Immunohistochemical localization of enkephalin and substance P in the nucleus caudalis of the spinal trigeminal V in the medulla oblongata of the human fetus. The distribution of enkephalin-positive neurons, substance P-positive and enkephalin-positive fibers was studied in the nucleus caudalis of the trigeminal spinal V in the medulla oblongata regions of developing humans (12 weeks gestation to 40 weeks gestation). Enkephalin-positive neurons were identified in all the subnuclei of the nucleus caudalis as early as 12 weeks of gestation and increased in number as the fetus aged. Substance P-positive neurons were absent in this area throughout development. On the other hand, substance P-positive and enkephalin-positive fibers were present in all the subnuclei, again commencing as early as 12 weeks of gestation. These fibers tended to be linked to each other in the different subnuclei and to the reticular formation in this area and to increase significantly in quantity by the latter quarter of pregnancy. These results show the early presence of these neurons and fibers in the first trimester of development.


Neonatal sciatic nerve section results in thiamine monophosphate but not substance P or calcitonin gene-related peptide depletion from the terminal field in the dorsal horn of the rat: the role of collateral sprouting. The expression of substance P, calcitonin gene-related peptide (CGRP) and thiamine monophosphatase in the sciatic nerve terminal field of the lumbar dorsal horn of the rat was examined following neonatal sciatic nerve section and ligation. The total terminal field from L3 to L5 was mapped from semi-serial sections on the treated side and compared to equivalent maps on the contralateral intact side. To obtain a detailed time course of events, data were obtained 4, 7, 10, 15-20 and 40-60 days after sciatic nerve section. At 4-7 days thiamine monophosphate was depleted from the cut nerve terminals resulting in a gap in dorsal horn thiamine monophosphate stain similar to that seen after adult nerve section. In contrast, substance P and CGRP-containing terminals showed only a transient fall in expression in the first week following nerve section and then staining was no different from that seen on the control side. The depletion of peptides normally observed after adult nerve section did not occur. This phenomenon was only observed if the sciatic nerve was cut at birth. Nerve section at 10 days of age resulted in the same pattern of peptide depletion as is observed in the adult. A week after neonatal sciatic nerve section, thiamine monophosphate-containing nerve terminals from nearby intact nerves begin to sprout into the sciatic nerve territory in the dorsal horn. This, together with some recovery of thiamine monophosphate from the remaining sciatic terminals themselves, results in a slow filling in of the gap in the thiamine monophosphate stain. Resection of the cut sciatic nerve, together with adjacent intact nerves, re-establishes the depletion. Substance P and CGRP terminals from nearby intact nerves also sprout into the deafferented sciatic field and this can be demonstrated by the larger than normal area of depletion following section of these nerves when adult. Furthermore, resection of the neonatally cut sciatic nerve when adult also causes some depletion of substance P and CGRP within the sciatic field, indicating a degree of recovery or up-regulation of peptides in surviving cut afferents. However, even after resection of the cut sciatic nerve and nearby intact nerves, substance P and CGRP staining remained in the terminal region. We conclude that while central collateral sprouting does take place in both substance P and CGRP-containing afferents following peripheral nerve section, it cannot account for the lack of depletion of peptides observed.(ABSTRACT TRUNCATED AT 400 WORDS)


Up-regulation of 5-hydroxytryptamine2 and neurokinin-1 receptors associated with serotonin/substance P hyperinnervation in the rat inferior olive. The fate of serotonin and substance P receptors following serotonin/substance P hyperinnervation of CNS tissue was investigated in the inferior olivary complex of adult rats subjected to earlier intraventricular administration of 5,6-dihydroxytryptamine. [3H]8-hydroxy-2-(Dl-n-propylamino)tetralin, [3H]5-hydroxytryptamine, [3H]ketanserin and [125I]Bolton-Hunter-substance P were respectively used to label 5-hydroxytryptamine1A, 5-hydroxytryptamine1B, 5-hydroxytryptamine2 and neurokinin-1 receptor sites for quantitative ligand binding autoradiography. Only 5-hydroxytryptamine2 and neurokinin-1 sites were detected in the normal or serotonin/substance P-hyperinnervated inferior olivary complex. In the normal inferior olivary complex, the density of [3H]ketanserin binding (5-hydroxytryptamine2 receptors) was relatively low, being the highest in pars a of the caudal medial accessory olive and the principal olive; moderate in pars c of the caudal medial accessory olive; truly low in the medial and the lateral dorsal accessory olive, nucleus b and pars b of the caudal medial accessory olive; and negligible in the middle medial accessory olive, rostral medial accessory olive and the smaller subnuclei. [125I]Bolton-Hunter-substance P binding (neurokinin-1 receptors) appeared denser, being highest in nucleus beta and the middle medial dorsal accessory olive; moderate in the three portions of the caudal medial accessory olive, the lateral dorsal accessory olive and the dorsal cap of Kooy; low in the rostral medial accessory olive, the ventrolateral outgrowth and the dorsomedial cell column; and very low or null in the principal olive and the medial dorsal accessory olive. After serotonin/substance P hyperinnervation, there were striking increases in the apparent density of both populations of receptor. [3H]Ketanserin binding was now stronger in the most olivary subnuclei, including some in which it had not been found in the normal, such as the middle and the rostral medial accessory olive. [125I]Bolton-Hunter-substance P binding showed even greater elevations in a few subnuclei, such as the principal olive and the dorsomedial cell column; it was now detectable in the medial dorsal accessory olive, unchanged in the dorsal cap of Kooy and the ventrolateral outgrowth, and slightly decreased in the lateral dorsal accessory olive. The normal and altered distributions of both ligands did not match the respective patterns of serotonin and substance P innervation and hyperinnervation previously demonstrated with immunocytochemistry. In some sectors of the inferior olivary complex where both transmitters are presumably co-localized, there was no overlap in the distribution of the respective binding sites either in the normal or in the hyperinnervated state.(ABSTRACT TRUNCATED AT 400 WORDS)


Kappa-/mu-receptor interactions in the opioid control of the in vivo release of substance P-like material from the rat spinal cord. The possible involvement of mu and kappa receptors in the opioid control of the spinal release of substance P-like material was assessed in vivo, in halothane-anaesthetized rats whose intrathecal space was continuously perfused with an artificial cerebrospinal fluid supplemented with various opioid receptor agonists and antagonists. Whereas the intrathecal perfusion with the mu agonist DAGO (10 microM) significantly enhanced (approximately + 50%) the spontaneous release of substance P-like material, that with the kappa agonist U 50488 H (10 microM) produced no change in the peptide outflow. The respective antagonists naloxone (10 microM) for the mu receptors and nor-binaltorphimine (10 microM) for the kappa receptors did not affect the spontaneous release of substance P-like material, indicating that endogenous opioids acting at mu and kappa receptors do not exert a tonic control on substance P-containing neurons in the spinal cord of halothane-anaesthetized rats. However, as expected from the involvement of mu receptors, the stimulatory effect of DAGO on the peptide outflow could be prevented by naloxone but not norbinaltorphimine. Furthermore, instead of an increase with DAGO alone, a significant decrease in the spinal release of substance P-like material was observed upon the intrathecal perfusion with DAGO plus U 50488 H. Additional experiments with the respective mu and kappa antagonists naloxone and nor-binaltorphimine demonstrated that this effect actually resulted from the simultaneous stimulation of mu and kappa receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Morphology and distribution of neuropeptide-containing neurons in human cerebral cortex. Biopsies of human cerebral cortex were fixed by immersion and immunostained for the detection of neuropeptides in neuronal cell bodies and axons. Four neuropeptides (neuropeptide Y, somatostatin, , substance P and cholecystokinin) were visualized in a series of adjacent sections. All populations of immunoreactive neurons had a morphology characteristic of interneurons, with variations in dendritic arborizations and laminar distribution. The cholecystokinin-immunoreactive neurons were most numerous in the supragranular layers, whereas neurons containing the other three peptides occurred mainly in infragranular layers, or even in neurons populating the subcortical white matter. Quantitatively, each population of neuropeptide-containing neurons accounted for 1.4-2.5% of the total neuronal population. The distribution of these neurons varied slightly between cytoarchitectonic divisions, with substance P- and somatostatin-immunoreactive neurons dominating in the temporal lobe and cholecystokinin-immunoreactive neurons in the frontal lobe. Neuropeptide Y-immunoreactive neurons dominated in the gray matter of the frontal half of the hemisphere and in the subcortical white matter of the caudal half of the hemisphere. Furthermore, co-existence of neuropeptide Y or substance P immunoreactivity within somatostatin-immunoreactive neurons could be demonstrated using double labeling immunofluorescence techniques. The axonal plexuses immunoreactive for neuropeptide Y, somatostatin, or substance P were distributed in all layers, with a strong predominance of horizontally oriented fibers in layer I, a moderate plexus of randomly oriented fibers in the supra- and infragranular layers, and a slightly weaker innervation of layer IV. Immunoreactive axons formed, in addition, complex terminal arbors, mostly in older subjects, suggesting that they resulted from an as yet undefined aging process. The present study underlines several aspects of the organization of the neuropeptide-containing neurons of the human cerebral cortex, which are of particular interest in the light of the involvement of these neurons in several neurodegenerative diseases.


GABAergic and catecholaminergic innervation of mediobasal hypothalamic beta-endorphin cells projecting to the medial preoptic area. In the absence of cellular estrogen receptors or proven direct estrogen action in the rat, it is assumed that estrogen indirectly regulates the secretory activity of the preoptic area luteinizing hormone-releasing hormone-producing cells. We have previously shown that pro-opiomelanocortin neurons in the arcuate nucleus of the rat send axons rostrally to connect with luteinizing hormone-releasing hormone neurons of the preoptic area. An experiment combining retrograde tracing and double-immunostaining was used to test the hypothesis that rat GABAergic and/or catecholaminergic neurons can influence luteinizing hormone-releasing hormone-producing cells via mediobasal hypothalamic beta-endorphin neurons. The retrograde tracer horseradish peroxidase was injected into the medial preoptic area; two days later, arcuate nucleus Vibratome sections were double-immunostained for beta-endorphin and glutamate decarboxylase or tyrosine hydroxylase. Light and electron microscopic analysis of these triple-labeled sections demonstrated that a population of beta-endorphin-immunoreactive neurons concentrated in the ventromedial arcuate nucleus contain retrogradely transported horseradish peroxidase granules and form synaptic contacts with glutamate decarboxylase- and tyrosine hydroxylase-immunoreactive axon terminals. The present data suggest that arcuate nucleus GABA and catecholamine fibers may influence luteinizing hormone-releasing hormone-containing neurons via projective pro-opiomelanocortin cells.


Protein kinase C activation by phorbol esters induces chromaffin cell cortical filamentous actin disassembly and increases the initial rate of exocytosis in response to nicotinic receptor stimulation. Nicotinic stimulation and high K+ depolarization of bovine chromaffin cells cause disassembly of cortical filamentous actin networks. Previous work from our laboratory has demonstrated that disassembly of actin filaments is Ca(2+)-dependent, precedes exocytosis and occurs in cortical areas of low cytoplasmic viscosity which are the sites of exocytosis. It has also been suggested that protein kinase C is involved in catecholamine secretion from chromaffin cells. Therefore, the possibility that protein kinase C activation might be implicated in cortical filamentous actin disassembly was investigated. Here we report that phorbol myristate acetate, a protein kinase C activator, causes cortical filamentous actin disassembly. Short-term phorbol ester treatment does not alter the morphology of chromaffin cells; however, 1 h after phorbol ester exposure an increase in cell flattening and membrane ruffling is observed. Phorbol ester-induced cortical filamentous actin disassembly is inhibited by protein kinase C activity inhibitors, is independent of extracellular Ca2+ and has a slower time course than that induced by either nicotinic receptor stimulation or K(+)-depolarization. Phorbol ester effects are likely to be mediated by activation of protein kinase C and not by any changes in intracellular Ca2+ levels, as indicated by measurements of Ca2+ transients. Pretreatment of chromaffin cells with phorbol myristate acetate increases the initial rate of nicotine-evoked catecholamine release. Nicotine-induced cortical actin filament disassembly and catecholamine secretion are partially (29-40%) inhibited by pretreatment of cells with either calphostin C, staurosporine or sphingosine. The results suggest that protein kinase C may be involved in the reorganization of the cortical actin filament network priming the cells for release by removing a barrier to secretory granule mobility. However, its role in exocytosis is modulatory but not essential.


An inhibitor of nitric oxide synthesis prevents memory formation in the chick. Memory formation is presumed to require retrograde communication across synaptic junctions. Nitric oxide (NO) is a putative retrograde messenger at N-methyl-D-aspartate (NMDA)-mediated synapses [8, 9]. Inhibitors of nitric oxide synthesis block initiation of long-term potentiation [2, 3, 19]. Memory for a one-trial passive avoidance task in the young chick involves an NMDA-linked intracellular cascade culminating in lasting modulation of synaptic morphology and [6, 18]. Here we show that injection of the nitric oxide synthase inhibitor N-nitro-L-arginine prior to training results in amnesia for the passive avoidance task; the amnesia can be overcome by injecting L-arginine along with the inhibitor. Thus we have verified for the first time experimentally that NO plays a role in memory formation.


Striatal neuropeptide levels in Parkinson's disease patients. Substance P (SP), Met-enkephalin (Met-enk) and cholecystokinin-8-S (CCK-8-S) were measured by a combined HPLC/RIA method in the caudate nucleus and anterior putamen from controls and from Parkinson's disease (PD) patients. SP levels were reduced in caudate in PD, but unchanged in putamen. No differences in Met-enk content were found in parkinsonians compared to controls. However, a significant correlation between DA and Met-enk levels in caudate nucleus from PD was observed. The concentration of CCK-8-S was unaltered in caudate nucleus or putamen in PD. The decrease in caudate nucleus SP levels might be related to the decrease in nigral SP levels in PD, while the reduction in Met-enk levels appears to be a feature of a subgroup of parkinsonian patients.


A subpopulation of hippocampal glial cells specific for the zinc-containing mossy fibre zone in man. The projection of the zinc-containing axons of granule cells of the fascia dentata, e.g. the mossy fibres, is restricted to the hilar region and sector CA3 of the hippocampus. Serial sections of human hippocampi were stained for zinc-containing fibres with a non-perfusion Timm method, while adjacent ones were stained with Darrow red and aldehydefuchsin. GFAP, glutamine synthetase immunocytochemistry and a specific silver stain were employed to label other subtypes of astrocytes. The distribution of Timm-stained areas correlates only with the distribution of aldehydefuchsin-positive glial cells, most probably astrocytes. Since glial cells regulate axonal outgrowth in a region-specific manner, it is temptative to speculate that the aldehydefuchsin-positive glial cell is a candidate for a specific neuron-glia interaction which is somehow involved in the control of outgrowth of mossy fibres.


Evidence for upregulation of galanin synthesis in rat glial cells in vivo after colchicine treatment. The localization of galanin (GAL) and GAL mRNA was studied in rat brain after colchicine or vinblastine treatment using in situ hybridization and immunohistochemistry. GAL-like immunoreactivity was found in glial cells, presumably activated microglia, in the cortex, caudate nucleus and septum, mainly on the injection side. GAL mRNA expression was found in small cells in the same areas with an overlapping distribution, including the adjacent white matter. The results suggest that the glial cells initiate synthesis of the peptide GAL in response to intraventricular injection of high doses of the 2 mitosis inhibitors.


Rationale of combining radiation and interferon for the treatment of cervical cancer. The recent surge of interest in the mechanisms of action of biomodulators, also known as biological response modifiers, offers a new avenue of approach in the treatment of cancer. The in vitro antitumor activities of these agents, such as interferons, when combined with chemo- or radiotherapy, have generated enthusiasm among clinicians for developing clinical trials. In recent years many antineoplastic agents have been investigated as neoadjuvant or adjuvant therapy for patients with cervical cancer in an attempt to improve local control and to decrease incidence of metastasis. Normal tissue tolerance limits the potential combinations of standard cytotoxic chemotherapeutic agents with radiation. Interferon used as a radiomodulator has been studied mainly for the treatment of lung cancer with promising results. In this paper we report the rationale of combining interferon and radiation for the treatment of patients with cervical cancer.


Morphologic and immunohistochemical observations on the mammary and extramammary Paget's disease: implications for the histogenesis. Using a panel of three anti-CK MoAbs, belonging to Gown's Classes II, III and IV, the Paget's cells shown a variable reactivity to such antibodies indicating a more frequent immunocytochemical similarity to the cells of the epidermal basal-ductal system than to the cells of the glandular secreting section of the epidermal derivatives. According these findings the P.C. not necessarily is the result of a cellular migration from ductal section toward the epidermis, but may arise from a epidermal basal-stem cell, from a ductal or more rarely from glandular secreting cell. This assumption is enforced also by the expression of the CEA from morphologically bonafide basal epidermal cells in proximity of the pagetic lesion.


Effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) on hemopoiesis and survival rate following allogeneic bone marrow transplantation in mice. The effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) on the survival rate and hemopoiesis after allogeneic bone marrow transplantation (BMT) were examined. Specific pathogen-free (SPF) C3H mice and germ-free C3H mice, kept in an isolator for germ-free animals, received 10 Gy of total body irradiation (TBI) and all the mice died by day 9 after TBI. These survival rates were improved by BMT. In the case of SPF mice, survival rates at 14 and 100 days were 33% (12/36), 17% (6/36) and in the case of germ-free mice they were 79% (15/19), 74% (14/19) respectively. When SPF mice received rhG-CSF (30 micrograms/kg/day) subcutaneously for 14 consecutive days following BMT, their survival rates were improved to 79% (30/38), 79% (30/38) respectively. The survival rate of rhG-CSF treated SPF mice were equal to that of germ-free mice. When the effect of rhG-CSF treatment on hemopoiesis of SPF mice after allogeneic BMT was examined various hematopoietic progenitor cells in the bone marrow and spleen increased until day 10 after, BMT, while only neutrophils increased in the peripheral blood during the period. No adverse effects of rhG-CSF were observed throughout the study period. It was suggested that in SPF mice treated with rhG-CSF after BMT, the neutrophil recovered in counts quickly and increased neutrophil prevented endogenous infections and improved the survival rate without apparent complications.


Human papillomavirus in oral verrucal-papillary lesions. A comparative histological, clinical and immunohistochemical study. The purpose of this study was to determine the presence of Human Papillomavirus (HPV) virions in the verrucal-papillary (OVP) lesions by clinical, histological and immunohistochemical [peroxidase-antiperoxidase (PAP) features]. Thirty seven positive cases were compared with 47 negative cases. Considerable overlapping of clinical and histological features can be seen among all these lesions. The study provides evidence that there are no clinical or histologic features with taken alone are strongly correlated with the presence of virions in oral lesions.


Primary papillary serous tumor of the peritoneum. Report of a case. In the present report we describe a rare papillary serous tumor of the peritoneum occurring in a woman without evidence of ovarian neoplasia. Histochemical, immunohistochemical and ultrastructural findings are reported, with particular emphasis on electron microscopic results and on their importance in the differential diagnosis with malignant mesotheliomas.


Suppression of the onset of myelination extends the permissive period for the functional repair of embryonic spinal cord. In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).


Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution. The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.


Regulation of mouse gamete interaction by a sperm tyrosine kinase. A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.


Cellular regulation of the iron-responsive element binding protein: disassembly of the cubane iron-sulfur cluster results in high-affinity RNA binding. The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.


