Biomedical subjects

G Ju

Publications and source records attributed to G Ju.

At least 91 records · Page 5Linked to original sources

PubMed1993

Human interleukin 2 analogues that preferentially bind the intermediate-affinity interleukin 2 receptor lead to reduced secondary cytokine secretion: implications for the use of these interleukin 2 analogues in cancer immunotherapy.

Cancer patients undergoing interleukin (IL)-2-based immunotherapy frequently experience dose-limiting side effects believed to be caused by the actions of such cytokines as IL-1 beta, tumor necrosis factor (TNF)-alpha and -beta, and interferon-gamma (IFN-gamma). Human peripheral blood mononuclear cells (PBMC) or monocyte-depleted peripheral blood lymphocytes were stimulated for up to 7 days by either of 2 IL-2 analogues (R38A or F42K) that bind to the intermediate-affinity IL-2 beta gamma receptor but have reduced abilities to bind the high-affinity IL-2 receptor. We previously reported that these IL-2 analogues retain the ability to generate lymphokine-activated killing by PBMC. In this study, we analyzed the cytokine content of supernatants from stimulated PBMC and peripheral blood lymphocyte cultures by enzyme-linked immunosorbent assay. The secretions of IL-1 beta, TNF-alpha, and -beta, and IFN-gamma induced by either R38A or F42K were markedly reduced compared with secretions produced in response to recombinant wild-type IL-2. In 4 experiments, secretion was reduced an average of 39% for IL-1 beta, 57% for TNF-alpha, 83% for TNF-beta, and 86% for IFN-gamma. Polymerase chain reaction analysis of recombinant wild-type IL-2 or analogue-stimulated PBMC did not reveal the presence of IL-2 mRNA; thus, differential production of endogenous IL-2 could not account for these findings. These data suggest the interaction of IL-2 and the high-affinity IL-2 receptor on human PBMC or peripheral blood lymphocyte is required for maximal secretion of IL-1 beta, TNF-alpha, TNF-beta, and IFN-gamma. Because such cytokines are believed to mediate the toxicity seen with IL-2-based immunotherapies, IL-2 analogues with reduced binding to the high affinity IL-2 receptor may prove to be an effective and less toxic means of cancer treatment.

Base Sequence↗

PubMed1993

Unexpected effects of the IL-2 receptor alpha subunit on high affinity IL-2 receptor assembly and function detected with a mutant IL-2 analog.

Binding of IL-2 to the high affinity IL-2R results in the formation of a stable complex consisting of IL-2 and at least three distinct, noncovalently associated receptor subunits: IL-2R alpha (p55), IL-2R beta (p70-75), and IL-2R gamma (p64). Ligand binding also stimulates the rapid endocytosis of this receptor complex. To gain further insight into the function of the various subunits of the multicomponent high affinity IL-2R complex, we have carried out binding, internalization, and proliferation assays using an IL-2 analog designated F42A that contains a single Phe for Ala amino acid substitution at position 42. This mutation markedly reduces the intrinsic affinity of the resultant IL-2 analog for the low affinity IL-2R alpha subunit although having little or no effect on binding to the IL-2R beta (or IL-2R beta/gamma) intermediate affinity receptor. We have confirmed that F42A does not bind to the IL-2R alpha chain when expressed alone on MT-1 cells or in the presence of the large or small excess of IL-2R beta chains present on either YT-1 cells or forskolin-induced YT-1 cells, respectively. However, although F42A does not interact with the large number of low affinity IL-2R alpha chains present on HUT 102B2 cells, this ligand does bind to the small number of IL-2R beta chains present on these cells with at least 10-fold higher than expected affinity. These findings indicate that excess IL-2R alpha chains may exert positive effects on binding perhaps by changing the conformation of IL-2R beta. In F42A-stimulated internalization assays on forskolin-induced YT-1 cells, the IL-2R alpha chain is consistently endocytosed together with the IL-2R beta subunit indicating that IL-2R alpha is stably associated with the F42A-IL-2R beta complex even though the alpha-subunit contributes little if any affinity to the F42A binding reaction. In proliferation assays on mouse IL-3-dependent pro-B BA/F3 cells stably coexpressing transfected human IL-2R alpha and IL-2R beta subunits (but a mouse IL-2R gamma subunit), F42A proved to be a more effective agonist of growth than wild-type IL-2. These results suggest that the interaction between wild-type IL-2 but not F42A and the IL-2R alpha subunit in this mixed species high affinity receptor complex may induce an unfavorable receptor conformation leading to diminished rather than enhanced growth signal transduction.

Animals↗

PubMed1993

Characterization of lymphokine-activated killing by human peripheral blood mononuclear cells stimulated with interleukin 2 (IL-2) analogs specific for the intermediate affinity IL-2 receptor.

Interleukin 2-stimulated human peripheral blood mononuclear cells (PBMC) generate lymphokine-activated killing (LAK). Using the IL-2 analogs R38A and F42K, which interact primarily with the beta and gamma subunits of the IL-2 receptor, we assessed the roles of IL-2R beta gamma and the high-affinity IL-2 receptor complex in LAK activation. Although the kinetics of LAK activation were identical, lytic activity was approximately 30% lower and proliferation was up to 55% lower in those PBMC stimulated by R38A or F42K than in those exposed to wild-type IL-2. The percentage of cells expressing cell-surface markers such as CD3, CD4, CD8, and CD16 was not significantly different after treatment with wild-type IL-2, R38A, or F42K; however, the proportion of cells expressing IL-2R alpha increased dramatically in response to stimulation by F42K (30%) compared to stimulation by either rIL-2 or R38A (15%). In addition, by Day 7 the concentration of soluble IL-2R alpha in analog-stimulated LAK culture supernatants was 50-75% less than that from wild-type IL-2-cultured cells. These findings suggest that interaction of IL-2 with IL-2R beta gamma alone is sufficient for both proliferation and the generation of LAK, and that stimulation with subunit-specific IL-2 analogs results in differential regulation of the IL-2R alpha on human LAK cells.

Binding, Competitive↗

PubMed1993

Effect of peripheral nerve cut on neuropeptides in dorsal root ganglia and the spinal cord of monkey with special reference to galanin.

Using the indirect immunofluorescence method and in situ hybridization, the localization and levels of immunoreactivities and mRNAs for several neuropeptides were studied in lumbar dorsal root ganglia and spinal cord of untreated monkeys (Macaca mulatta) and after unilateral transection of the sciatic nerve. Immunoreactive galanin, calcitonin gene-related peptide, substance P and somatostatin and their mRNAs were found in cell bodies in dorsal root ganglia of untreated monkeys and on the contralateral side of the monkeys with unilateral sciatic nerve lesion. After axotomy there was a marked decrease in the number of calcitonin gene-related peptide-, substance P- and somatostatin-positive neurons in dorsal root ganglia ipsilateral to the lesion, whereas the number of galanin positive cells strongly increased. A few neuropeptide tyrosine-positive cells were seen in after axotomy, whereas no such neurons were found in controls. No vasoactive intestinal polypeptide-, peptide histidine isoleucine-, cholecystokinin-, dynorphin-, enkephalin-, neurotensin- or thyrotrophin releasing hormone-positive cell bodies were seen in dorsal root ganglia of any of the groups studied. In the dorsal horn of the spinal cord all peptide immunoreactivities described above, except thyrotropin releasing hormone, were found in varying numbers of nerve fibres with a similar distribution in untreated monkeys and in the contralateral dorsal horn in monkey with unilateral sciatic nerve lesion. Two cholecystokinin antisera were used directed against the C- and N-terminal portions, respectively, showing a distinctly different distribution pattern in the dorsal horn. Somatostatin- and dynorphin-like immunoreactivities were also observed in small neurons in the dorsal horn. No certain effect of axotomy on these interneurons could be seen. However, marked changes were observed after this type of lesion for some peptide containing fibres in the ipsilateral dorsal horn. Thus, there was a marked increase in galanin-like immunoreactivity, whereas calcitonin gene-related peptide-, substance P-, somatostatin-, peptide histidine isoleucine neurotensin- and cholecystokinin-like immunoreactivities decreased. No changes could be observed in neuropeptide tyrosine or enkephalin-positive fibres. The present results demonstrate marked ganglionic and transganglionic changes in peptide levels after peripheral axotomy. When compared to published results on the effect of axotomy on peptides in dorsal root ganglia and spinal cord of rat, both similarities and differences were encountered.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Calcitonin gene-related peptide- and substance P-like-immunoreactive innervation of the anterior pituitary in the rat.

In our previous studies substantial amounts of substance P- and calcitonin gene-related peptide-like-immunoreactive nerve fibers have been identified in the anterior pituitary of the monkey and the dog. They were found to be in close proximity to the gland cells, even making synaptic contacts with some types of the gland cells. The present study investigated in detail the calcitonin gene-related peptide- and substance P-like immunoreactivities of the anterior pituitary in the rat. Though the immunoreactive fibers were not as abundant as in the anterior pituitary of the monkey and the dog, they still appeared in notable amounts. The calcitonin gene-related peptide- and substance P-like-immunoreactive nerve fibers occurred mostly as thin, tortuous, and densely varicose fibers, weaving among the gland cells. They are widely distributed, more in the central part of the gland. Double-immunostaining proved nearly complete co-localization of these two peptides in the nerve fibers. It is hypothesized that the anterior pituitary can be regulated by direct neural factors as well as humoral factors.

Animals↗

PubMed1993

Structure-function analysis of human IL-1 alpha: identification of residues required for binding to the human type I IL-1 receptor.

Using oligonucleotide-directed mutagenesis, the binding site on human interleukin-1 alpha (IL-1 alpha) for the human type I IL-1 receptor (IL-1R) has been analyzed. Substitution of seven amino acids (Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resulted in a significant loss of binding to the receptor. Based on crystallographic information, the side chains of these residues are clustered in one region of IL-1 alpha and exposed on the surface of the protein. Five of the residues in the IL-1 alpha binding site align with the binding residues previously determined in human IL-1 beta, demonstrating that the type I IL-1R recognizes homologous regions in both ligands. Unexpectedly, only three of the aligned residues are identical between IL-1 alpha and IL-1 beta. These observations suggest that the composition of contact residues in the binding site is unique for each ligand-receptor complex in the IL-1 system.

Animals↗

PubMed1992

Responses of neurons in the rat anterior hypothalamic periventricular zone to osmotic stimulation and angiotensin II in vitro.

Extracellular recordings were made in vitro from neurons in slices of the rat periventricular zone surrounding the anterior portion of the third ventricle. Spontaneous unit activity of these neurons was decreased in 39.5% of the neurons tested, increased in 32.9%, and biphasically changed (i.e., excitation-inhibition) in 10.5%, in response to bath application of hypertonic artificial cerebrospinal fluid (aCSF) containing elevated levels of NaCl (330 mOsm). Similar responses were observed following bath application of hypertonic aCSF prepared with additional mannitol. Angiotensin II in the perfusion medium dramatically increased spontaneous unit activity in 73.7% of neurons tested, while, unexpectedly, 5.3% were inhibited. These results suggest that some neurons in the periventricular zone are themselves osmosensitive, and may be involved in the regulation of water balance.

Angiotensin II↗

PubMed1992

An electron microscopical study of calcitonin gene-related peptide-like immunoreactive innervation of the anterior pituitary in the dog.

Our previous studies have demonstrated the presence of a considerable number of substance P-, calcitonin gene-related peptide (CGRP)-, and galanin-like immunoreactive (LI) nerve fibers in the anterior pituitary in several mammalian species. The present study investigated the ultrastructure of the CGRP-LI innervation of this gland in the dog. The CGRP-LI nerve fibers were unmyelinated, with a wealth of varicosities containing both small clear synaptic vesicles and large dense-cored vesicles. They were found to be in direct contact with every cell type of the anterior pituitary. However, only on corticotropes and somatotropes were CGRP-LI synaptic contacts identified. Most of them were asymmetrical in type. Occasional symmetrical synaptic contacts were also found. It is considered likely that direct neural factors may play a role in the regulation of the anterior pituitary.

Animals↗

PubMed1992

Neuronal responses of the anterior commissural nucleus to osmotic stimulation and angiotensin II in hypothalamic slices in the rat.

The firing rate and pattern of activity of neurons in the anterior commissural nucleus (ACN), which was rich in oxytocin-containing neurons, were studied electrophysiologically in hypothalamic slices. Extracellular recording showed that most ACN neurons exhibited irregular or regular continuous spontaneous unit activity. