonary baroreflex function. These physiologic responses may contribute to the relatively low incidence of symptomatic orthostatic hypotension observed during chronic treatment with this agent.


Renal structure and function in rats after suprapharmacologic doses of quinapril, an angiotensin-converting enzyme inhibitor.

Angiotensin-converting enzyme (ACE) inhibitors have adverse effects on renal function in some hypertensive patients, and some of them produce renal tubular lesions in animals at high doses. To assess the effect of quinapril on renal function and structure, a 4-week time-course study was conducted in male Wistar rats with daily oral gavage doses of 0, 10, 100, or 400 mg/kg. Glomerular filtration rate (GFR) estimated as creatinine clearance and fractional electrolyte excretion values were derived from urinalysis and blood chemistry data obtained at days 1, 7, 14, and 28. Renal sections were collected on these days for histopathologic evaluation, and cortical slices were obtained to assess organic ion transport in vitro. Expected pharmacologic effects of an ACE inhibitor were observed at all doses and included increased urine output, increased water consumption, decreased serum aldosterone (65 or 25% of control at 10 or 400 mg/kg, respectively, on day 28), increased plasma renin activity (PRA, up to two- to threefold higher than controls at day 28), and hypertrophy of the juxtaglomerular apparatus. Despite these expected class effects, quinapril administration to male rats for 28 days produced no functional alterations or renal tubular lesions suggestive of renal toxicity at doses up to 400-fold higher than the effective antihypertensive dose in rats.


Electrophysiologic effects of ambasilide (LU 47110), a novel class III antiarrhythmic agent, on the properties of isolated rabbit and canine cardiac muscle.

Electrophysiologic effects of ambasilide in canine isolated cardiac muscle driven at 1 Hz and in rabbit sinoatrial (SA) node preparations were determined by standard microelectrode technique. Ambasilide (10(-7)-10(-5) M) produced concentration-dependent increases in action potential duration measured at -80 mV (APD-80) repolarization time in canine ventricular muscle and in Purkinje fibers. APD measured at -20 mV (APD-20) was also increased in ventricular muscle, but it shortened with 10(-5) M in Purkinje fibers; at this concentration, there was a negligible change in the amplitude and the maximum upstroke velocity (Vmax) of action potentials and in the resting membrane potential. With the stimulation frequency between 30/min and 120/min, ambasilide (10(-5) M) produced a parallel increase in APD-80 as well as in APD-20 of ventricular muscle. In Purkinje fibers, the prolonging effect of ambasilide on APD-80 was more pronounced at lower stimulation frequency, whereas APD-20 shortened at all stimulation frequencies. Ambasilide at 10(-5) M also produced a prolongation of the effective refractory period (ERP) in Purkinje fibers. The drug produced no significant change in the frequency-dependent relationship between ERP and APD-80. A small but significant frequency-dependent inhibition of Vmax was noted in both ventricular muscle and Purkinje fibers. When the stimulus cycle length was reduced from 1,000 to 300 ms, Vmax in ventricular muscle decreased by 8.4 +/- 3.4% in control solution but by 22.6 +/- 5.6% after 10(-5) M ambasilide (n = 8, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)