In vivo treatment of human leukemia in a scid mouse model with c-myb antisense oligodeoxynucleotides. The c-myb protooncogene encodes proteins that are critical for hematopoietic cell proliferation and development. Disrupting c-myb function might, therefore, prove an effective therapeutic strategy for controlling leukemic cell growth. Antisense oligodeoxynucleotides have been utilized for this purpose in vitro, but their in vivo efficacy has not been reported. We therefore established human leukemia-scid mouse chimeras with K562 cells and treated diseased animals with phosphorothioate-modified antisense oligodeoxynucleotides. K562 cells express the c-myb protooncogene, which served as the target for the antisense DNA. They also express the tumor-specific bcr-abl oncogene that was utilized to track the human cells in the mouse host. Once circulating leukemic blast cells had been detected, the survival of untreated control mice was 6 +/- 3 days (mean +/- SD). The survival of animals treated for 7 or 14 days with either sense or scrambled-sequence c-myb oligodeoxynucleotides was not statistically different from the control animals. In distinct contrast, animals treated for similar lengths of time with antisense c-myb oligodeoxynucleotides survived at least 3.5 times longer than the various control animals. In addition, animals receiving antisense c-myb DNA had significantly less disease at the two sites most frequently manifesting leukemic cell infiltration, the central nervous system and the ovary. These results suggest that phosphorothioate-modified antisense DNA may be efficacious for the treatment of human leukemia in vivo, and by analogy, for the treatment of other human neoplasias.


Cell cycle-dependent changes in the organization of an intermediate filament-associated protein: correlation with phosphorylation by p34cdc2. During mitosis in BHK-21 baby hamster kidney cells the hyperphosphorylation of the type III intermediate filament (IF) protein vimentin is accompanied by the disruption of the IF network into punctate, protofilamentous structures. In this study, the morphological and biochemical changes of IFAP 300, a 300-kDa IF-crossbridging protein, are examined during mitosis. Double-label immunofluorescence shows that the distribution of IFAP 300 coincides with the typical filamentous pattern displayed by vimentin in interphase cells, whereas in mitotic cells it is reorganized into a punctate, nonfilamentous pattern. Accompanying these latter morphological changes, IFAP 300 is phosphorylated at a unique, mitosis-specific site. Comparison of the sites phosphorylated in cultured cells with those phosphorylated in vitro by various kinases suggests that IFAP 300 is phosphorylated by the same two kinases that phosphorylate vimentin during mitosis. One of these is p34cdc2 protein kinase, which appears to be responsible for the phosphorylation of the mitosis-specific site. The other kinase phosphorylates IFAP 300 in vitro at a site that is also found in the protein immunoprecipitated from either mitotic or interphase cells. In contrast to vimentin, the phosphorylation levels of IFAP 300 are not obviously altered between interphase and mitosis. Our results show that IFAP 300 is a physiological substrate for p34cdc2 and that this kinase may be involved in the mitotic reorganization of IFAP 300 by phosphorylating a mitosis-specific site. Taken together with our previous results, this study suggests that the activation of p34cdc2 coordinates the mitotic reorganization of the vimentin IF network both by severing IF-IF connections mediated by IFAP 300 and by disassembling individual IFs into protofilaments.


Immunohistochemical localization of the D1 dopamine receptor in rat brain reveals its axonal transport, pre- and postsynaptic localization, and prevalence in the basal ganglia, limbic system, and thalamic reticular nucleus. D1 dopamine receptor localization was examined by immunohistochemistry using a polyclonal anti-peptide antibody which (i) immunoprecipitated a protein fragment encoded by a D1 receptor cDNA and (ii) on Western blots of solubilized striatal and hippocampal membranes recognized two proteins of approximately 50 kDa and 75 kDa, corresponding to reported sizes of D1 receptor proteins. Immunoreactivity overlapped with dopamine-containing pathways, patterns of D1 receptor binding, and mRNA expression. Staining was concentrated in prefrontal, cingulate, parietal, piriform, entorhinal, and hippocampal cortical areas and subcortically in the basal ganglia, amygdala, septal area, substantia inominata, thalamus, hypothalamus, and neurohypophysis. Prominent labeling was seen in the thalamic reticular nucleus, a region known to integrate ascending basal forebrain inputs with thalamocortical and corticothalamic pathways and in fiber bundles interconnecting limbic areas. In striatal neuropil, staining appeared in spines (heads and necks), at postsynaptic sites in dendrites, and in axon terminals; in the pars reticulata of the substantia nigra, labeling was prevalent in myelinated and unmyelinated axons and dendrites. These data provide direct evidence for the regional and subcellular distribution of D1 receptor protein in the brain and for its pre- and postsynaptic localization in the basal ganglia. The prominent immunoreactivity seen in the limbic system and thalamic reticular nucleus supports an important role for this receptor subtype in mediating integrative processes involved with learning, memory, and cognition.


Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Tubular-fluid reabsorption by specialized cells of the nephron at the junction of the ascending limb of the loop of Henle and the distal convoluted tubule, termed the macula densa, releases compounds causing vasoconstriction of the adjacent afferent arteriole. Activation of this tubuloglomerular feedback response reduces glomerular capillary pressure of the nephron and, hence, the glomerular filtration rate. The tubuloglomerular feedback response functions in a negative-feedback mode to relate glomerular capillary pressure to tubular-fluid delivery and reabsorption. This system has been implicated in renal autoregulation, renin release, and longterm body fluid and blood-pressure homeostasis. Here we report that arginine-derived nitric oxide, generated in the macula densa, is an additional intercellular signaling molecule that is released during tubular-fluid reabsorption and counters the vasoconstriction of the afferent arteriole. Antibody to rat cerebellar constitutive nitric oxide synthase stained rat macula densa cells specifically. Microperfusion of the macula densa segment of single nephrons with N omega-methyl-L-arginine (an inhibitor of nitric oxide synthase) or with pyocyanin (a lipid-soluble inhibitor of endothelium-derived relaxation factor) showed that generation of nitric oxide can vasodilate the afferent arteriole and increase glomerular capillary pressure; this effect was blocked by drugs that prevent tubular-fluid reabsorption. We conclude that nitric oxide synthase in macula densa cells is activated by tubular-fluid reabsorption and mediates a vasodilating component to the tubuloglomerular feedback response. These findings imply a role for arginine-derived nitric oxide in body fluid-volume and blood-pressure homeostasis, in addition to its established roles in modulation of vascular tone by the endothelium and in neurotransmission.


Expression cloning of a human dual-specificity phosphatase. Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase. Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody. Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro. The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1. The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro. Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein. These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.


Transcription of the tumor necrosis factor alpha gene is rapidly induced by anti-immunoglobulin and blocked by cyclosporin A and FK506 in human B cells. The human tumor necrosis factor alpha (TNF-alpha) gene encodes a cytokine whose activities have been implicated in many immunopathological processes, including the activation and differentiation of lymphocytes. Originally identified as a monocyte factor, our studies and those of others have demonstrated that B and T lymphocytes produce TNF-alpha when stimulated by a variety of inducers. We report here that TNF-alpha gene transcription is rapidly and highly induced in three independently derived human Burkitt lymphoma cell lines, as well as in freshly isolated human splenic B cells, activated by antibodies to surface immunoglobulin. This burst in TNF-alpha gene transcription is associated with an induction of TNF-alpha bioactivity in the culture supernatants from stimulated splenic B cells. Moreover, induction of TNF-alpha gene transcription by anti-immunoglobulin was blocked by the immunosuppressants cyclosporin A and FK506. These studies demonstrate that TNF-alpha production is an early event in B-cell activation and they establish the efficacy of using immunosuppressants as probes in dissecting transcriptional activation pathways in human B cells.


Action to avoid an unnecessary outcome. Identifying iron deficiency in children. Although mainly associated with anaemia, there are several other physical and psychological manifestations of iron deficiency. Awareness of community screening programmes would do much to reduce the incidence of this problem.


Tumor markers.

Tumor markers. The past decade has seen many advances in the detection, characterization, and clinical applications of tumor markers. Although cancer screening applications have been limited by low disease prevalences in asymptomatic populations, tumor markers may be of diagnostic value in specific situations. The major use of tumor markers in primary care is in monitoring disease recurrence and the response to therapy. These uses may obviate the need for second-look surgery as it has with CA 125 elevations in ovarian carcinoma. On the other hand, tumor markers may indicate a need for second-look surgery as when CEA levels are elevated in colorectal carcinoma. Despite the apparent usefulness of markers in detecting cancer recurrence, the clinician is reminded of the conditions justifying treatment with the discovery of an abnormal laboratory value as put forth by Fries and Holman: (1) the treatment should be known to be effective, (2) early treatment should be known to be more effective than therapy given after the disease is clinically apparent (or early treatment carry the risk of less toxicity), and (3) prediction of impending deterioration must be consistently accurate. When these criteria are met, the planning of therapeutic regimens on the basis of marker levels may be rationally considered. Until these criteria are met, elevated markers can only stimulate the clinician to be more vigilant in the search for response to therapy or recurrence. Exciting developments are on the horizon with respect to the use of tumor markers in radionuclide imaging, radioimmunoguided surgery, and development of drug delivery systems. Further research into the structure and function of these substances may provide further insights into the phenomena of malignant transformation, tumor invasion, metastasis, and the development of new therapeutic options. With new advances in molecular biology and with the identification of oncogenes, it may be possible in the future to detect mutant oncoproteins that are specific for early cancers and premalignant conditions. Delecting these oncoproteins may provide a basis for development of truly sensitive and specific markers that can be used to detect cancer at an early and curable stage.


Interleukin-8 as a macrophage-derived mediator of angiogenesis. Angiogenic factors produced by monocytes-macrophages are involved in the pathogenesis of chronic inflammatory disorders characterized by persistent angiogenesis. The possibility was tested that interleukin-8 (IL-8), which is a cytokine that is chemotactic for lymphocytes and neutrophils, is also angiogenic. Human recombinant IL-8 was potently angiogenic when implanted in the rat cornea and induced proliferation and chemotaxis of human umbilical vein endothelial cells. Angiogenic activity present in the conditioned media of inflamed human rheumatoid synovial tissue macrophages or lipopolysaccharide-stimulated blood monocytes was equally blocked by antibodies to either IL-8 or tumor necrosis factor-alpha. An IL-8 antisense oligonucleotide specifically blocked the production of monocyte-induced angiogenic activity. These data suggest a function for macrophage-derived IL-8 in angiogenesis-dependent disorders such as rheumatoid arthritis, tumor growth, and wound repair.


Activation of transcription by IFN-gamma: tyrosine phosphorylation of a 91-kD DNA binding protein. Interferon-gamma (IFN-gamma) induces the transcription of the gene encoding a guanylate binding protein by activating a latent cytoplasmic factor, GAF (gamma-activated factor). GAF is translocated to the nucleus and binds a DNA element, the gamma-activated site. Through cross-linking and the use of specific antibodies GAF was found to be a 91-kilodalton DNA binding protein that was previously identified as one of four proteins in interferon-stimulated gene factor-3 (ISGF-3), a transcription complex activated by IFN-alpha. The IFN-gamma-dependent activation of the 91-kilodalton DNA binding protein required cytoplasmic phosphorylation of the protein on tyrosine. The 113-kilodalton ISGF-3 protein that is phosphorylated in response to IFN-alpha was not phosphorylated nor translocated to the nucleus in response to IFN-gamma. Thus the two different ligands result in tyrosine phosphorylation of different combinations of latent cytoplasmic transcription factors that then act at different DNA binding sites.


A clinicopathological and immunohistochemical study of osteofibrous dysplasia, differentiated adamantinoma, and adamantinoma of long bones. A clinicopathological and immunohistochemical study of 12 cases of osteofibrous dysplasia (OFD), two cases of differentiated adamantinoma, and five cases of adamantinoma of long bones is presented. Although OFD and differentiated adamantinoma showed similar radiologic findings, differentiated adamantinoma was more likely to be a recurrent lesion than osteofibrous dysplasia and seemed to require a more extensive surgical procedure. Immunohistochemically, cytokeratin- and vimentin-positive cells were seen in both OFD and differentiated adamantinoma. The positive cells were scattered in the former, and were both scattered and nest-like in the latter. Both these lesions, however, were negative for epithelial membrane antigen. Excluding two cases of Ewing-like adamantinoma, the other three cases of adamantinoma were also positive for cytokeratin and vimentin. These results suggest that these three lesions share the same histogenetic origin. The two cases of Ewing-like adamantinoma differ from tibial adamantinoma in their radiological, histological and immunohistochemical aspects, and seem to constitute a distinct variant of adamantinoma with a different histogenesis.


Homing of CD8CD57 T lymphocytes into acutely rejected renal allografts. This study compares the cellular events in excised rejected renal allografts (RKT), with concurrent data in the peripheral blood (PB) in the same patients. The kidney transplants were obtained from recipients after rejections crises that were refractory to treatment. Two-color flow cytometry data was used for quantitation of cell subset profiles and quantitation of the density of individual lymphocyte surface antigen(s). The level of CD3DR-positive T cells in RKT was significantly higher than in PB. This difference ranged from 3 to 19-fold increases. There was an even more pronounced increase in CD8CD57 cell subset levels in RKT versus PB. In parallel with these findings, there was a 15-fold greater mean density of the CD8CD57 markers on T cells in RKT, when compared with the same cells in PB. The density of CD8 and of DR markers on CD3+ cells was also significantly higher (4-fold in each instance) in RKT. These data point to a homing of CD8CD57 cells with corresponding increases in the density of these markers in acutely rejected renal allografts, with only a relative decrease of this cell subset in peripheral blood. The appearance of a high preponderance of CD8CD57 cells in the renal allograft at the time of a rejection crisis may constitute a particularly severe prognostic sign regarding the reversibility of the response after treatment with steroids and/or monoclonal antibodies.


In situ expression of tumor necrosis factor-alpha, interferon-gamma, and interleukin-2 receptors in renal allograft biopsies. The production and release of cytokines and their receptors are of critical importance in mediating graft injury. In order to evaluate the expression of cytokines in renal allograft biopsies, we performed immunocytochemical studies to detect activated cells positive for TNF-alpha, IFN-gamma, and IL-2R, using an alkaline phosphatase anti-alkaline phosphatase technique (APAAP). Sixty-one biopsy specimens from renal transplant patients were analyzed and were classified according to both clinical and conventional morphological criteria. There was a significant correlation between the number of positive cells reactive with monoclonal antibodies directed against TNF-alpha, IFN-gamma, and IL-2R and the presence of acute cellular rejection. The mean number of infiltrating cells (cells/mm2) positive for TNF-alpha (9.2 +/- 1.1), IFN-gamma (6.7 +/- 1.7), and IL-2R (31.2 +/- 4.8) was significantly greater in acute cellular rejection episodes compared with nonrejecting kidneys (0.9 +/- 0.2, 1.2 +/- 0.4, and 8.8 +/- 2.9 positive cells/mm2 for TNF-alpha, IFN-gamma, and IL-2R, respectively). No significant expression of these cytokines was found in the majority of biopsies with chronic rejection. In two cases, in which acute cellular rejection was not sustained on clinical grounds but was diagnosed on histology, the expression of TNF-alpha, IFN-gamma, and IL-2R was similar to that observed in typical cellular rejection. We conclude that TNF-alpha, IFN-gamma, and IL-2R are markedly expressed by activated mononuclear infiltrating cells in acute cellular rejection, and that these cytokines play an important role in allograft rejection. The immunocytochemical evaluation of cytokine expression is a simple and rapid method that is helpful in differentiating acute cellular rejection from other causes of graft disfunction.


[Single-fraction radiotherapy of painful bone metastases]. We analyzed first results obtained in 92 patients treated with single-dose fraction radiotherapy for painful bone metastases. Tumour doses was 10 Gy in a single fraction. Response was obtained in 59/92 (64%) patients. Thirty nine patients had complete response and 20 had partial response. Pain recurred in 18/39 patients who initially responded. Toxicity of this radiotherapeutic treatment is acceptable, and can easily be managed with standard therapy.


The distinct effects of FK506 on the activation, proliferation, and differentiation of human B lymphocytes. We examined the effect of FK506 on the activation, proliferation and differentiation of human B lymphocytes in vitro. FK506 inhibited the proliferative response of resting B cells induced by Staphylococcus aureus Cowan strain I (SAC) and phorbol myristate acetate (PMA) in a dose-dependent manner. Inhibition of cell proliferation by FK506 was caused by a selective block of G0 to G1 phase transition leading to cell arrest. In addition, the proliferative response of in vivo-activated B cells and lymphokine-driven B cell proliferation were also found to be sensitive to FK506. Interestingly, FK506 did not affect the expression of activation antigens such as CD23, IL-2 receptor (CD25), and transferrin receptor (CD71). Finally, FK506 had little effect on B cell antibody generation in a T cell-independent system. Conversely, FK506 suppressed neither proliferation nor immunoglobulin secretion in a human B lymphoblastoid cell line. These results indicate that FK506 has discrete effects on the different stages of the B cell maturation.


FK506-induced kidney tubular cell injury. Some renal changes associated with cyclosporine, such as tubular vacuolization and glomerular thrombosis, have also been reported with FK506. Furthermore, FK506 therapy is associated with a decrease in glomerular filtration rate and renal plasma flow and an increase in renal vascular resistance. We studied the in vitro tubular cell sensitivity to FK506 in comparison with CsA, the ultrastructural changes induced by FK506 and CsA, and the effect of both drugs on tubular cell growth in vitro. We also investigated whether FK506 and CsA induced endothelin-1 (ET-1) secretion of cultured tubular cells and whether this stimulatory effect coincided with a change in the endothelin systemic synthesis. Exposure of tubular cells to high concentrations of FK506 or CsA (10, 50, 100 microM) induced a time- and dose-dependent cell injury in vitro. The damage induced by FK506 and CsA was characterized by a direct cytotoxic effect on tubular cells, as expressed by release of 3H thymidine from prelabeled cells, N-acetyl-beta-D-glucosaminidase release, and cell detachment. Ultrastructural changes (vacuolizations, swelling, and mitochondrial enlargement) and inhibition of the growth of cultured tubular cells were also observed at high concentrations of FK506 and CsA. Low concentrations of FK506 and CsA (1, 0.1, 0.01, 0.001 microM) were not cytotoxic and induced only a minimal inhibitory effect on the growth of tubular cells in vitro. We demonstrated that FK506 (1, 0.1, 0.01 microM) time-dependently stimulated the secretion of endothelin by cultured tubular cells. CsA 10, 1, 0.1, 0.01 also exerted an enhancing effect on ET-1 secretion in cultured tubular cells. We observed that the concentration of CsA that induced the most important enhancing effect was 10 or 100 times higher than that required for FK506 to observe the same effect. The concentrations of FK506 or CsA that induced ET-1 secretion were not cytolytic for tubular cells in vitro. FK506- or CsA-treated rats showed an increase in serum level of ET-1 in comparison with the control. Through the stimulatory effect on endothelin secretion by tubular cells, FK506 and CsA may induce a perturbation of renal hemodynamics. Concentrations of FK506 and CsA, higher than established serum levels but close to those reached in tissues, are cytotoxic for tubular cells and induced ultrastructural changes and a significant delayed regeneration.


Heterogeneity in the anti-HLA-DR immune response elicited by the antiidiotypic monoclonal antibody F5-444. Analysis at the clonal level. A total of 630 hybridomas were generated from a BALB/c mouse immunized with the anti-id mAb F5-444, which binds an idiotope within the antigen-combining site of mAb AC1.59. The latter recognizes a determinant shared by HLA-DR1, DRw8, DR9 antigens and subtypes of HLA-DR4 and DR6 that is poorly expressed by HLA-DRw16 and DRw17 antigens. Eight anti-anti-id mAb were shown with serological and immunochemical assays to react with HLA-DR antigens. Detailed analysis of these anti-HLA-DR anti-anti-id mAb showed that they differ from the original anti-HLA-DR mAb AC1.59 and among themselves in either isotype, fine specificity, extent of reactivity with the nominal antigen, differential reactivity with soluble or cell-bound antigen, and/or idiotype profile. These observations emphasize the need to characterize a panel of antigen-binding anti-anti-id mAb in order to evaluate the degree of similarity existing between antigen-binding antibodies induced by an anti-id mAb and the original antigen-binding mAb.


Induction of long-term survival of rat skin allografts by a novel, highly efficient anti-CD4 monoclonal antibody. The new monoclonal antirat CD4 antibody RIB 5/2, which detects another epitope than those covered by W3/25 and MRC OX35, was tested for its immunosuppressive potency following skin allografting by using strain combinations with different genetic barriers in the MHC and genetic low- or high-responder background. High-dose and long-term therapy of the grafted rats led to a significant delay of the acute rejection (P < 0.01) in the strain combination Wistar Furth-to-BDX as well as in LEW1W-to-LEW1A. No significant prolongation of the mean allograft survival time was obtained for the high-responder rats (LEW1A-to-LEW1W). Cytofluorometric analysis revealed that RIB 5/2 exerts the immunosuppressive activity predominantly by modulation of the CD4 glycoprotein. Furthermore, the dependence of the humoral immune response against the mouse-globulins upon the administered protein quantity could be demonstrated.


Prolongation of graft survival in primate allograft transplantation by yttrium-90-labeled anti-Tac in conjunction with granulocyte colony-stimulating factor. High-affinity IL-2 receptors are expressed by T cells activated in response to foreign histocompatibility antigens but not by normal resting T cells. To exploit this difference in IL-2R expression, anti-Tac, a murine monoclonal antibody specific for the IL-2R alpha subunit, was used to inhibit organ allograft rejection. To enhance its effector function, anti-Tac was armed by chelation with yttrium-90, a pure beta-emitting radionuclide. Animals received no immunosuppression (n = 5, group I, controls), unmodified anti-Tac (n = 5, 1 mg/kg q.o.d., group II), or 90Y-anti-Tac (n = 5, 1.6 mCi/kg divided into four doses, group III). The animals in group IV (n = 4) were treated identically to those in group III with the exception that 5 micrograms/kg/dose of granulocyte colony-stimulating factor was administered intramuscularly on the days when the yttrium-90 was given and on postoperative days 12 through 35 in order to reduce hematopoietic toxicity. Mean graft survival +/- S.E.M. for the control group was 8.2 +/- 0.5 days as compared with 13.8 +/- 2.1 days (P < 0.05) for those monkeys treated with unmodified anti-Tac. Graft survival was further prolonged in animals of group III that received 90Y-anti-Tac, with a mean graft survival of 45.0 +/- 11.8 days; however, three of the five monkeys retained viable grafts within this group but died secondary to bone marrow suppression. In comparison, the monkeys in group IV that were treated with G-CSF in conjunction with 90Y-anti-Tac had a mean graft survival of 49.2 +/- 2.9 days. In contrast to group III there were no deaths in the group (IV) receiving G-CSF. Furthermore, animals in group IV had a reduced magnitude and shortened duration of irradiation-induced neutropenia when compared with that observed in group III animals that did not receive G-CSF. Thus, treatment with 90Y-anti-Tac in conjunction with G-CSF may have potential applications in organ transplantation and the treatment of IL-2 receptor-expressing neoplastic diseases.


Identification of natural suppressor cells in long-term renal allograft recipients. We have previously reported an abnormal expansion of CD3+Leu7+ (CD57+) large granular lymphocytes in long-term renal allograft recipients. These cells lacked NK activity, T-helper activity and did not respond to T cell mitogens. The following studies were done in order to further define the functional characteristics of these cells. We sorted CD3+Leu7+ T cells from the peripheral blood of 45 recipients (all with good renal allograft function), and found that these cells suppress mixed lymphocyte culture responses and pokeweed mitogen-induced IgG secretion in a non-genetically restricted manner. PWM-induced IgG secretion assays are suppressed by 60-100%, and MLC responses are suppressed by 20-85% at a ratio of 1:10 CD3+Leu7+ cells to responder/effector cells. Supernatants from CD3+Leu7+ cell cultures are also suppressive. On the other hand, unsorted cells and non-CD3+Leu7+ sorted cells either enhance responses or produce less than 10% suppression under the same conditions. Patients who were tested more than once showed a relatively stable percentage and suppressive effect of their CD3+Leu7+ cells over an interval of 6-12 months. These nonspecifically suppressive CD3+Leu7+ large granular lymphocytes are similar in many ways to the natural suppressor cells that have been identified in hematopoietic tissues, in graft-vs.-host disease, and in the lymphoid organs of neonates.


Enteral pancreatic enzyme feedback inhibition of the exocrine secretion of the human transplanted pancreas. The mechanism of regulation of negative feedback inhibition of the exocrine pancreas and its possible role in decreasing the exocrine secretion of the grafted human pancreas is unknown. To evaluate this we studied the effect of oral pancreatic enzymes on the stimulated transplanted pancreatic exocrine secretion in eight patients with allograft pancreaticocystostomies. After an 8-hr fast, all graft exocrine secretions via graft stent, fistula, and urinary anastomosis were collected for a 1-hr basal period. A standard 300-ml Lundh test meal was then ingested, and all exocrine secretions were collected in 30-min intervals for 3 hr. This test was repeated with 6 capsules of pancrelipase (24,000 units of lipase, 120,000 units of amylase, and 150,000 units of protease) given with the Lundh test meal. Stent, urine and fistula volume, amylase, and pH were measured for each collection period. The total 3-hr amylase secreted after the test meal and the test meal plus pancrelipase were compared. The period of peak amylase secretion after the test meal alone was compared with the same period after the test meal plus pancrelipase and the premeal basal period. The total amylase decreased 34% from 5550 +/- 1000 to 3680 +/- 740 IU/3 hr (P < .03) with pancrelipase. The peak amylase secretion decreased 63% from 1520 +/- 271 to 567 +/- 185 IU/30 min (P < .02) with the addition of pancrelipase to the test meal. Pancrelipase eliminated all meal-stimulated amylase secretion with the mean secretion 16% below the basal secretion of 674 +/- 117 IU/30 min. We conclude that pancreatic negative feedback inhibition significantly decreases meal-stimulated and basal exocrine secretion in the transplanted human pancreas.


Silver-binding nucleolar organizer regions in benign and malignant prostatic lesions. Thirty-six transvesical or transurethral prostatectomy cases were selected from the histopathology files of the Laiko General Hospital. Among the 36 cases, there were 10 with benign prostatic hyperplasia (8 patients having distinct areas of adenosis) and 26 cases of prostatic adenocarcinoma (6 grade I, 13 grades II and III and 7 grade IV carcinomas). From each case, silver-binding nucleolar organizer regions (AgNORs) have been counted in sections of routinely processed paraffin-embedded tissue blocks. The mean AgNOR count per case was calculated. For the cases of prostatic hyperplasia, the mean AgNOR count was found to be 2.95 +/- 0.42, for adenosis 3.45 +/- 0.56, for grade I adenocarcinoma 4.97 +/- 0.74, for grades II and III 7.31 +/- 0.81 and for grade IV adenocarcinomas 11.41 +/- 1.68. This difference in the mean AgNOR count was found to be of statistical significance (p < 0.001) between adenosis and grade I adenocarcinomas and between grade II and III and grade IV adenocarcinomas. It appears that AgNOR counting may prove to be of benefit in differentiating between some benign and malignant prostatic lesions and that it might provide information concerning the biological behavior of prostatic adenocarcinomas.


Treatment of castration-induced menopausal symptoms with low dose diethylstilbestrol in men with advanced prostate cancer. Menopausal symptoms manifesting as hot flashes and sweats occur in up to 75 percent of patients following either orchiectomy or treatment with a luteinizing hormone-releasing hormone agonist for prostate cancer. As many as one third of these patients will experience symptoms severe enough to seek palliation. We treated 12 such patients with low dose diethylstilbestrol (1/3 mg daily). Nine patients demonstrated both objective and subjective improvement in their menopausal symptoms. Five patients experienced toxicity including new onset of gynecomastia or breast soreness although no patient discontinued treatment on this basis. No cardiovascular complications were noted. We conclude that low dose diethylstilbestrol is an inexpensive, effective means of controlling troublesome postorchiectomy menopausal symptoms in carefully selected patients.


Benign prostatic hyperplasia: appearance on magnetic resonance imaging. Benign prostatic hyperplasia (BPH) has a variable appearance on magnetic resonance imaging (MRI). This study reviews and categorizes the MRI findings of this entity in a sample of 26 men. Cases where atypical or suspicious changes are incidental findings should be reported to the urologist for further investigation. In addition, familiarity with both the appearance of the normal prostate and the changes caused by BPH is necessary when staging patients with known prostate cancer.


Humoral responses in rabbits immunized with two fractions of Haemonchus contortus: the antigen specificity of such antibodies. Humoral responses were examined in rabbits immunized with either 28-40 kDa (Fraction 1) or a 19-24 kDa (Fraction 2) antigenic fraction from soluble antigens (Sol L3 Ag) from infective larvae (L3) of Haemonchus contortus. These fractions were eluted from electrophoretically separated Sol L3 Ag. Immunoblots revealed antibodies to Fraction 1 (fr. 1) or Fraction 2 (fr. 2) polypeptides as well as to several other molecular weight polypeptides of the Sol L3 Ag. The latter antibodies were shown by absorption studies not to be Sol L3 Ag cross-reactive anti-bacterial rabbit antibodies. When Sol L3 Ag was affinity-purified using monoclonal antibody to phosphorylcholine (PC) and the resulting fractions were further analysed by immunoblotting using rabbit anti fr. 1 or anti fr. 2 antiserum, the PC antigen was found to be shared between fr. 1 and other polypeptides of Sol L3 Ag. Using the rabbit antibody fractions eluted from nitrocellulose membranes containing fr. 1 or 2 polypeptides, it was found that these fractions contained antibody that bound mainly to fr. 1 and only to fr. 2 polypeptides of Sol L3 Ag. It is concluded that, from the present immune rabbit sera, antibodies specific for either fr. 1 or fr. 2 may be isolated and then used to purify small amounts of the corresponding antigens.


Development and characterization of monoclonal antibodies to first-generation merozoites of Eimeria bovis. Merozoites of Eimeria bovis were harvested from bovine monocyte cell cultures and used to immunize BALB/C mice. Spleens from immunized mice were removed and the cells fused with mouse myeloma cells. Supernates from resulting hybridoma cell lines were examined for antibodies to first-generation E. bovis merozoites using an indirect immunofluorescent antibody (IFA) assay. Three positive cell lines were identified and cloned by limiting dilution. All three cell lines produced immunoglobulins of the IgG1 isotype that recognized antigens in the anterior half to two-thirds of the merozoites. Specificity of the monoclonal antibodies was examined with the IFA assay against sporozoites of E. bovis, sporozoites and merozoites of Eimeria papillata from mice and Eimeria tenella from chickens, sporozoites of Isospora suis from pigs, and tachyzoites of Toxoplasma gondii and Neospora caninum from cell cultures. Monoclonal antibodies from the three clones reacted with the anterior end of E. bovis sporozoites, but did not react with the other parasites examined. None of the monoclonal antibodies reacted with merozoite antigens in immunoblots.


Specific alterations of the basement membrane and stroma antigens in human pituitary tumours in comparison with the normal anterior pituitary. An immunocytochemical study. Our report is the first immunocytochemical study of the principal elements of the basement membrane (BM) and connective tissue in normal and adenomatous human anterior pituitaries. In normal tissues, both the parenchymatous BM limiting the endocrine cell cords and the endothelial BM around the capillaries were continuous and were stained with anti-laminin (LM), anti-type IV collagen (CIV) and anti-fibronectin (FN) antisera. Antiserum to type I collagen (CI) stained the connective tissue only. The same antigens were investigated in 23 human pituitary adenomas, 6 of them having been diagnosed as locally invasive by the radiologist and the neurosurgeon. In all cases a lack of cordal structure was observed and the parenchymatous BM was completely absent (9 cases) or fragmented (14 cases). No correlation could be established between the extent of parenchymatous BM alterations and the invasive behaviour of the tumour. In contrast, a continuous endothelial BM was observed around the blood vessels in all cases and its presence was confirmed in double immunofluorescence experiments using anti-von Willebrand factor and anti-LM or anti-CIV antisera. Anti-FN and CI also stained the wall of the vessels. The tumours showed arterial development, in addition to the capillaries found in normal tissue. The present results favour the hypothesis of a decreased synthesis of parenchymatous BM by human adenomatous pituitary cells in comparison with normal cells and show that these tumours are the site of an active arterial neovascularization.


Carcinosarcoma of the urinary bladder: a distinct variant characterized by small cell undifferentiated carcinoma with neuroendocrine features. The clinicopathological features of two carcinosarcomas of the urinary bladder are reported. The tumours occurred in a 64- and a 66-year-old patient presenting with haematuria and both were polypoid. The epithelial component was consistent with small cell undifferentiated carcinoma with neuroendocrine differentiation, whereas the sarcomatous component did not display specific features. The carcinomatous component showed immunohistochemical reactivity for different epithelial markers as well as for chromogranin and neuron specific enolase. Conversely, the sarcomatous cells stained strongly for vimentin and in one case for muscle actin and smooth muscle actin. The differential diagnosis of biphasic tumours of the bladder is discussed and the literature reviewed.


Differential uptake of systemic fluorochrome Hoechst 33342 in lung and liver metastasis of B16 melanoma. The growth and vascularization patterns of B16 melanoma colonies in the liver and lungs were measured and compared by histological techniques and dye diffusion patterns after injection of the fluorochrome Hoechst 33342. In the liver, the fluorescent pattern of dye diffusion revealed that uninodular tumours measuring up to 146 microns in diameter were not functionally vascularized. However, when the nodules fused to give rise to multinodular tumours measuring between 256 and 366 microns in diameter, a reticular dye diffusion pattern revealed functional tumour vascularization. In the lungs, subpleural, parenchymal and peritubular (i.e. surrounding blood vessels and airways) tumours were observed. The two former classes were vascularized down to thicknesses and diameters of 49 and 24 microns respectively. In contrast, dye diffusion was never seen in peritubular tumour cuffs up to 609 microns in thickness. The results indicate differences in vascularization patterns in B16 tumours in the liver and lungs, and differences between tumours growing in different sites within the lungs. If these results are applicable to metastases in these two organs, they indicate potential diffusion-mediated resistance to chemotherapy, and potential hypoxia-mediated resistance to radiotherapy of both metastases and micrometastases.


Adrenocortical oncocytoma: case report with immunocytochemical and ultrastructural study. An adrenocortical oncocytic neoplasm was detected incidentally in a 58-year-old man. The tumour weighted 315 g, showing haemorrhagic areas and broad fibrous bands. It was composed exclusively of large eosinophilic cells packed with mitochondria showing flat and infrequent tubulovesicular cristae and regression of steroid-related organelles. Occasional annulate lamellae and mitochondrial osmiophilic inclusions were present. Vimentin was diffusely expressed, whereas AE1/AE3 cytokeratin was detected in half of the cells; a focal punctate pattern of staining was exclusively observed for cytokeratin peptides 8 and 18. The patient had no evidence of disease 21 months after surgery.


[The antigenic relationships of Morganella]. Interspecies relationships in the genus Morganella have been studied in the agglutination and adsorption tests. New data on the antigenic relationships of O groups O43 and O49 and more precise data on the antigenic relationships of O groups O1 to O42 have been obtained. For the first time the relationships of antigens 40 and 42 with 4, 35 and 31, 38 and 39, 9 and 45, 1a, d and 49; 45 and 47, 24 and 45; 5, 23, 26 with 47; 1, 19, 46 with 41, 48 have been described and the complete identity of O antigens 33 and 35; 34 and 36; 40 and 42; 3, 13 and 17 has been established. These data must be taken into consideration in the preparation of polyvalent agglutinating sera, necessary for finding out the serological picture of Morganella isolated from patients and the environment.


[The effect of the bronchoalveolar fluid of mice infected with the influenza virus on hematopoietic bone marrow precursors]. The influence of bronchoalveolar washing fluid (BAWF), as well as BAWF cells, obtained from mice infected with influenza virus, on the formation of exogenic spleen colony-forming units (CFUs) of lethally irradiated syngeneic recipients was studied. BAWF and BAWF cells of intact syngeneic mice stimulated the growth of CFUs. BAWF of mice infected with nonpathogenic strain A/PR/8/34 lost its capacity for stimulating the growth of colonies, and BAWF cells greatly suppressed colony formation in the spleen of recipients. The participation of interferon, colony-stimulating factor and the virus itself in the process of the modulation of colony formation is discussed.


Immunohistochemical localization of alpha 2-macroglobulin receptors in human skin. The localization of receptors for the proteinase inhibitor alpha 2-macroglobulin was studied in human skin by immunohistochemistry using a monoclonal mouse antibody. No epidermal staining was identified. alpha 2-Macroglobulin receptors were identified on dermal fibroblasts and dermal dendritic cells. Endothelial cells did not stain with the antibody, but positive staining cells were concentrated around dermal vessels. The myoepithelial layer of eccrine glands exhibited receptors; however, there was no staining of the eccrine epithelial layer. The distribution of alpha 2-macroglobulin receptors correlates with the reported distribution of alpha 2-macroglobulin: both are present in the dermal connective tissue and absent in epithelium and endothelium. The distribution of alpha 2-macroglobulin and its receptor in the dermis is consistent with a possible role in regulation of dermal proteolytic activity.


A continuous spectrum of hypercoagulability exists in acute nonlymphoblastic leukemia. A consumption coagulopathy syndrome has frequently been reported in association with some cases of acute nonlymphoblastic leukemia (ANLL) and mainly in acute promyelocytic leukemia (M3). Eighteen cases of ANLL have been studied on admission, before chemotherapy was started. Levels of antithrombin III (AT-III), protein C (PC), protein S (PS), thrombin-antithrombin complex (T-AT-III), tissue plasminogen activator, plasminogen (Pg), alpha-2-antiplasmin (alpha-2-AP), D-dimer (DD) and fibrinogen (Fg) were determined. The results showed normal levels of AT-III and PS, decreased levels of PC, alpha-2-AP, Pg and Fg in some cases, and an elevation of DD and T-AT III complex in almost all patients. There was a continuous evolution of data from M1 cases in which only slight alterations were seen up to M3 cases where all those pathologic data were observed.


Effects of interleukin-3 and interleukin-6 on peripheral blood cells from multiple myeloma patients and their clinical significance. The effects of interleukin-3 (IL-3) and interleukin-6 (IL-6) on nonadherent mononuclear cells (NMC) from the peripheral blood of 28 patients with multiple myeloma (MM), 3 patients with monoclonal gammopathy of undetermined significance (MGUS), and 3 normal controls were investigated. In 15 of 27 evaluable patients with MM, monoclonal-cytoplasmic-immunoglobulin (cIg)-positive plasma cells appeared from the T-cell-depleted NMC after 10 days of culture in the presence of IL-3 and IL-6. These changes were not observed in the T cell fraction of myeloma blood or in the T-cell-depleted NMC obtained from cases of MGUS or from normal controls. The percentage of cIg-positive plasmacytoid cells after 10 days of culture was significantly higher in the presence of both IL-3 and IL-6 than with each interleukin alone or the control medium. Furthermore, these changes were often observed in untreated patients. These findings suggest that myeloma precursor cells exist in the peripheral blood of MM patients, especially at diagnosis, and differentiate into cIg-positive cells in the presence of IL-3 and IL-6. This assay may be useful in discriminating the early stage of myeloma from MGUS.


DNA polymorphism in the beta-globin gene cluster in Saudi Arabs: relation to severity of sickle cell anaemia. Significant DNA polymorphisms have been reported in the beta-globin gene cluster of epsilon-G gamma-A gamma-psi beta-delta-beta-gene region, in normal (Hb AA) individuals and in patients with sickle cell anaemia (SCA). Investigations of the extent of the DNA polymorphisms in the beta A- and beta S-globin gene cluster using Hind III, Hinc II, Ava II, Xmn I, and Hpa I, revealed several associations with mild SCA. The correlation of the presence (+) or absence (-) of the restriction endonuclease site to clinical severity in patients homozygous for beta S-gene showed that the mild form of SCA was associated mainly (> 90%) with the Xmn I polymorphic site 5' to G gamma, and to a lesser extent with Hinc II polymorphic site 5' to epsilon and in the psi beta-gene, with Hind III polymorphic site in G gamma and Hpa I polymorphic site 3' to the beta-globin gene, while in the severe form of SCA these polymorphic sites were absent in most patients. The polymorphism in the beta-globin gene cluster was significantly related to the expression of the beta S-gene and clinical severity of SCA.


Heterogeneity and variation of clinical and haematological expression of haemoglobin S in Saudi Arabs. Sickle cell haemoglobin (Hb S) occurs at a high frequency in the Eastern (EP), South-Western (SWP) and North-Western (NWP) Provinces of Saudi Arabia and the presentation of the Hb S is believed to exhibit clinical diversity in the different regions. Three areas of Saudi Arabia were screened to determine the frequency of Hb S and alpha- and beta-thalassaemias and glucose-6-phosphate dehydrogenase deficiency genes. Furthermore, in an attempt to investigate and compare the clinical and haematological presentation of sickle cell disease (SCD) in the different regions of Saudi Arabia, the individuals identified as Hb S homozygotes were investigated further. The patients were further classified on the basis of whether there was associated alpha- or beta-thalassaemia. A severity index (SI) was calculated for each patient and the clinical presentations and laboratory findings were compared. The results showed significantly variable severity of SCD in patients from different regions. The patients from the EP generally had a mild clinical presentation, while in the SWP and NWP majority of the patients suffered from a severe disease as judged by the SI. No correlation could be established between Hb F level and SI, though the WBC level correlated positively with the SI. The lowest SI values were encountered in patients with associated alpha-thalassaemia who also had the lowest WBC count and MCV and the highest RBC count and packed cell volume.


Hb H disease caused by a homozygosity for the AATAAA-->AATAAG mutation in the polyadenylation site of the alpha 2-globin gene: hematological observations. We have identified 7 patients with Hb H disease as homozygotes for a mutation in the polyadenylation site (AATAAA-->AATAAG) and have compared their hematological data with those of Hb H patients having other types of alpha-thalassemia determinants. All 7 patients exhibited moderate anemia with microcytosis and hypochromia being similar to that observed in the other patients. Relatives with a heterozygosity for this mutation are borderline microcytic and hypochromic without a significant anemia but with a low in vitro alpha/beta chain synthesis ratio. Analyses of the hemoglobin components identified low levels of Hb A2 and Hb H that were comparable to those found in other patients with Hb H disease; the level of the zeta-chain was low (average 0.14%).


Nucleolar organizer regions in malignant salivary gland tumors. Proliferative activity of carcinomas arising from salivary glands was analyzed by enumeration of argyrophilic nucleolar organizer regions (AgNORs). The mean numbers of AgNORs in the various tumors were as follows: mucoepidermoid carcinoma, 2.20; acinic cell carcinoma, 2.51; adenoid cystic carcinoma (ACC), 2.57; carcinoma in pleomorphic adenoma, 1.00 (benign component) and 3.99 (cancer-bearing area); salivary duct carcinoma, 4.49; polymorphous low-grade adenocarcinoma, 3.37; sebaceous carcinoma, 2.57; oncocytic carcinoma, 4.63; adenocarcinoma, 4.53. Cells of most tumors showed heterogeneous activity within the same tumor. In mucoepidermoid carcinoma, the mucous cells had suppressed activity in comparison with the epidermoid cells and intermediate cells. In ACC, the activity of the tumor cells increased according to growth pattern in the order tubular, glandular and solid. In carcinoma in pleomorphic adenoma, vigorous proliferative activity was observed in the malignant component, whereas less active cells were seen in the myxoid or chondroid matrix. AgNOR staining was useful for distinguishing benign from malignant regions in carcinoma in pleomorphic adenoma. Our results suggest that mucoepidermoid carcinoma, acinic cell carcinoma and ACC, except for that with a solid growth pattern, may be considered as low-grade malignancies, whereas solid-type ACC, the cancer component in carcinoma in pleomorphic adenoma and some of the other carcinomas have high-grade malignant behavior.


A histochemical study of acid phosphatases in medullary bone matrix and osteoclasts in laying Japanese quail. Acid phosphatase activity in medullary bone matrix and osteoclasts of laying Japanese quail was examined histochemically. To avoid nonspecific staining, the reactivity of the enzyme was evaluated using both the azo dye method and the lead salt method and nonembedded thick sections and resin-embedded thin sections. The pH of the incubation medium was also varied from the acid range (pH 5.0 and 6.5) to the alkaline range (pH 8.5). Medullary bone osteoclasts contain both tartrate-resistant acid phosphatase (TRAP) activity and fluoride-resistant acid phosphatase (FRAP) activity, and no significant difference in intensity was detected between active and inactive osteoclasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts and suggest that medullary bone matrix includes TRAP throughout the matrix.


Preparation and characterization of a mouse osteoclast-like multinucleated cell population. We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.


Effects of stem cell factor on osteoclast-like cell formation in long-term human marrow cultures. Stem cell factor (SCF) is a newly described hematopoietic growth factor that stimulates the growth of primitive hematopoietic progenitors and mast cells. Since the osteoclast precursor is hematopoietic in origin, we tested SCF for its capacity to stimulate the formation of osteoclast-like multinucleated cells (MNC) in long-term human marrow cultures. These MNC express an osteoclast phenotype and form resorption lacunae on calcified matrices. Addition of SCF alone (0.1 pg/ml to 100 ng/ml) to long-term marrow cultures did not increase MNC formation. However, treatment of these cultures sequentially with SCF for 1 week followed by 1,25-(OH)2D3 for the second and third weeks of culture significantly enhanced MNC formation. [3H]Thymidine incorporation studies showed that SCF increased the proliferation of MNC precursors. These data suggested that SCF was acting on early MNC precursors. We then tested the capacity of SCF to stimulate the formation of colonies of committed precursors for osteoclast-like MNC. SCF (20 pg/ml to 20 ng/ml) enhanced osteoclast precursor formation in unfractionated bone marrow mononuclear cells but was unable to increase osteoclast precursor formation when a highly purified population of hematopoietic precursors was used as the target cells for SCF. These data suggest that SCF works in concert with other factors produced by nonhematopoietic marrow cells to increase the precursor pool for osteoclasts and that other factors, such as 1,25-(OH)2D3, complete the differentiation process to the mature osteoclast.


Computerized immunoblot analyses (CIBA) of the distribution of prekallikrein and its activation products in vivo and in vitro. The distribution of prekallikrein and the complexes of kallikrein with C1 inhibitor (C1INH), alpha 2-macroglobulin and approximately 58-kDa protein(s) (e.g. antithrombin III), differs in normal and C1INH-deficient human plasma, activated in vitro, due to different C1INH levels. Different distribution in the deficient plasma activated in vivo or in vitro, suggests different rates of complex clearance.


Determination of human low molecular weight kininogen by immunoassay. It was the aim of the present investigation to develop a convenient method for determination of human kininogens. Using mono- and polyclonal antibody preparations an ELISA-system for specific determination of LMWK could be developed. In addition, the specificity of the monoclonal antibody suggests that their epitope in HMWK and its heavy chain is different to that in LMWK.


Kininogen deficiency in the rat.

Kininogen deficiency in the rat. The Brown Norway Katholiek (B/N-Ka) strain rat is the only animal strain that demonstrates deficiency in plasma HMW- and LMW-kininogens with a low level of prekallikrein. We developed an RIA for rat HMW-kininogen, LMW-kininogen, and T-kininogen, and using them measured these proteins in B/N-Ka and normal strain (B/N-Ki) rats. Plasma level of immunoreactive as well as kinin-releasing HMW-kininogen and LMW-kininogen in B/N-Ka rats was either around 3% of their levels in the normal B/N-Ki rats. The cause of the plasma deficiency of kininogens in the B/N-Ka strain was examined by 35S-methionine uptake of primary cultures of hepatocytes from the B/N-Ki and B/N-Ka strains. The results indicated that the kininogens were synthesized in the B/N-Ka liver but not secreted into the medium. Northern blot analysis of poly A(+)RNA extracted from the livers of both strains demonstrated that the band corresponding to mRNA of HMW-kininogen was present in the mRNA from B/N-Ka liver as well as in that from the B/N-Ki one. The band was similar in size and intensity in both cases. This result confirmed the data that immunoreactive HMW-kininogen was found in the liver of B/N-Ka rats (12). Thus, the cause of plasma deficiency of HMW-kininogen in the mutant appears to be secretory defect in nature. The B/N-Ka rats showed less reactivity to the inflammatory stimulus, such as carrageenin or kaolin, but the strain expressed almost the same response as normal rats to phorbol ester (PMA) or zymosan for pleurisy induction. These results indicate that kinin may play an important role in exudation in carrageenin- and kaolin-induced edema but not in that induced by PMA or zymosan. The deficient rat strain could be useful for differentiation of the inflammatory model which shows involvement of the kinin system.


T-kininogen, processing and functions.

T-kininogen, processing and functions. Studies are presented which indicate that T-kininogen, the acute phase kininogen of the rat, could be a healing protein because of its properties as a cysteine protease inhibitor. Evidence is also presented that mRNA of T-kininogen synthesis may be a function of interleukin 6 production. A regulatory mechanism is postulated by which SH cofactors could determine if T-kinin is released or whether the T-kininogen molecule would remain intact. Evidence is also presented that T-kinin acts through kinin B2 receptors. No specific binding of bradykinin or T-kinin could be detected in rat heart preparations.


Further characterization of endopeptidase H2 a serine proteinase from human urine. A human urine serine proteinase chymotrypsin like hydrolyzes the peptide bonds: Phe-Ser (kinin); Gly-Gly, Leu-Arg, Phe-Lys (neuropeptides) and Gln-Gln (substance P). Endopeptidase H2 hydrolyzes better oligopeptides with 4 to 18 aminoacid residues than larger peptides, it does not hydrolyzes kininogen or proenkephalin. The enzyme behaves as an oligoendopeptidase.


Anti-idiotypic antibodies against the kinin receptor. Three sets of monoclonal antibodies against bradykinin (MBK1, MBK2, MBK3) were generated by somatic cell fusion, characterized by their peptide specificity and compared to the known ligand specificity of the kinin receptor subtypes. By these criteria the paratope of MBK3 resembled the B2 receptor binding site whereas MBK1 shared principal binding characteristics with the B1 recrptor. Anti-idiotypic antibodies against MBK1, MBK2 and MBK3 were raised in rabbit and sheep. Specificity of the network components was verified by inhibition experiments on the level of peptide, idiotype and anti-idiotype. Anti-idiotypic antibodies against MBK3 recognized a conformation-dependent epitope which was binding site-related. Binding studies on human foreskin fibroblasts and guinea pig ileum showed mutual displacement of the anti-idiotypic antibody and bradykinin at the binding site pointing to a specific interaction of the antibody with the receptor from various species. An agonist activity of the antibodies, demonstrated in human (inositolphosphate pathway) and mouse (prostaglandin pathway) fibroblasts indicated that the anti-idiotypes bear an internal image of the ligand epitope. This molecular mimicry which was further substantiated by the detection of bradykinin specific anti-idiotypic antibodies, provides the structural basis for the observed cross-reactivity over species borders.


Identification of proteins of the kallikrein family by isoelectrofocusing and immunoblotting. We have found that kallikrein-like proteins differ in their isoelectric point but share antigenic determinants. For identification of kallikrein-like proteins an initial separation was carried out in flat-bed isoelectrofocusing gels. The kallikrein-like nature was demonstrated by an immunological similarity to kallikrein-like proteins by immunoblotting using antiserum against a kallikrein family member for staining. We used this system to identify different kallikrein-like proteins during purification of both known as well as new enzymes.


Selective expression of sialyl-Lewis x and Lewis a epitopes, putative ligands for L-selectin, on peripheral lymph-node high endothelial venules. High endothelial venules (HEV) lined by the high endothelium are the sites where leukocytes enter into the lymph nodes from the blood. Lymphocyte homing into lymph nodes is organ-selective, i.e., different molecules are involved in the lymphocyte homing to peripheral nodes compared with mucosa associated lymphoid tissue. The traffic into peripheral nodes is regulated by the expression of L-selectin on leukocytes and its ligand on HEVs. The ligand for L-selectin is suggested to be a 50, 90, or 105 kd glycoprotein, which is sulfated, fucosylated, and sialylated. The two other members of the selectin family (E- and P-selectin) recognize sialyl-Lewis x and -Lewis a (sLex and sLea, respectively) carbohydrate motifs, and there is preliminary data suggesting that this would also be the case for L-selectin. We have initiated a study to identify the expression of these sialylated structures on endothelial surfaces. We present data that show that HEVs in peripheral nodes, but not in the mucosa-associated lymphoid tissue, express large quantities of sLex and sLea identified by MAbs in immunohistology. Endothelium in capillaries or larger vessels in non-lymphoid tissues do not react with anti-sLex or -Lea mAbs. Only 1-2% of the lymphocytes in the peripheral blood express sLex and so far only the skin-homing lymphocytes are known to be sLex positive in larger quantities. We show that in many occasions the B cells in the peripheral lymph-node germinal centers are also sLex-, but not sLea-positive, and provide evidence of the restricted pattern of sLex and sLea expression on peripheral lymph-node HEVs. We propose that they are at least parts of the ligand for L-selectin.


Interleukin-8. A corneal factor that induces neovascularization. A rabbit corneal pocket model was used to demonstrate that physiologic concentrations of human recombinant (r) IL-8 may induce corneal neovascularization. Computer-assisted analysis of sequential fluorescein angiograms showed that rIL-8 doses ranging from 2 to 40 ng/cornea (P = 0.01), but not high dose rIL-8 (400 ng/cornea), results in neovascularization within 14 days. Repeat fluorescein angiograms 6 weeks after placing angiogenic doses of rIL-8 demonstrated significant regression (P = 0.01) of the vascularity present at 2 weeks, suggesting that IL-8 angiogenesis undergoes dynamic modulation similar to that normally seen in wound healing. To our knowledge, this is the first study showing an angiogenic role for IL-8, a finding that emphasizes the interplay between inflammation and wound healing. Our results imply that corneal-derived IL-8 may be important in corneal neovascularization, in particular, and that IL-8 may modulate wound healing in general. Finally, these results raise the possibility that corneal-derived cytokines, such as IL-8, may obfuscate the effects of agents tested in experimental corneal pocket models.


Development and characterization of an experimental model of brain abscess in the rat. Brain abscesses were produced in the rat by direct intracerebral injection of agarose beads laden with Staphylococcus aureus. The method proved to be easy, reproducible, effective and was associated with a low mortality rate. The histopathologic features of the experimental abscess are similar to other animal models and to human abscesses. The encapsulation of the lesion, macrophage/microglial response, astrocytic activation, and changes in the surrounding brain parenchyma were studied by immunohistochemistry. Edema, diffuse microglial activation and diffuse astrocytosis characterized the early reaction of the injected cerebral hemisphere. After day 10, edema subsided, and the microglial and astrocytic responses became restricted to the area around the lesion. Fibronectin deposition in the capsule preceded the appearance of myofibroblasts, which was concurrent with the beginning of collagen deposition on day 9. Hypervascularity of the capsule appeared as early as day 6 and persisted through day 28. This study suggests that brain abscess formation can be separated into three components: an initial period of edema and glial activation; an intermediate phase of neovascularization and fibronectin deposition; and a final phase of collagen deposition and progressive fibrosis. This new model offers an excellent paradigm for the analysis of neural tissue reaction and de novo fibrous tissue deposition.


Regulated expression of vascular cell adhesion molecule-1 in human malignant melanoma. Expression of the endothelial adhesion molecule VCAM-1 was studied in human malignant melanoma lines by flow cytometry. Clones 2/4 and 2/14 (derived from the same lesion) had appreciable levels of VCAM-1 expression, whereas clone 2/21 and the lines A2058, Mel24, and A375 were negative. Clone 2/14 was selected for further analysis. Exposure to tumor necrosis factor (TNF) markedly augmented VCAM-1 on melanoma cells. Surface VCAM-1 was associated with expression of specific transcripts that were augmented by TNF. Analysis by reverse transcriptase and polymerase chain reaction using appropriate primers revealed that TNF-stimulated melanoma cells expressed both 7 and 6 immunoglobulin domain transcripts with predominance of the longer species. Tumor necrosis factor--stimulated melanoma cells bound more VLA-4-expressing cells (melanoma and monocytes) than resting tumor cells and anti-VCAM-1 monoclonal antibodies significantly inhibited binding, thus suggesting that surface VCAM-1 on melanoma is functional. Analysis of melanoma tissue sections demonstrated that VCAM-1 is not a marker of transformation of melanocytes because it can be detected in benign nevi. Although, unlike ICAM-1, VCAM-1 is not correlated with tumor progression, its expression in a fraction of primary melanomas indicates that it may play a role in regulating host immune response and homotypic interactions in some malignant melanomas.


Cutaneous expression of Thy-1 in mycosis fungoides. Dermal dendritic cells from eleven cases of mycosis fungoides (MF) (six patch and five plaque stage), two cases of pre-MF, and five specimens of normal human skin, were characterized immunohistochemically using a panel of antibodies including anti-human Thy-1, intercellular adhesion molecule-1 (ICAM-1; CD54), endothelial leukocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD1a, CD2, CD14, CD18, CD34, MAC387, KP-1, EBM-11, factor XIIIa, factor XIIIs, and S100. Thy-1 expression in normal skin was limited to the microvascular endothelium and perivascular dendritic cells. An extensive interstitial network of Thy-1+ dendritic cells was seen in the papillary dermis of all cases of MF, whereas no epidermal cells were Thy-1+. The mean +/- standard deviation of interstitial Thy-1+ cells per high power field in the dermis was: normal skin, 2.86 +/- 0.34; pre-MF, 15; patch stage MF, 13.4 +/- 7.08; plaque stage MF, 49.96 +/- 21.29. Thy-1+ dendritic cells morphologically resembled the factor XIIIa+ "dermal dendrocyte" (DD) and shared their VCAM-1+, ICAM-1+, CD1a, CD2-, CD14+, CD18+, EMB11+, factor XIIIa+, factor XI-IIs-, S100-, MAC387- and KP-1-immunophenotype in MF. Double labeling studies revealed up to 50% of Thy-1+DD were also factor XIIIa+ in MF. Immediately beneath these cells was a similar network of CD34+, Thy-1-, factor XIIIa- dendritic cells limited to the reticular dermis. Strong microvascular endothelial cell expression of Thy-1 and VCAM-1, and focal vascular ELAM-1 expression were also seen in MF. Distinct cellular compartmentalization (papillary dermis versus reticular dermis versus epidermis) of dendritic cells is demonstrated by the differential expression of Thy-1, factor XIIIa, and CD34 antigens. The extensive number and prominent dermal dendritic network in the papillary dermis juxtaposed between epidermal keratinocytes (KC) and dermal/epidermal T cells, suggests an important pathophysiologic role for this newly recognized and immunophenotypically distinctive cell population in MF.