Comparison of microenvironmental CO concentrations in two cities for human exposure modeling. The microenvironmental components of the CO concentration in two cities are compared by subtracting the ambient background concentration from personal exposures measured in Denver, Colorado, and Washington, DC. Two surrogate measures for the ambient background concentration are tested. Both improve the similarity of the means in the two cities, but the Denver standard deviations are higher than those in Washington, DC. Microenvironments containing the internal combustion engine have both higher means and standard deviations in Denver compared with Washington, DC. The Washington, DC, mean concentration for automobiles, for example, was 59% of the Denver mean (2.0 ppm versus 4.9 ppm). Washington, DC, had approximately 57% of the Denver emissions, and the difference in mean CO concentrations is roughly consistent with the lower emissions in Washington, DC, due to lower elevation. A surprising finding is that mean CO exposure levels caused by cooking with gas stoves in Washington, DC, were only 58% of the levels in Denver (1.9 ppm and 3.3 ppm, respectively). This result suggests that elevation may exert an influence on gas stove emissions that is similar to its influence on internal combustion engines. Using an averaging time model, analysis of the autocorrelation of sleeping and office microenvironments suggests that considerable serial dependency exists. The microenvironmental data and findings in this paper have important implications for constructing human exposure-activity pattern models. For future human exposure field studies, the findings emphasize the importance of measuring background values in a location that is extremely close to each microenvironment studied.


Ultrastructural immunodetection of a Pichia anomala killer toxin: a preliminary study. A monoclonal antibody (mAb KT4), produced against a Pichia anomala killer toxin, was used to study the secretion process of toxin producing cells. The indirect immunofluorescence assay, performed with large concentrations of mAb KT4, showed a homogeneous distribution of the epitope at the cell surface of the P anomala cells. When increasing dilutions of mAb KT4 were employed, a 'punctuated' labeling appeared on the yeast's cell wall which suggested a heterogeneous secretion of the killer toxin. Similar labeling was also observed by immunodetection on live yeast cells held in buffered suspension. These results confirmed that 'punctuated' labeling was not an artefact due to a distortion of the cell's shape by having been dried on glass slides. Indirect immunodetection was performed in electron microscopy on ultra-thin sections of cells embedded in Araldite resin. The labeling thus obtained showed both the presence of the epitope in the cytoplasm and its sensitivity to strong glutaraldehyde fixation. Indirect immunodetection, performed on ultra-thin frozen sections, showed a cytoplasmic and cell wall labelling. However, the amount of gold particles observed in the cell wall was too low to confirm the heterogeneous killer toxin secretion observed in immunofluorescence. In this case, killer cells were fixed with a low concentration of glutaraldehyde which preserved the structure of the epitope complementary with mAb KT4.


Cellular analysis of the kinetics of alpha-fetoprotein and nuclear oncogene activation in primary cultures of adult rat hepatocytes stimulated by epidermal growth factor. Alpha-fetoprotein (AFP), a fetal gene normally inactivated in quiescent adult hepatocytes, is re-expressed in hepatocytes during the proliferating response induced by liver regeneration in vivo, or epidermal growth factor (EGF) in vitro. The nuclear oncogenes c-jun, c-fos and c-myc are 'immediate early' genes also activated during the proliferative response of hepatocytes. Whether AFP gene activation is linked to oncogene activation is not known. As a first step in answering this question, we have analysed the cellular kinetics of nuclear oncogene and AFP gene activation in primary cultures of adult rat hepatocytes stimulated by EGF. Gene activation was evaluated at the mRNA level by dot blot and in situ hybridization, and at the protein level by immunoperoxidase. C-jun, c-fos and c-myc mRNA steady state levels in total cellular RNA were increased from 30 min-2 h after EGF stimulation. In situ hybridization analysis showed that transcripts of the three oncogenes increased in all hepatocytes after EGF stimulation. While unstimulated cultures did not immunostain for Fos and Myc proteins. Fos immunostaining was visible in the majority of hepatocyte nuclei 1 and 2 h after EGF addition, and Myc cytoplasmic immunostaining of the majority of hepatocytes was observed at 2 h of stimulation. AFP mRNA increased in total cellular RNA 2 and 4 h after EGF stimulation, with elevated in situ hybridization signal for AFP mRNA in all hepatocytes. No hepatocytes immunostained for AFP in unstimulated cultures, but a cytoplasmic labeling of 20-30% of the hepatocytes was observed 6 h after stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Bombesin-like immunoreactivity in eosinophils. A light and electron microscopic study: effect of fixatives and cross-reactivity with various compounds. Eosinophils immunopositive for bombesin tetradecapeptide were detected by means of light and electron microscopy in human and rat gastrointestinal tract and pulmonary tissue. This immunoreaction was only evidenced after the use of acetic acid-containing fixative such as Bouin's fluid. The dependence of this immunostaining on fixatives and time course were extensively studied. This immunoreaction promotes mainly one epitope probably associated with the C-terminal sequence. This epitope seems also to be present in other neuropeptides such as substance P (SP) and, to a lesser extent in chemotactic factors like formyl peptide (fMLP) or eosinophil chemotactic factor of anaphylaxis (ECF-A). At the electron microscopic level, the immunopositivity was associated with eosinophil membranes.


Do forebrain structures compete for behavioral expression? Evidence from amphetamine-induced behavior, microdialysis, and caudate-accumbens lesions in medial frontal cortex damaged rats. The neurochemical basis of behavioral changes following medial frontal cortex damage were investigated. Experiment 1 examined locomotion in response to D-amphetamine (1.5 and 5 mg/kg) in rats that had received bilateral aspirative lesions of the medial frontal cortex alone or in combination with 6-hydroxydopamine (6-OHDA) lesions of the nucleus accumbens or caudate-putamen. Relative to controls, medial frontal cortex rats were initially hypoactive (day 1 postoperative) but rapidly became hyperactive (days 5-15 postoperative). Locomotor-time profiles and stereotypy ratings showed that amphetamine produced a selective enhancement of locomotion at the expense of stereotyped behavior. Nucleus accumbens lesions blocked the locomotion but enhanced stereotyped behavior in the medial frontal cortex damaged rats, suggesting that amphetamine-enhanced locomotion is dependent upon the integrity of the nucleus accumbens. In Experiment 2, intracerebral microdialysis was used to examine whether alterations in dopamine (DA) or monoamine metabolites in the nucleus accumbens or caudate-putamen accompanied the lesion-induced changes in locomotion. There were no differences in extracellular DA or monoamine levels between control rats and medial frontal cortex rats when tested on day 1 or day 15 postsurgery, either when they were at rest, while they walked on a motor-driven belt, or after amphetamine treatment. Therefore, it seems unlikely that changes in amphetamine-induced locomotion following medial frontal cortex lesions are related to underlying modifications in dopaminergic activity in the nucleus accumbens. It is suggested that neural structures compete for behavioral expression and that postlesion behavioral alterations reveal the competitive advantage of remaining intact neural systems.


Competitive interactions during dendritic growth: a simple stochastic growth algorithm. A simple growth algorithm is presented that deals with one feature of dendritic growth, the distance between branches. The fundamental assumption of our growth algorithm is that the lengths of dendritic segments are determined by the branching characteristics of the growing neurite. Realistic-appearing dendritic trees are produced by computer simulations in which it is assumed that: (1) growth of individual neurons occurs only at the tips of each growing neurite; (2) the growing neurite can either branch (as a bifurcation) or continue to elongate; (3) events at any one growing tip do not affect the events at any other growing tip; and (4) the probability of branching is a function only of the distance grown either from the cell body (if branching has not occurred) or from the previous branch point. An analytic solution of a differential equation based on these same assumptions produces a distribution of dendritic segment lengths that accurately fits an experimentally determined distribution of dendritic segment lengths of reconstructed neurons, accounting for about 89% of the sample variance. Our analysis indicates that, immediately following branching, the temporary suppression of further branching during dendritic growth may be an important mechanism for regulating the distance between branches.


Presence of the binding of a variety of ligands related to ionotropic excitatory amino acid receptors in rat retina. The binding of [3H]L-glutamic (Glu), [3H](+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic and [3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acids was detected in rat retinal membranes extensively washed and treated with a low concentration of Triton X-100, in addition to the binding of both [3H]glycine (Gly) and [3H]5,7-dichlorokynurenic acid. Furthermore, retinal membranes exhibited the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne in the presence of Glu, Gly and spermidine irrespective of the incubation period employed. Rat retina also contained the binding of [3H]kainic acid as well as the binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid which was sensitive to potentiation by potassium thiocyanate. In addition, the binding of both [3H](+)-3-(hydroxyphenyl)-N-(l-propyl)piperidine and [3H]1,3-di-o-tolyl-guanidine was found in rat retinal membranes extensively washed but not treated with Triton X-100. These results give support for the proposal that the rodent retina contains subclasses of ionotropic brain excitatory amino acid receptors including the N-methyl-D-aspartate receptor ionophore complex as well as sigma sites.


Distinct populations of neurons in the ventromedial periaqueductal gray project to the rostral ventral medulla and abducens nucleus. Following microinjections of a colloidal gold complex into the nucleus paragigantocellularis (PGi) of the ventral medulla, and of latex microspheres into the nucleus abducens (Abd) of the same animal, retrogradely labeled neurons were identified in the region of the contralateral supraoculomotor nucleus (SOM) in the ventromedial periaqueductal gray (PAG). Neurons labeled from the Abd were found in the SOM in the ventromedial PAG throughout the midbrain, as well as scattered ventrally in the oculomotor nucleus. Neurons in the SOM area retrogradely labeled from the PGi were most numerous rostral to the dorsal raphe nucleus and extended throughout the level of the oculomotor nucleus. Direct comparison of the two labels revealed that the neurons that project to the Abd were located slightly more ventrally than PGi-projecting neurons. Almost no doubly labeled neurons were identified, although singly labeled neurons formed adjacent but separate populations. These results indicate that neurons in the SOM area projecting to the PGi are distinct from those projecting to the Abd, and that the PGi-projecting neurons are probably not pre-oculomotor neurons. Given their more dorsal location and the nature of their target neurons in the rostral ventrolateral medulla, PGi-projecting neurons may be related to visceromotor, as opposed to oculomotor functions.


TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase. Intracerebral hemorrhage occurs in tumors, stroke and head trauma. Proteolysis of the extracellular matrix around cerebral capillaries by naturally occurring mammalian 72-kDa type IV collagenase may initiate this pathologic event. To investigate this hypothesis adult rats underwent intracerebral injection of type IV collagenase purified from human melanoma cells. Histologically, at 4 h there was perivascular cellular infiltration with hemorrhage, and by 24 h there was infarction with necrosis, edema and hemorrhage. Ultrastructurally, the basal lamina of endothelial cells was disrupted at 2 h. Brain uptake of [14C]dextran and [3H]sucrose increased after intracerebral injection of type IV collagenase compared to controls (P less than 0.0001). Tissue inhibitor of metalloproteinase-2 (TIMP-2) reduced the tracer uptake (P less than 0.02). Metalloproteinase inhibitors reduce extracellular matrix proteolysis and protect the blood-brain barrier.


Intracellular electrophysiological and morphological study of the medullary inspiratory neurons of the decerebrate rat. Intracellular recordings and labelings with horseradish peroxidase (HRP) of inspiratory neurons were performed in decerebrate, paralyzed and ventilated rats. A total of 58 neurons were located within the ventrolateral medulla. They were identified as bulbospinal neurons (n = 15), cranial motoneurons (n = 9) and not antidromically activated (NAA) neurons (n = 34) by antidromic stimulation or HRP labeling, or both. These inspiratory neurons had rhythmical changes in membrane potentials similar to those reported in cats, i.e. an abrupt depolarization at the onset of phrenic discharge followed by trajectories of depolarization which evolved into augmenting I, bell-shaped I or decrementing I patterns until a rapid repolarization at the start of expiration. All types were hyperpolarized during expiration by chloride-dependent inhibitory postsynaptic potentials (IPSPs) which were demonstrated in 13 neurons from which the reversal was obtained. Such IPSPs were apparent in two waves throughout expiration, an early one in post-inspiration (stage I of expiration) and a late one in late expiration (stage II of expiration). These properties are also similar to those of feline inspiratory medullary neurons. Four labeled bulbospinal neurons had axonal collaterals which were ipsi- and contralateral to the site of their somata. Two of 6 labeled NAA neurons exhibited profuse axonal arborizations within various medullary nuclei. No medullary axonal collateral was seen from 6 labeled motoneurons. These results indicate that even though in the rat a single concentration of inspiratory neurons within the ventrolateral medulla has been demonstrated, there is no fundamental difference in the organization of the inspiratory neuronal network compared to that of the cat.


Blockade of acute hypertensive response does not prevent changes in behavior or in CSF acetylcholine (ACH) content following traumatic brain injury (TBI). There is evidence that the blood-brain barrier (BBB) is breached following traumatic brain injury (TBI), allowing the unregulated entry of circulating neuroactive substances into the central nervous system. As the traumatic episode is typically associated with an acute hypertensive event, which in itself may alter BBB status, the effects of the blockade of TBI-associated hypertension on injury-associated behavioral and cerebrospinal fluid (CSF) neurochemical changes were assessed in rats. Animals were injected with either saline or hexamethonium 15 min prior to a moderate fluid percussion injury while under light methoxyflurane anesthesia. This dose of hexamethonium was demonstrated to block the hypertensive response to TBI. Pretreatment with hexamethonium prevented neither acute nor more enduring behavioral deficits observed after TBI. Hexamethonium did not prevent TBI-associated increases in CSF acetylcholine (ACh) content in separate group of rats sampled 12 min following TBI. Furthermore, histological inspection indicated that hexamethonium did not prevent TBI-induced disruption of the BBB, as assessed by intravascular horseradish peroxidase (HRP). Thus, blockade of the hypertensive response to TBI does not afford behavioral protection nor does it prevent changes in the BBB or CSF ACh content following TBI. TBI is in itself sufficient to modify behavior, neurochemistry and BBB function in the absence of hypertension.


Demonstration of a neuronal projection from the entopeduncular nucleus to the substantia nigra of the rat. The two neuronal tracers, Phaseolus vulgaris leucoagglutinin (PHA-L) and Cholera toxin subunit B (CHB) were used in order to study a possible neuronal projection from the entopeduncular nucleus to the ventral mesencephalon in the rat. Both tracers were identified in brain sections by means of immunohistochemistry. After injection of PHA-L in the entopeduncular nucleus PHA-L-positive nerve fibers observed in the mesencephalon were moderate in number and mostly restricted to the mediodorsal part of the substantia nigra, pars reticulata. In addition, a low number of PHA-L-immunoreactive nerve fibers was found in the substantia nigra, pars compacta, the rostral part of the ventral tegmental area, and in the deep mesencephalic nucleus. In agreement with these observations, several labeled neurons were observed in the ipsilateral entopeduncular nucleus after injections of CHB in the substantia nigra. These results indicate the presence of a direct neuronal projection between the two major output channels of the basal ganglia.


AMPA glutamate receptor activation in the posterior zona incerta inhibits amphetamine- and apomorphine-induced stereotypy. Previous work demonstrated that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor antagonism in the zona incerta (ZI) dorsal to the subthalamic nucleus inhibits stereotypy in rats. The current investigation was undertaken to determine if AMPA receptors in a more caudal portion of the ZI have a role in the expression of stereotyped behavior. Rats were injected bilaterally with AMPA into the posterior ZI dorsal to the substantia nigra, and immediately given a systemic injection of d-amphetamine (10 mg/kg, s.c.) or apomorphine (1 mg/kg s.c.). AMPA produced a dose-dependent inhibition of stereotypy induced by both drugs which was prevented by the coadministration of the AMPA/kainic acid antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) (0.5 microgram/0.5 microliter). A dose of AMPA as low as 62.5 ng completely abolished the oral component of stereotypy induced by both apomorphine and amphetamine. This dose of AMPA alone had no significant effect on spontaneous locomotor activity but enhanced the locomotor response stimulated by amphetamine (10 mg/kg, s.c.) due to an inhibition of stereotypy. The finding that activation of AMPA receptors in the posterior ZI inhibits stereotypy shows a contrast to results in the neighboring medial ZI dorsal to the subthalamic nucleus, where blockade of AMPA/kainic acid glutamate receptors with DNQX inhibits stereotypy.


Mechanotransducing ion channels in astrocytes. Ion channels present on the soma of neonatal rat astrocytes in primary cell culture were studied using the single channel recording technique. Ion channels were activated by changing the pressure in the back of the pipette. The morphological structure of the patch membrane was examined while recording channel activity. One class of channel was activated by increasing the pipette pressure (curvature-sensitive or CS channels). CS channels were observed in 150 mM KCl, 150 mM NaCl, or 150 mM sodium gluconate. At constant pressure the closed times decreased with depolarization. CS channels had a conductance of 50 pS in 150 mM NaCl, and displayed an inwardly rectifying current-voltage relationship. CS channel activity was found only in cell-attached patches, and were active only when the patch membrane curved towards the soma. The other class of channel was found to be activated by both suction and pressure (stretch-activated or SA channels). Four SA conductance levels were found: 360, 230, 144, and 70 pS in 150 mM KCl. Each conductance displayed a linear current-voltage relationship. At negative membrane potentials SA channels were inhibited by Cs+, Ba2+ or Na+. The relationship between average mechanosensory current and pressure was biphasic for SA channels and monophasic for CS channels. Combinations of SA and CS channels could be observed in the same patch. We propose that CS channels are non-specific cation channels which sense membrane tension only when the patch membrane is in a specific, permissive curvature. SA channels appear to be K(+)-selective channels that sense membrane tension independent of the direction of curvature.


Guanethidine sympathectomy increases substance P concentration in the superior sympathetic ganglion of adult rats. Adult rats received intraperitoneal injections of guanethidine or saline for 5 weeks. Six to 8 weeks following completion of treatment, concentrations of substance P and neuropeptide Y (NPY) were measured by radioimmunoassay in the superior cervical ganglion (SCG) and thoracic spinal cord. The SCG was also immunostained for NPY and substance P. No differences were observed in thoracic spinal cord content of either NPY or substance P. We observed depletion of NPY immunoreactive neurons and NPY levels in the SCG, and pharmacologic evidence of postganglionic denervation in guanethidine-treated rats. In guanethidine-treated rats, there was a marked increase of substance P levels in the SCG, where substance P was localized in fibers, but not cell bodies. Thus, sprouting of substance P-containing sensory fibers in the sympathetic ganglia occurs following postganglionic sympathectomy in adult rats.


Serum alpha-L-fucosidase. A useful marker in the diagnosis of hepatocellular carcinoma. The value of serum alpha-L-fucosidase activity in the diagnosis of hepatocellular carcinoma (HCC) was investigated by determining the enzyme activity levels in 21 patients with HCC, 76 patients with cirrhosis, 22 patients with other malignant neoplasms, and 23 healthy subjects. The serum alpha-L-fucosidase activity level in patients with HCC (575.76 +/- 212.86 nmol/ml/h) was significantly higher than that found in patients with cirrhosis (274.55 +/- 138.97 nmol/ml/h; P less than 0.001) or other neoplasms (257.91 +/- 128.12 nmol/ml/h; P less than 0.001) and in controls (221.23 +/- 114.45 nmol/ml/h; P less than 0.001). No significant differences were found between controls and patients with cirrhosis and between controls and patients with other malignant neoplasms. When 443 nmol/ml/h is taken as the cutoff value (mean value of controls plus 2 standard deviations), alpha-L-fucosidase sensitivity and specificity were 76% and 90.9%, respectively. These results suggest that alpha-L-fucosidase is a useful marker for detecting HCC, in conjunction with alpha-fetoprotein and ultrasonography.


A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis. The purpose of this study was to determine the histogenesis of jejunoileal and appendiceal carcinoids and to ascertain whether this could be useful in further explaining the pathology of these neoplasms. Eight cases each of multiple jejunoileal carcinoids and appendiceal carcinoids together with their respective age-matched and sex-matched controls were stained with silver stains, chromogranin A, serotonin, and S-100. Histomorphometric evaluations of the endocrine cells in the mucosa adjacent to the carcinoids were carried out and compared with the respective controls using the Student's t test. All the carcinoids from both groups stained for argyrophilia, argentaffinity, chromogranin A, and serotonin. Histomorphometric evaluations showed intraepithelial endocrine cell hyperplasia (IECH) in the jejunoileal carcinoid group (P = 0.007, chromogranin; P = 0.004, serotonin) but not in the appendiceal carcinoid group. On the other hand, subepithelial endocrine cell aggregates that were separate from the main tumor were seen in two cases of appendiceal carcinoids. With S-100, all appendiceal carcinoids showed intrinsic tumor positivity whereas the jejunoileal carcinoids did not. The finding of IECH with multiple jejunoileal carcinoids suggests that these carcinoids arise from a field effect. The absence of IECH with appendiceal carcinoids as well as their association with subepithelial endocrine cell aggregates and their intimate relationship with Schwann cell processes suggests that appendiceal carcinoids arise from a more discrete unit, the subepithelial neuroendocrine complex.


Immunohistochemical identification of tissue factor in solid tumors. Patients with cancer experience a much higher than expected incidence of thromboembolic disorders, commonly referred as Trousseau syndrome. Although this association has been well documented, the etiology of the hypercoagulable state is not known. The expression on tumor cells of tissue factor (TF), a membrane-bound lipoprotein that functions as a cofactor to factor VIIa in the initiation of the extrinsic pathway of blood coagulation, has been postulated as a possible mechanism. Whereas the distribution of TF in normal tissues is known, no large survey of TF expression in malignant tissues has been reported. In this study a polyclonal, monospecific rabbit anti-human TF IgG was used for immunohistochemical localization of TF antigen in 85 different tumor specimens. In general, cell types which normally express TF continued to do so after malignant transformation (41 of 60 epithelial tumor specimens were positive for TF). Tumors of nonepithelial origin frequently lacked TF, with only 3 of 19 specimens containing evidence of TF antigen. In addition five of six benign tumors did not express TF. Many tumor types commonly associated with Trousseau syndrome, for example lung, pancreatic, breast, colon and gastric carcinomas, stained positively for TF. Based on this survey, it appears that TF expression by tumors may be an important factor in the pathogenesis of a hypercoagulable state in some patients with cancer.


A novel Arg-Gly-Asp containing peptide specific for platelet aggregation and its effect on tumor metastasis: a possible mechanism of RGD peptide-mediated inhibition of tumor metastasis. A novel cyclic tetrapeptide containing L-arginine-glycine-L-aspartic acid-L-phenylglycine (cyclo-RGDPhg) was synthesized and found to be a potent inhibitor of platelet aggregation induced by highly metastatic murine squamous cell carcinoma (SCCVII) cells (IC50 = 3.3 microM) as well as ADP (1.5 microM). This cyclic peptide, however, showed similar or less inhibitory activities on adhesion of SCCVII cells to fibronectin, vitronectin and type IV collagen as compared with those of parent linear tetrapeptide, RGDS. These results show that cyclo-RGDPhg peptide is a highly specific antagonist for gpIIb/IIIa on platelets. Moreover, this peptide failed to suppress pulmonary metastasis of SCCVII cells in an experimental metastasis model. These results indicate that RGD peptide-mediated inhibition of tumor metastasis is attributed to the suppression of cell adhesion but not platelet aggregation. These also suggest that platelet aggregation is not an essential step during blood circulation of tumor cells for the completion of metastasis.


Induction of intercellular adhesion molecule 1 on small cell lung carcinoma cell lines by gamma-interferon enhances spontaneous and bispecific anti-CD3 x antitumor antibody-directed lymphokine activated killer cell cytotoxicity. The interaction between LFA-1 and its natural ligand, ICAM-1, plays an important role in leukocyte adhesion and signal transduction. LFA-1-mediated T-cell adhesion is generally activated by CD3-mediated signal in association with T-cell receptor-mediated recognition of the antigen/major histocompatibility complex on antigen-presenting cells. In the present study, we compared spontaneous or bispecific antibody (BsAb)-directed LAK cell cytotoxicity against ICAM-1+ or ICAM-1- small cell lung cancer (SCLC) cell lines. gamma-Interferon (IFN-gamma)-induced ICAM-1 expression on ICAM-1- SCLC cell lines, and susceptibility to LAK cells was increased simultaneously. Increased cytolysis of the IFN-gamma-treated SCLC was inhibited by an anti-ICAM-1 monoclonal antibody (mAb). Furthermore, LAK cell cytotoxicity directed by BsAb, which was composed of OKT3 and anti-SCLC mAb, was also increased by the IFN-gamma treatment of SCLC, and this increase was inhibited by an anti-ICAM-1 mAb but not by anti-Class I or anti-CD2 mAb. These results suggest that a prior administration of IFN-gamma would enhance the efficacy of the following specific targeting therapy utilizing BsAb and LAK cells by up-regulating the ICAM-1 expression on tumor target cells. The combinational use of IFN-gamma and anti-CD3 x anti-tumor BsAb might be a promising way of enhancing LAK cell-mediated adoptive immunotherapy in small cell lung cancer patients.


A new tumor-associated antigen useful for serodiagnosis of hepatocellular carcinoma, defined by monoclonal antibody KM-2. After immunization of mice with the human hepatocellular carcinoma (HCC) cell line PLC/PRF/5, we produced monoclonal antibody KM-2, which allowed us to characterize a new HCC-associated antigen (KM-2 antigen) and to develop a sandwich-type radioimmunoassay. The KM-2 antigen was strongly expressed on the cell surface of HCC cell lines. Immunofluorescence staining of frozen sections of different tissues and tumors confirmed its specific expression on the cell surface of a group of HCC. The antigen was also detected in the bile canaliculi of normal liver. Its biochemical characterization revealed a high molecular weight (M(r) approximately 900,000) glycoprotein with an N-linked carbohydrate chain close to the peptide epitope recognized by the KM-2 monoclonal antibody. By the radioimmunoassay for the KM-2 antigen, the antigen was detected in sera of 72 (47%) of 154 patients with HCC and 3 (3%) of 102 patients with liver cirrhosis; it was not detected in 96 patients with chronic hepatitis or in 100 healthy control individuals. The positive rate of KM-2 antigen (72 of 154, 47%) was significantly (P less than 0.01) higher than that (51 of 154, 33%) of alpha-fetoprotein (AFP) when the cut-off level of AFP was taken as the widely accepted 400 ng/ml. No significant correlation was recognized between serum levels of the KM-2 antigen and AFP (r = 0.15; P greater than 0.05). In addition, among 103 patients with HCC whose AFP levels were less than 400 ng/ml, 31 (30%) were positive for the KM-2 antigen. Determination of the serum KM-2 antigen would be useful for the serodiagnosis of patients with HCC, particularly in cases with normal or low AFP levels.


Acidic fibroblast growth factor-Pseudomonas exotoxin chimeric protein elicits antiangiogenic effects on endothelial cells. It has recently been shown that chimeric toxins composed of acidic fibroblast growth factor fused to mutant forms of Pseudomonas exotoxin (aFGF-PE) are cytotoxic to a variety of tumor cell lines with FGF receptors. Although aFGF-PE might be considered as a possible chemotherapeutic toxin, limited knowledge is available concerning its effect on endothelia. This study investigates whether one of the aFGF-PE fusion proteins, aFGF-PE664GluKDEL, can function as an anti-angiogenic agent. Protein synthesis studies using rat epididymal fat pad microvascular endothelial cells (RFCs) indicated that after 24 h in culture, aFGF-PE had a significant inhibitory effect on protein synthesis at concentrations greater than 100 ng/ml. In cultures incubated with 1000 ng/ml aFGF-PE, RFC protein synthesis was inhibited as much as 83%. RFCs were also cultured in a 3-dimensional type I collagen gel and incubated with either transforming growth factor beta 1, aFGF-PE, or a combination of both. Transforming growth factor beta 1 elicits in vitro angiogenesis in these 3-dimensional cultures which consist of rapid formation of complex tubular structures. Transforming growth factor beta 1-treated RFCs incubated with aFGF-PE were unable to produce this angiogenic response, nor were they able to migrate out of the 3-dimensional culture to form a monolayer as shown by controls. Cell viability analyses showed that aFGF-PE produced a dose-dependent toxic effect which ranged from 10 to 90% cell death. Competition assays in which the chimeric toxin was preincubated with antibodies to aFGF resulted in an 89% reversal of the inhibitory effects of aFGF-PE on endothelial cells. Acidic FGF-PE with a mutation in the ADP ribosylation domain of PE was inactive in both 2-dimensional and 3-dimensional cultures. These data show that aFGF-PE has specific in vitro cytotoxic, antiangiogenic, and antimigratory effects on microvascular endothelia.


Inhibition of angiogenesis by suramin.

Inhibition of angiogenesis by suramin. In this study, we have determined the ability of suramin to inhibit angiogenesis in the chick chorioallantoic membrane. Suramin alone showed significant angiostatic activity in a dose-related manner. Suramin also potentiated the activity of the angiostatic steroids, cortisol-21-phosphate, 17 alpha-hydroxyprogesterone, tetrahydrocortisol, and tetrahydrocortexolone. The presence of heparin decreased the angiostatic activity of suramin. These results suggest that suramin may decrease tumor growth by inhibiting angiogenesis. These novel findings indicate that suramin, perhaps in combination with angiostatic steroids, may be the basis for important new therapeutic approaches for diseases of neovascularization.


IGFBP-3 gene expression and estrogen receptor status in human breast carcinoma. Insulin-like growth factors (IGF) I and II are potent mitogens for breast carcinoma proliferation. IGF-mediated proliferative activity can be markedly enhanced by the presence of specific IGF-binding proteins (IGFBPs). IGFBP-3 has been shown to enhance IGF-mediated growth in a number of systems. Studies have demonstrated IGFBP-3 secretion only in estrogen receptor (ER)-negative breast carcinoma cell lines while IGFBP-3 could not be detected in media conditioned by ER-positive cell lines. We investigated whether a relationship exists between ER status and IGFBP-3 mRNA expression in human breast carcinoma biopsy specimens. We have detected IGFBP-3 mRNA in breast carcinoma tissue obtained from patients utilizing in situ hybridization. Quantitation of IGFBP-3 mRNA levels was performed utilizing image cytometry. There was a significantly higher expression of IGFBP-3 mRNA in ER-negative breast carcinoma specimens when compared to the ER-positive specimens. Whether this higher expression of IGFBP-3 mRNA and presumed secretion of IGFBP-3 by ER-negative tumors play a role in the rapid proliferation and poor prognosis of these tumors remains to be determined.


The use of porcine liver (2----3)-alpha-sialyltransferase in the large-scale synthesis of alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc, the epitope of the tumor-associated carbohydrate antigen CA 50. alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-D-GlcpNAc (2) and, alpha-Neup5Ac-(2----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn+ ++ were prepared on a large scale by the action of beta-D-Galp-(1----3)-D-GalpNAc (2----3)-alpha-sialyltransferase (partially purified from porcine liver) on beta-D-Galp-(1----3)-D-GlcpNAc and beta-D-Galp-(1----3)-beta-D-GlcpNAcOMBn, respectively. The trisaccharide 2 is the epitope of the tumor-associated carbohydrate antigen CA 50, highly expressed in human pancreatic adenocarcinoma.


Further definition of the size of the blood group-i antigenic determinant using a chemically synthesised octasaccharide of poly-N-acetyllactosamine type. In earlier studies, the minimum structure which inhibited the binding of anti-i to an i-active glycoprotein was the linear trisaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-D-Gal. There was an increasing hierarchy of inhibitory activities in the linear tetrasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D -GlcNAc , its methyl beta-glycoside, and in the methyl beta-glycoside of the hexasaccharide. The linear octasaccharide methyl beta-glycoside in this series is approximately only half as active as the hexasaccharide methyl beta-glycoside. Analyses by high resolution 1H-n.m.r. of these two oligosaccharides indicated that they have similar conformations in solution, and there is no evidence for the occurrence of inter-molecular interactions which might partially hinder the binding of anti-i to the octasaccharide methyl beta-glycoside. These results are consistent with the size of the i antigen being in the region of a hexasaccharide. It is proposed that the methyl aglycon group of the hexasaccharide methyl beta-glycoside confers an above normal activity by presenting a hydrophobic area for additional contact in the vicinity of the antibody-combining site.


Synthesis of allyl 6-O-(3-deoxy-alpha- and -beta-D-manno-oct-2- ulopyranosylonic acid)-(1----6)-2-deoxy-2-[(3R)-3-hydroxytetradecanamido]- beta-D-glucopyranoside 4-phosphate and of the copolymer of the alpha anomer with acrylamide. The title disaccharides were synthesized by mercuric cyanide-catalyzed condensation of methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-oct-2-ulopyranosyl bromide)onate and allyl 2-[(3R)-3-acetoxy-tetradecanamido]-3-O-benzyl-2-deoxy-beta-D- glucopyranoside, followed by phosphorylation of the alcoholic function of the amino sugar. The phosphorylated anomers were separated by chromatography and deprotected by conventional methods. Polymeric material was obtained by copolymerisation, catalyzed by peroxosulphate and N,N,N',N'-tetramethylethylenediamine, of the alpha anomer with acrylamide; it contained ca. one disaccharide unit for 18 acrylamide residues.


Stereoselective syntheses of a di-, tri-, and tetra-saccharide fragment of Shigella dysenteriae type 1 O-antigen using 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride as a glycosyl donor. Methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (1) furnished a crystalline 3-O-bromoacetyl derivative that was treated with the dichloromethyl methyl ether-ZnCl2 reagent to give 2,4-di-O-benzoyl-3-O-bromoacetyl-alpha-L-rhamnopyranosyl chloride (3). Compounds 1 and 3 were condensed under the conditions of base-deficient, silver trifluoromethanesulfonate-mediated glycosylation to give a fully protected rhamnobioside, which on O-debromoacetylation afforded the disaccharide nucleophile 10. Similar condensation of 3 with methyl 3-O-benzoyl-4,6-O-benzylidene-alpha-D-galactopyranoside, followed by O-debromoacetylation and condensation of the thus formed methyl O-(2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl)-(1----2)-4,6-O-benzylidene- 3-O-benzoyl-alpha-D-galactopyranoside again with 3, gave the trisaccharide glycoside. Subsequent O-debromoacetylation gave 17, having only HO-3(3) unsubstituted. Silver perchlorate-mediated glycosylations of 1, 10, and 17 with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride afforded, with high alpha stereoselectivity, protected di-, tri-, and tetra-saccharide glycosides. Subsequent hydrogenation, followed by N-acetylation and O-deacylation, afforded three oligosaccharide glycosides having nonreducing terminal 2-acetamido-2-deoxy-alpha-D-glucopyranosyl residues and comprising successively larger portions of the repeating unit of Shigella dysenteriae type 1 O-antigen.


Protein synthesis in pulmonary, cardiac, and skeletal muscle in acute hypertension induced by aortic constriction in the rat. The aim was to investigate nucleic acid composition and rates of protein synthesis in cardiopulmonary tissues and skeletal muscle in response to hypertension induced by aortic constriction. After five days of abdominal aortic constriction, protein, RNA, and DNA contents were measured in the lung, the left and right atria, the left and right ventricles, and gastrocnemius muscle from young male Wistar rats weighing 120-140 g. Rates of protein synthesis were also measured in these tissues with L[4-3H]phenylalanine. Aortic constriction significantly increased the right atrial weight and in contrast reduced the lung weight, compared to pair fed and sham operated controls. The wet weights of all other tissues were unaffected. The concentrations of right atrial proteins, RNA, and DNA were also significantly reduced though total protein, RNA, and DNA contents were unaltered. The left ventricular RNA concentration increased and there were variable alterations in protein and DNA composition. The protein, RNA, and DNA compositions of the other tissues showed patterned responses, which included reductions in lung and skeletal muscle DNA concentrations, reductions in the skeletal muscle RNA/DNA ratio, and a decrease in the lung protein/DNA ratio. In response to aortic constriction there were increases in the left ventricular fractional rate of protein synthesis in mixed, high salt (myofibrillar), and low salt (sarcoplasmic) fractions. Rates of protein synthesis in all other regions of the heart, lung and skeletal muscle were not significantly changed. We conclude that in abdominal aortic constriction, the left ventricles display early adaptive responses without any concomitant change in mass. Those regions of the rat cardiopulmonary system which are not directly exposed to the acute pressure overloading, ie, right atrium, lungs, and skeletal muscle, also show disturbances.


New reactions catalyzed by a group II intron ribozyme with RNA and DNA substrates. Here we describe three novel reactions of the self-splicing group II intron bI1 (the first intron of the COB gene of yeast mitochondria) demonstrating its catalytic versatility: reversal of the first step of the self-splicing reaction catalyzed by a linear form of the intron utilizing the energy of a phosphoanhydride bond for transesterification, ligation of a single-stranded DNA to an RNA, and cleavage of a single-stranded DNA substrate. These results have the following evolutionary implications: use of the alpha-beta bond of a terminal triphosphate for transesterification suggests that an RNA RNA replicase could use mononucleotide triphosphates as precursors, and cleavage of single-stranded DNA and DNA-RNA ligation suggests that excised group II introns might integrate directly into DNA without prior reverse transcription.


The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes. Epidermolytic hyperkeratosis (EH) is a skin disease characterized by keratin filament clumping and degeneration in terminally differentiating epidermal cells. We have discovered that the genetic basis for EH resides in mutations in differentiation-specific keratins. Two of six distinct incidences of EH had a keratin 10 (K10) point mutation in a highly conserved arginine. Remarkably, this same residue is mutated in the basal epidermal K14 in three incidences of another skin disease, epidermolysis bullosa simplex (EBS). By genetic engineering, gene transfection, and 10 nm filament assembly, we show that this mutation is functionally responsible for the keratin filament clumping that occurs in basal (EBS) or suprabasal (EH) cells. These studies strengthen the link between filament perturbations, cell fragility, and degeneration first established with EBS. They also suggest a correlation between filament disorganization and either cytokinesis or nuclear shape, giving rise to the seemingly binucleate cells typical of EH.


A leucine----proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Epidermolytic hyperkeratosis is an autosomal dominant disorder affecting the structural integrity of the suprabasal layers of human epidermis. We have recently documented in one family linkage of the disease phenotype to the cluster of type II keratins. We have now identified a leucine----proline amino acid substitution in the conserved H1 subdomain of keratin 1 that is present only in affected family members. Using a quantitative assay and electron microscopy with synthetic peptides, we show that, whereas the wild-type H1 peptide rapidly disassembles preformed keratin filaments in vitro, the mutant peptide does this far less efficiently. Therefore the mutation in keratin 1 is likely to cause defective keratin filaments and hence a defective cytoskeleton in the epidermal cells in vivo.


Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Steel factor (SF) and LIF (leukemia inhibitory factor) synergistically promote the proliferation and survival of mouse primordial germ cells (PGCs), but only for a limited time period in culture. We show here that addition of bFGF to cultures in the presence of membrane-associated SF and LIF enhances the growth of PGCs and allows their continued proliferation beyond the time when they normally stop dividing in vivo. They form colonies of densely packed, alkaline phosphatase-positive, SSEA-1-positive cells resembling undifferentiated embryonic stem (ES) cells in morphology. These cultures can be maintained on feeder layers for at least 20 passages, and under appropriate conditions give rise to embryoid bodies and to multiple differentiated cell phenotypes in monolayer culture and in tumors in nude mice. PGC-derived ES cells can also contribute to chimeras when injected into host blastocysts. The long-term culture of PGCs and their reprogramming to pluripotential ES cells has important implications for germ cell biology and the induction of teratocarcinomas.


An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes. Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.


Expression of cystic fibrosis transmembrane regulator Cl- channels in heart. Cyclic AMP (cAMP)-dependent chloride channels modulate changes in resting membrane potential and action potential duration in response to autonomic stimulation in heart. A growing body of evidence suggests that there are marked similarities in the properties of the cAMP-dependent chloride channels in heart and cystic fibrosis transmembrane regulator (CFTR) chloride channels found in airway epithelia or in cells expressing the CFTR gene product. We isolated poly A+ mRNA from rabbit ventricle and converted it to cDNA for amplification using the polymerase chain reaction (PCR). A fragment corresponding to the nucleotide-binding domain 1 (NBD1) of the CFTR transcript was cloned. Comparison of the amino acid sequence of NBD1 of human CFTR with the deduced sequence of the rabbit heart PCR product indicated 98% identity. Northern blot analysis, using the heart amplification product as a cDNA probe, demonstrated expression of homologous transcripts in human atrium, guinea pig and rabbit ventricle, and dog pancreas. Xenopus oocytes injected with poly A+ mRNA extracted from rabbit and guinea pig ventricle or dog pancreas expressed robust time-independent chloride currents in response to an elevation of cAMP. We conclude that CFTR chloride channels are expressed in heart and are responsible for the observed cAMP-dependent chloride conductance.


Thrombin receptor 14-amino acid peptide mediates endothelial hyperadhesivity and neutrophil adhesion by P-selectin-dependent mechanism. Thrombin cleaves its receptor at arginine-41, resulting in the generation of a new receptor NH2-terminus with the sequence SFLLRNPNDKYEPF. This peptide (TRP-14) may signal a variety of thrombin's responses. We examined the effects of TRP-14 in inducing endothelial cell hyperadhesivity and neutrophil (PMN) adhesion to endothelial cell monolayers. Human umbilical vein endothelial cells (HUVECs) challenged with TRP-14 (10(-4) to 10(-5) M) produced concentration-dependent increases in endothelial adhesivity to PMN. In contrast, position 1 to 2 inverted peptide (FSLLRNPNDKYEPF) did not induce the response. The adhesion response was transient; that is, PMN adhesion increased within 15 minutes and decreased by 75 minutes after TRP-14 challenge of HUVECs. The transient endothelial adhesiveness paralleled the time course of P-selectin expression. TRP-14-induced release of P-selectin from intracellular stores may be a critical determinant of the response since treatment of endothelial cells with anti-P-selectin monoclonal antibody (mAb) G1 prevented the increase in PMN adhesion. Control nonneutralizing anti-P-selectin mAb S12 and mAb RR1/1 directed against intercellular adhesion molecule-1 (ICAM-1) on HUVECs were ineffective. The results indicate that the "tethered ligand" of the thrombin receptor created by the proteolytic action of thrombin on its receptor (i.e., TRP-14) signals increased endothelial adhesiveness by a P-selectin-dependent mechanism. Thrombin-induced PMN adhesion may involve formation of a new NH2-terminus of the endothelial thrombin receptor with the sequence SFLLRNPNDKYEPF followed by activation of endothelial second messenger pathways and the transient expression of P-selectin.


Ionic currents in single smooth muscle cells of the canine renal artery. Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)


Decreased collagen gene expression and absence of fibrosis in thyroid hormone-induced myocardial hypertrophy. Response of cardiac fibroblasts to thyroid hormone in vitro. The regulatory effects of thyroid hormone on biosynthesis of myocardial proteins that originate from cardiac myocytes are well established. Little is known, however, of regulatory effects of thyroid hormone on interstitial proteins. In this study we examined the effects of thyroid hormone on collagen gene expression in thyroid hormone-induced myocardial hypertrophy and the response of cardiac fibroblasts to thyroid hormone in culture. Adult male Sprague-Dawley rats were treated intraperitoneally with L-thyroxin (10 micrograms/100 g body wt) for 2 hours or 1, 2, 3, 6, 12, or 14 days. Northern blot analysis of RNA from total ventricular tissue showed that after 2 hours of treatment, the abundance of mRNA for pro alpha 2(I) collagen decreased by 53% (p less than 0.05) and reached the lowest level (60% decrease, p less than 0.02) at day 1, remained diminished at day 3, and then gradually returned toward normal levels. After transient transfection of chimeric DNA containing collagen type I promoter-chloramphenicol acetyl transferase (CAT) gene into the thyroxin-treated cardiac fibroblasts, the level of CAT activity decreased significantly. Treatment of cardiac fibroblasts in culture (10 nM L-thyroxin) resulted in a 33% (p less than 0.005) decrease in the abundance of mRNA for pro alpha 2(I) collagen. The stability of the mRNA for pro alpha 2(I) collagen in cardiac fibroblasts, as measured by mRNA half-life, was slightly (16.6%) decreased by thyroid-hormone treatment. Collagen synthesis as shown by immunofluorescent staining of intracellular collagen in cultured fibroblasts and in frozen sections of myocardium was also diminished.(ABSTRACT TRUNCATED AT 250 WORDS)


Epicardial excitation during ventricular pacing. Relative independence of breakthrough sites from excitation sequence in canine right ventricle. In a previous investigation, epicardial recordings with 1,124 closely spaced electrodes revealed 20-35 breakthrough (BKT) sites and an equal number of separate wave fronts on the ventricular surface of exposed dog hearts during normal sinus rhythm. In the present study we tried 1) to determine whether ventricular pacing also produced multiple BKTs and wave fronts and 2) to determine whether the number and location of BKTs were, to some degree, independent of pacing site. The study mainly focused on right ventricular BKTs observed during right ventricular pacing. To test hypotheses 1 and 2 we identified many breakthrough sites during sinus rhythm in seven exposed dog hearts and then paced the heart from several BKT and non-BKT sites on the right ventricle. Epicardial potential maps and excitation time maps were obtained by using 1,124 epicardial electrodes covering the anterior right ventricle and part of the anterior left ventricle. A primary wave front spread radially for several centimeters from the pacing site, and no BKTs appeared in the areas covered by the primary wave front. In the remaining areas (secondary areas), multiple BKTs appeared; their number was close to that observed during sinus rhythm in the same areas (113 versus 115, respectively, in 12 paced beats). The majority of paced BKTs (83 out of 115, or 72%) occurred exactly at the same locations where they appeared during sinus rhythm. However, 30 right ventricular BKTs observed during sinus rhythm disappeared in the secondary areas and were replaced by approximately the same number of new BKTs. Many areas without BKTs in normal beats remained so in paced beats.(ABSTRACT TRUNCATED AT 250 WORDS)


Electrophysiological effects of myocardial stretch and mechanical determinants of stretch-activated arrhythmias. Although the existence of myocardial mechanoelectrical feedback is well established, the mechanism of arrhythmia induction by ventricular dilatation or stretch remains insufficiently defined. In particular, controversy exists when comparing the arrhythmogenic potential of chronic versus acute myocardial stretch. Also, assessment of cellular electrophysiological effects of myocardial stretch has been incomplete. To evaluate the electrophysiological and arrhythmogenic effects of slow versus rapid ventricular wall stretch, we developed an isolated Langendorff-perfused rabbit heart model in which left ventricular (LV) volume can be changed by a computer-controlled servopump. Cellular electrophysiological effects and premature ventricular excitations (PVEs) and their origin produced by the volume increases were assessed by a multiple-site monophasic action potential (MAP) recording system and by volume-conducted ECGs obtained by immersing the entire preparation in a saline-filled tank. Volume was increased either gradually with slow volume ramps (0.1 ml/sec) or suddenly by volume pulses of varying pulse waveforms (three different amplitudes and five different rise velocities) applied randomly 250-350 times to each of eight hearts. Gradual LV volume loading caused gradual decreases in MAP resting and action potential amplitude, whereas rapid, transient volume pulses caused transient depolarizations. Despite similar membrane potential effects of stretch, gradual volume increases rarely (11%) produced PVEs, even with large volume loads, whereas rapid volume pulses of moderate amplitudes regularly triggered PVEs (45-100% of interventions). Logistic regression analysis showed that the probability of PVE occurrence increased independently with both the amplitude and the velocity of the volume increase, with the greatest sensitivity to stretch velocity exhibited at low and intermediate pulse amplitudes. Faster volume pulse rise velocities triggered PVEs at a lower instantaneous pulse amplitude than lower rise velocities, further corroborating the dependence of stretch-activated arrhythmias on the velocity of stretch. In contrast, an increase in the basic ventricular volume had no effect on the probability of PVE occurrence during the volume pulses. The MAP recordings demonstrated spatial variability in the extent of local depolarizations and site of PVE origin; transient depolarizations occurred, and PVEs originated most often in the posterolateral region of the left ventricle. Membrane depolarization is caused by both gradual and rapid ventricular stretch, but PVEs are more easily elicited by rapid stretch. Regions of greater myocardial compliance that experience greater relative stretch may act as "foci" for stretch-activated arrhythmias during dynamic ventricular loading. These whole-heart data corroborate the existence of stretch-activated membrane channels in ventricular myocardium and may help explain ventricular ectopy under conditions of differential ventricular loading, as in ventricular dyskinesia, or regional muscle traction, as in mitral valve prolapse syndrome.


Protective effects of cyclophosphamide, cyclosporin A and FK506 against antigen-induced lung eosinophilia in guinea-pigs. A close association has been recognized between activated T cells and eosinophils in asthma, albeit circumstantial. The present study attempted to investigate this relationship in an animal model of lung eosinophilia using the new generation of T cell-selective immunosuppressants, cyclosporin A and FK506, compared with the myelotoxic immunosuppressive agent cyclophosphamide. Antigen challenge of ovalbumin-sensitized guinea-pigs resulted in a lung eosinophilia which was assessed by bronchoalveolar lavage. All three agents caused a marked suppression of lung eosinophilia at 24 h post-challenge when the compounds were administered at the time of sensitization but not when administered for 3 days before lavage. However, the lung eosinophilia at 72 h post-challenge was reduced significantly by FK506 and by cyclophosphamide, but not by cyclosporin A, when the drugs were administered for 3 days, before lavage. These results strongly suggest the involvement of T cells in antigen-induced late phase (72 h) eosinophilia in guinea-pigs but not at 24 h. The effects of cyclophosphamide were always associated with a reduction in circulating white cell counts, whereas cyclosporin A and FK506 showed no myelotoxic properties. These results suggest the potential therapeutic use of selective, non-cytotoxic immunosuppressive agents in asthma.


Increased LAK activity against HIV-infected cell lines in HIV-1+ individuals. The role of natural killer (NK) cells and their inducible counterparts, lymphokine-activated killer (LAK) cells in AIDS with regard to HIV-1 viral immunosurveillance and the control of secondary opportunistic disease has yet to be established. In this study, we have demonstrated that LAK cells derived from all HIV-1+ groups showed striking increases in their capacity to lyse HIV-1-infected U-937 cells relative to their uninfected U-937 counterparts. Surprisingly, similarly derived LAK cells from healthy seronegative controls showed no differences in their lysis of HIV-1-infected versus uninfected U-937 cells. The differential ability of LAK effectors from seropositive donors to lyse HIV-1-infected targets was demonstrable using a number of U-937 subclones and their HIV-1-infected counterparts. Again, no differences in LAK cell-mediated lysis of HIV-1-infected and uninfected U-937 subclones were observed in seronegative individuals. Our findings that HIV-1+ individuals show selective expansion of non-MHC restricted, HIV-1-directed cytotoxic LAK cells indicate that natural immunity may indeed play a role in HIV-1 viral immunosurveillance.


Characterization of an anti-idiotypic MoAb bearing an internal image of the receptor-binding epitope of cholera toxin. A mouse anti-cholera toxin (CT) MoAb, mAb1, specific for the GM1-binding epitope of CT, was used to raise a syngenic anti-idiotypic MoAb, mAb2. Purified mAb2 was specific for mAb1 as shown by latex particle counting immunoassay and ELISA. Several experiments of competition between mAb2 and CT for binding to mAb1 demonstrated that mAb2 bore an internal image of the GM1-binding epitope of CT. Binding of mAb2 to GM1 unambiguously corroborated the mAb1-paratopic specificity of mAb2. Furthermore, mAb2 acted as a CT-surrogate antigen: rabbits injected with mAb2 produced some anti-CT antibodies, Ab3, which resembled mAb1 in specificity as expected. The potential use of this mAb2 as vaccine or as prophylactic agent to prevent CT from binding to its cellular receptor is discussed.


Characterization of B cell epitopes on the 16K antigen of Mycobacterium tuberculosis. To characterize the antigenic parts of the 16K protein of Mycobacterium tuberculosis, overlapping peptides according to the amino acid sequence of the 16K protein were synthesized. In total, 14 peptides of 20 amino acids in length with an overlap of 10 amino acids and two additional decapeptides (amino acids 31-40 and 61-70) were tested with eight anti-16K MoAbs and human sera. The common recognition site of MoAbs F67-8 and F67-16 was LRPTFDTRLM (amino acids 31-40) and of MoAbs F159-1 and F159-11 DPDKDVDIMV (amino acids 61-70). However, for binding of the MoAbs to these peptides additional amino acids were required at either the N- or C-terminus, suggesting that some kind of conformation is required. The recognition sites of the MoAbs F23-41, F23-49, F24-2 and TB68 could not be identified using the peptides, indicating that the MoAbs only bound to conformational epitopes and not to peptides which may contain parts of these epitopes. The MoAbs bound to beta-galactosidase fusion proteins comprising parts of the 16K protein, indicating that some kind of native conformation is present on the recombinant proteins. Sera from 14 of 19 patients with tuberculosis and none from 19 controls reacted with the purified 16K protein. Sera from four of these 14 patients reacted with two overlapping peptides (amino acids 71-100). Apparently, antibodies in patients' sera against the 16K protein are predominantly directed against conformational epitopes.


Cells with natural killer activity in human rabies. Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P greater than 0.05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P greater than 0.05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-alpha) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown.


Hepatitis C: a possible etiology for cryoglobulinaemia type II. Out of 15 successive patients with mixed essential cryoglobulinaemia type II (monoclonal IgM kappa/IgG), 13 had serological evidence for hepatitis C infection as shown by specific enzyme immunoassays and immunoblot. RNA was purified from the serum of seven patients and hepatitis C sequences were identified in five following reverse transcription and DNA amplification. The liver histology showed chronic active hepatitis with or without cirrhosis in the 12 patients with hepatitis C who had a liver biopsy. The two patients without serological evidence of hepatitis C suffered from haematological malignancies. Hepatitis C may be a major etiological agent of cryoglobulinaemia type II.


Induction of a pharmacologically active clonotypic B cell response directed to an immunogenic region of the human beta 2-adrenergic receptor. It has been reported that autoantibodies against the beta 2-adrenergic receptors are involved in the pathology of allergic disorders and of Chagas' disease. Therefore, the immune response against a peptide (H26Q) corresponding to the putative second extracellular loop of the human beta 2-adrenergic receptor, which could be a target for autoantibody attack, was analysed in view of its possible immunogenicity. The free peptide induced a T cell-mediated humoral response in the context of three different murine MHC haplotypes. The T cell epitope was found to be localized in the N-terminal region of the peptide. Highly specific T helper cells were capable of stimulating B cells with the potential to generate a large antibody repertoire reactive with the loop peptide. MoAbs were screened to analyse this B cell response for antibodies potentially interfering with receptor function and a MoAb was found that impaired ligand binding to the receptor.


Expression of CD56 (NKH-1) differentiation antigen in human thyroid epithelium. Leu-19 (CD56) MoAb is well known to recognize gp220 expressed on natural killer (NK) cells and is widely used as a NK cell marker. The expression of CD56 antigen was tested by means of sensitive alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemical technique and the above mentioned MoAb as a primary antibody, on frozen sections of various fresh human tissues. Out of 11 organs examined only thyroid gland provided a distinct reaction confined to cell membranes of epithelial follicular cells. The reaction had a diffuse pattern in cases of Graves' disease and colloidal goitre while in Hashimoto's thyroiditis presented as a focal pattern. Other anti-NK cell MoAbs such as VD4 (CD16) and Leu-7 (CD57) reacted only with single cells of thyroid stroma. The results of APAAP staining were confirmed by the cytofluorimetric assessment of isolated thyroid cells. It is speculated that CD56 expression on thyroid cells may have a functional significance, perhaps related to neural-endocrine interactions.


The prognostic significance of very low frequency ventricular ectopic activity in survivors of acute myocardial infarction. BHAT Study Group. Survivors of myocardial infarction with less than 2 PVC/h on 24-h ambulatory electrocardiography were followed up for an average of 25 months (11 to 40 months) while receiving a placebo (1,222 patients) or propranolol, 180 or 240 mg/day (1,234 patients). Three quarters of the participants with PVCs had an average of less than 2 PVC/h. Only 16 percent did not have any ventricular ectopic activity during the 24 h. Analysis of total mortality according to the number of premature ventricular complexes per hour showed that patients who had PVCs with a very low frequency (less than 0.5/h) had 49 percent higher mortality than patients who did not have any PVC. Patients who had greater than 0.5 PVC/h but less than 1 PVC/h had a statistically significant higher mortality rate, 11.7 vs 4.1 percent (p less than 0.0001) than patients who had no PVC. These data indicate that low ventricular ectopic activity frequency is associated with increased mortality in survivors of acute myocardial infarction.


The thermal transition in crude myelin proteolipid has a lipid rather than protein origin. Myelin proteolipid has been isolated from bovine brain and purified using organic solvents according to conventional procedures. The protein content of the purified sample, or crude proteolipid, contains a minimum of 75% w/w of proteolipid, with DM-20, a proteolipid molecule with an internal deletion of 35 out of 276 amino acid residues, as the only other component. Biochemical analysis has shown the differences in lipid composition between brain white matter, myelin and crude proteolipid preparations. The latter contained practically no cholesterol, while the other two samples had about 22-23% w/w. High-sensitivity differential scanning calorimetry experiments with both crude proteolipid and its extracted pool of lipids have shown similar reversible thermal transitions at 52 degrees C and 48 degrees C. The effect of increasing amounts of cholesterol on the two calorimetric transitions led in both cases to a continuous decrease in the melting temperature and in the transition enthalpy. Parallel Fourier-transform infrared spectroscopy studies of crude proteolipid have detected a reversible, co-operative lipid transition centred at 49 degrees C, with no detectable change in the amide region between 20 degrees C and 60 degrees C. Once more an increase in cholesterol content led to a decrease in the sharpness of this transition. It is concluded that the thermal transition detected in crude proteolipid, which has in the past been attributed to proteolipid thermal denaturation (Mateo et al. 1986), actually corresponds to a thermotropic phase transition of the lipids included in the crude proteolipid sample.


The inhibition of rat adrenal cytochrome P-45011 beta gene expression by androgens. The synthetic androgen methylandrostenediol (MAD) and the naturally occurring one, testosterone, both bring about hypertensive cardiovascular disease when chronically administered to rats. The pathogenesis of this form of experimental hypertension is thought to result from inhibition of steroid 11 beta-hydroxylase activity. In contrast to the above androgens, 19-nortestosterone, androstenedione and dehydroepiandrosterone (DHEA) have been reported to be without effect in elevating blood pressure. To examine the mechanism(s) involved, we have in this study compared the effects of a number of androgens on adrenal cytochrome P- 45011 beta enzyme and mRNA steady state levels. These parameters were also correlated with the ability of adrenal mitochondria isolated from these groups to hydroxylate 11-deoxycorticosterone (DOC) to corticosterone and 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC). Rats treated for seven days with 10 mg per day of dihydrotestosterone, testosterone, 19-nortestosterone or MAD showed a profound decrease in cytochrome P-45011 beta mRNA levels (to less than 20% of controls). This was accompanied by similar changes in both the level and activity of the enzyme. Androstenedione and DHEA were less potent in effecting these changes. In addition, for MAD and testosterone we tested the dose dependence of these changes and found that increasing doses (0.1 mg to 10 mg per day) of either androgen caused progressive decreases in the parameters measured. To assess selectivity we also determined the steady state level of cytochrome P-450scc mRNA in rats treated with the various androgens. In contrast to what was found with cytochrome P-45011 beta, the mRNA transcript for cytochrome P-450scc was equal to or above control levels. We conclude that, in general, the extent of inhibition of cytochrome P-45011 beta enzyme and mRNA level by a given androgen correlates with its reported facility in producing hypertension in rats. Increased secretion of DOC continues to be a likely mechanism for the development of this hypertension but with some androgens extra-adrenal effects may be involved.


Altered patterns of T cell migration through lymph nodes and skin following antigen challenge. Antigen challenge has profound effects on a regional lymph node (LN); it leads to an increase in blood flow to the node, and a marked increase in lymphocyte output through the efferent lymphatics. We used the isolated LN model developed in the sheep to see if antigen challenge in a LN resembled inflammation in peripheral tissues. Following stimulation with an antigen (purified protein derivative of tuberculin), lymphocyte output from the LN showed the typical periods of "lymphocyte shutdown" and "recruitment". The shutdown phase, when cell numbers in efferent lymph dropped by approximately 80%, affected almost exclusively the naive-type (adhesionlo, L-selectin+) T cell population. The large increase in T cell traffic through the node during the recruitment phase was mostly due to CD4+ memory-type T cells and, moreover, the majority of these T cells were L-selectin-, indicating that these cells were crossing from the blood by a molecular mechanism other than L-selectin interaction with its ligand, the "lymph node vascular addressin" (MECA-79). Examination of LN high endothelial venules revealed the presence of vascular cell adhesion molecule-1 (VCAM-1), an endothelial adhesion molecule which has been reported to bind preferentially memory-type T cells in inflammatory lesions. Within the skin, antigen challenge also induced the rapid expression of VCAM-1 on vascular endothelium. It was purely memory-type T cells (beta 1+, L-selectin+/-) that collected in lymph draining from this tissue. However within chronically inflamed skin, the MECA-79 determinant appeared on vascular endothelium, and a small proportion of T cells draining from chronically inflamed skin were of naive-type. The present results illustrate that there are similarities in the cellular and molecular events that characterize antigen stimulation of a LN and inflammation in a peripheral tissue.


Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus-1-infected mothers. Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV-1-infected mothers. These CTL killed HLA-matched HIV-1-infected H9 target cells or doubly transfected P815-A2-env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA-A1/A3 or HLA-A2, respectively. HIV-1-specific CTL were detected early after birth (less than 2 months) and remained present during the asymptomatic phase of the infection. As in HIV-1-infected adults, HIV-specific CTL declined with disease progression. Surprisingly, HIV-1-specific CTL were detected in the PBMC of three children who subsequently became seronegative.


Production of 17.2.25 mu transgenic and endogenous immunoglobulin in X-linked immune deficient mice. In M54 mice transgenic for a completely rearranged mu(a) heavy chain there is a decrease in total B cells and the rearrangement of endogenous immunoglobulin genes is partially inhibited. Surprisingly, however, endogenous immunoglobulin gene rearrangement and significant heavy chain polypeptide production does occur. We tested the hypothesis that only CD5+ B cells produce endogenous immunoglobulin by taking advantage of the fact that X-linked immune deficient (xid) mice normally are deficient in CD5+ B cells. We found that the frequency of CD5+ splenic B cells was similar in XxidY transgenic and non-transgenic F1 males, and in XxidX transgenic and non-transgenic F1 females. In both XxidX and XxidY transgenic F1 mice some, but not all, splenic B cells are CD11b+. There was a striking deficit of splenic B cells expressing endogenous immunoglobulin in XxidY transgenic mice, although this was not true for peritoneal cells. Thus, the introduction of the 17.2.25 mu transgene does not prevent the development of CD5- B cells nor does it limit endogenous immunoglobulin gene arrangement and expression solely to CD5+ B cells. However, in mice capable of expressing B cell surface CD5 or CD11 this transgene can lead to expansion of the fraction of B cells positive for these molecules. We conclude that while the introduction of the 17.2.25 mu transgene alters the frequencies of B cell populations, maturation is not limited to one subpopulation.


The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. This study addressed the question of whether the mucosal adjuvant property of cholera toxin (CT) and the structurally closely related Escherichia coli heat-labile toxin (LT) requires the enterotoxic and adenylate cyclase/cAMP activating property of these molecules. Therefore, we investigated the cytotoxic and adjuvant abilities of the enterotoxins and compared the results with those obtained with the non-toxic CT and LT derivatives; recombinant CTB (rCTB) and a mutated LT (mLT), which had a single amino acid substitution in position 112 (Glu----Lys) of the A subunit. Detailed functional studies revealed that, in contrast to the enterotoxins, both rCTB and mLT lacked ADP-ribosylating and cAMP-stimulating abilities. However, similar membrane ganglioside GM1-receptor binding ability of all the putative adjuvants was demonstrated. When the probe antigen, keyhole limpet hemocyanin (KLH), was given perorally together with CT or LT strong gut mucosal anti-KLH immune responses were stimulated, whereas no or very low anti-KLH responses were seen in the groups which received antigen admixed with rCTB or the mLT. Moreover, the specific serum antibody responses to the various immunization protocols closely paralleled the local anti-KLH response in the gut. From these results it appears that the adjuvant mechanism of LT, and probably also of CT, is linked to the ability to ADP-ribosylate and to stimulate cAMP formation. However, this study does not unequivocally rule out other possibilities such as interactions by the A1 fragment of CT or LT with other G-proteins than Gs alpha or events that parallel or precede the effects on the adenylate cyclase/cAMP system. Thus, the levels of ADP-ribosylation and cAMP-induction that are required and the key event or target cell that is responsible for the adjuvant effect of CT and LT remain to be elucidated. Studies are underway to address these issues.


Mouse tumor rejection antigens P815A and P815B: two epitopes carried by a single peptide. Mouse mastocytoma P815 expresses several distinct tumor rejection antigens recognized by syngeneic cytolytic T lymphocytes (CTL). Two of these tumor rejection antigens, P815A and P815B, are encoded by gene P1A, the sequence of which was reported previously. Tumor cell variants having lost one or both of these antigens were isolated by in vitro selection with CTL and also by collecting tumor cells that progressed in vivo after escaping a nearly complete immune rejection process. The structure of gene P1A in these antigen-loss variants was examined. Several A-B- variants presented a complete or partial deletion of the gene. One variant that had lost only antigen A displayed a point mutation in the first exon. Peptides were synthesized that corresponded to the normal sequence located in the region of this point mutation. They sensitized target cells to both anti-A and anti-B CTL. The homologous peptide encoded by the mutated gene of the P815 A-B+ variant sensitized cells only to anti-B CTL. We conclude that anti-A and anti-B CTL recognize on the same peptide two distinct epitopes that are affected differently by the mutation.


Discrimination of human CD4 T cell clones based on their reactivity with antigen-presenting T cells. In this report, we describe the discrimination of human T cell clones based on their reactivity with activated T cells as antigen-presenting cells (APC). CD4+ T cell clones specific for peptide P30 of tetanus toxin (amino acids 947-967) and restricted to the DP4 molecule were established and tested for proliferation to peptide presented either by peripheral blood mononuclear cells (PBMC), Epstein-Barr virus (EBV)-transformed B cells or major histocompatibility complex (MHC) class II-expressing T cells. We found two sets of T cell clones: one set proliferated to peptide presentation by PBMC, EBV-transformed B cell lines (EBV-B cells) and MHC class II+ T cells (termed T-responder clones), while the other set of clones was only stimulated to proliferate, if the peptide was presented by PBMC or EBV-B cells, but not by T cells (T-nonresponder clones). Nevertheless, these T-nonresponder clones recognized P30 also on T cells, as revealed by Ca2+ influx. The discrimination of the clones was not due to different avidities of the T cell receptors (TcR) of individual clones for the MHC-peptide complex as T-responder and T-nonresponder clones had similar dose-response curves to P30 presented by fixed EBV-B cell lines. Addition of cytokines [interleukin (IL)-1, IL-2, IL-4 and interferon gamma] did not change the proliferative response of the clones, which was consistent throughout an observation period of greater than 4 months. T-nonresponder clones, exposed to P30 on MHC class II-expressing T cells, became not anergic, as they could be restimulated by P30 presented on EBV-B cells. The measurement of a panel of T cell activation markers and adhesion molecules on T-responder and T-nonresponder clones revealed a higher expression of the CD28 molecule on the T-nonresponder clones. The data suggest that freshly cloned T cells can be differentiated by peptide presentation on classical (PBMC, EBV-B cells) or non-classical APC (class II+ T cells), and that this discrimination is further underlined by different levels of adhesion molecules.


Enhancers of nonspecific immunity induce nitric oxide synthase: induction does not correlate with toxicity or adjuvancy. A range of bacterial products and related synthetic compounds, either alone or in combination, enhance nonspecific resistance to infection and tumors. These compounds, which vary in their other properties such as pyrogenicity, toxicity and adjuvancy, were used to assess the hypothesis that nonspecific resistance is mediated by induction of the L-arginine: nitric oxide (NO) pathway. The results obtained show that agents which stimulate nonspecific immunity, such as endotoxin, muramyl dipeptide (MDP) and combinations of monophosphoryl lipid A (MPL) with either trehalose dimycolate (TDM) or MDP cause a substantial induction of Ca(2+)-independent NO synthase in murine lung. In contrast, agents which do not stimulate nonspecific resistance, such as either MPL or TDM alone or threonyl MDP (ThrMDP), do not induce NO synthase. This difference in the ability of MDP and ThrMDP to induce NO synthase in the lung in vivo was also manifest in peritoneal macrophages in vivo as well as being evident in the greater than 100-fold greater potency of MDP in inducing the enzyme in vitro in lung slices. In contrast to the good correlation between induction of NO synthase and induction of nonspecific resistance, no correlation was observed with either the toxic effects of these agents or their adjuvancy.


Protective effect of anti-interleukin (IL)-6 antibody against endotoxin, associated with paradoxically increased IL-6 levels. Two rat monoclonal antibodies (6B4 and 20F3) against mouse interleukin (IL)-6 were studied for their effects on the generalized Shwartzman reaction and on cytokine production elicited by endotoxin injections. Both antibodies were found to protect mice against the generalized Shwartzman reaction. Production of interferon and tumor necrosis factor in these animals, as assessed from serum levels, were not consistently affected by the antibody treatment, although rather increased levels were occasionally noted. Paradoxically, however, endotoxin-induced serum levels of IL-6 in anti-IL-6-treated mice were consistently found to be markedly increased and also to persist for longer time periods. The more vigorous and persistent response may have been due to slower elimination, increased synthesis, or a combination of both. Endogenous production of IL-6 in mice may be sufficiently large to supersede the neutralizing potential of an excess of antibody, as was evident from the fact that ascites fluid of the anti-IL-6-producing 6B4 hybridoma was biologically active in the IL-6 assay.


Netwhat?

Netwhat? The hallmark of an antibody response is considered to be its specificity for the immunogen. Nonetheless, most antibody elicited by an immunogen has been reported to be unreactive with it. We have evaluated specificity by characterizing the primary antibody response of mice to heat-aggregated human gamma-globulin with four approaches: (a) quantification of antibody-producing cells; (b) hybridoma analysis; (c) in situ antigen binding and (d) analysis of secreted antibody. The results show that the nonspecific component of this response is negligible. These observations suggest that such a component is not a basic feature of the antibody response and it is discussed that nonantigen-specific antibody may arise from a variety of causes many of which are artifactual.


Actions of novel bombesin receptor antagonists on pancreatic secretion in rats. Recently synthesized highly specific and potent bombesin receptor antagonists permit study of the role of endogenous bombesin-like peptides in the physiological regulation of pancreatic secretion. We now tested the action of three novel pseudononapeptide bombesin/gastrin releasing peptide (GRP) antagonists (RC-3095, RC-3100 and RC-3120) on amylase release in vitro from isolated rat pancreatic acini and on protein secretion in vivo in chronic pancreatic fistula rats. In isolated pancreatic acini, all three bombesin receptor antagonists inhibited the amylase secretion induced by bombesin by shifting to the right the amylase response to bombesin without altering the maximal response. These antagonists alos reduced concentration dependently the near-maximal amylase response to bombesin, the concentration required for 50% reduction (IC50) being about 10(-7) M for RC-3095 and RC-3100 and 10(-6) M for RC-3120. None of the bombesin/GRP antagonists used affected the amylase response to CCK, pentagastrin or urecholine. In conscious rats with a chronic pancreatic fistula, all three bombesin antagonists shifted to the right the pancreatic protein response to graded doses of bombesin without changing the maximal response. These antagonists inhibited the protein response to constant background stimulation with bombesin in a dose-dependent manner, the ID50 being about 20 nmol/kg per h for RC-3095 and RC-3100 and about 160 nmol/kg per h for RC-3120. None of the antagonists significantly affected basal pancreatic secretion or secretion induced by sham-feeding, ordinary feeding or the diversion of pancreatic juice from the duodenum. These results indicate that exogenous bombesin is a potent direct stimulant of pancreatic enzyme secretion.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage in guinea pig airways. To study the effect of maturation on histamine-induced airflow obstruction and airway microvascular leakage, we measured concomitant changes in lung resistance (RL) and in extravasation of Evans Blue dye in the airways of anesthetized immature (aged 14 +/- 2 days) and adult guinea pigs (aged 60 +/- 12 days). RL was measured for 6 min after iv. histamine (0, 5, 15, 30 and 50 micrograms/kg). For comparison, responses after 1 microgram/kg substance P were also examined. After measurement of RL, microvascular leakage in trachea, main bronchi, and proximal and distal intrapulmonary airways was also examined in the same animal. Immature animals required a larger dose of histamine than adults to achieve a similar degree of maximal bronchoconstriction after histamine. In contrast, equal doses of histamine (15 and 30 micrograms/kg) induced a significantly greater extravasation of dye in immature airways in both proximal and distal intrapulmonary airways, although not in trachea or main bronchi. Substance P did not cause any age-related differences in dye extravasation at any airway level. These results suggest that i.v. histamine specifically causes a greater degree of airway microvascular leakage in peripheral airways but induces less smooth muscle contraction in the airways of immature guinea pigs than in the airways of adult animals.


Activity of peptide and non-peptide antagonists at peripheral NK1 receptors. We investigated the affinity of several tachykinin antagonists reportedly selective for NK1 receptors at various tachykinin receptors and NK2 receptors subtypes. The four antagonists tested were: L 668,169, Spantide II, Ac-Thr-DTrp(for)-Phe-NMeBzl (FR 113680) and the novel nonpeptide antagonist (+/-)-CP-96,345. The four antagonists were found to be effective against NK1 receptor-mediated responses in the guinea-pig ileum with the following rank order of potency (pKB values in parentheses): (+/-)-CP-96,345 (8.11) greater than Spantide II (7.08) greater than FR 113680 (6.61) greater than or equal to L 558,169 (6.44). (+/-)-CP-96,345, Spantide II and FR 113680 were distinctly more potent at NK1 receptors than at NK2 receptors (NK2A in the rabbit pulmonary artery, NK2B in the hamster trachea). L 668,169 antagonized neurokinin A-induced contractions in the hamster trachea with an affinity similar (pKB value 6.16) to that found in the guinea-pig ileum for NK1 receptors (pKB value 6.44). All antagonists were inactive at NK3 receptors of the rat portal vein. In a second series of experiments, the affinities of test antagonists for NK1 receptors in the guinea-pig ileum were compared to those for NK1 receptors in the guinea-pig vas deferens, the rabbit jugular vein and the rat urinary bladder. For each antagonist, the affinity measured in the guinea-pig vas deferens and the rabbit jugular vein was comparable to that found in the guinea-pig ileum. In the rat urinary bladder, (+/-)-CP-96,345 was about 100 times less potent in blocking NK1 receptor-mediated contractions than in the guinea-pig ileum.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of Chilean, Bolivian, and Argentinian Trypanosoma cruzi populations by restriction endonuclease and isoenzyme analysis. Ninety-one Chilean, 15 Bolivian, and 9 Argentinian Trypanosoma cruzi stocks, isolated from various hosts and vectors, were characterized by schizodeme analysis with EcoRI and MspI endonucleases. The three major similar pattern groups that emerged from this sample correlated with results of isoenzyme analysis. This result confirms previous work and supports the hypothesis of the clonal structure of natural populations of T. cruzi, fully defined at the level of isoenzyme analysis, quantitative kinetoplast DNA restriction fragment length polymorphism, and kinetoplast DNA hybridization analysis. In Chile, sylvatic and domestic cycles of T. cruzi transmission appear to be mainly independent: genetically different families of natural clones are specific to these cycles. Nevertheless, the possibility of overlap remains unclear. Results described here indicate that natural clones inhabiting Chilean regions appear genetically related to the natural clones identified in neighboring countries. In Chile the more frequently sampled parasite types are natural clone 39 and a genetically closely related clone NP13. In this work an evaluation of T. cruzi natural clone mixtures in T. cruzi stocks from Chile was performed for the first time by schizodeme analysis before and after serial transfer in mouse maintenance. The results indicate that six of nine stocks are composed of two or more natural clones. This observation raises the relevant question of whether specific T. cruzi natural clones generate different clinical features of Chagas' disease.


Brain factor from Galleria mellonella (Lepidoptera) stimulating silk gland activity. Brain extracts from day 1-4 last instar larvae of Galleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [3H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.


A marine algal Na(+)-activated ATPase possesses an immunologically identical epitope to Na+,K(+)-ATPase. Immunological homology was investigated between Heterosigma akashiwo (a marine algae) Na(+)-activated ATPase and animal Na+,K(+)-ATPase. The former polypeptide [(1989) Plant Cell Physiol. 30, 923-928] reacted with anti-serum raised against the amino-terminal half of the pig kidney Na+,K(+)-ATPase alpha subunit. It is suggested that the Na+,K(+)-ATPase epitope within the amino-terminal region is conserved in the plant Na(+)-activated ATPase, and the region containing the epitope may be important for Na ion transport.


Endothelial nitric oxide synthase is myristylated. The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which 'anchors' the enzyme into the membrane either directly or via another membrane-bound protein.


[Structure-function studies of galanin]. Galanin is widely distributed in the central and peripheral nervous system and exerts a variety of physiological effects. This review briefly describes the chemical structure, tissue distribution, physiological effects, receptors and structure-function relationships of galanin. It is worth noting that the inhibitory effect of newly synthesized galanin (1-15)-ol on guinea pig ileum contractions was of the same magnitude as that of galanin. This observation gives us an important clue as to the discovery of antagonists of galanin for neural systems.


Analysis of DNA encoding 23S rRNA and 16S-23S rRNA intergenic spacer regions from Plesiomonas shigelloides. Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.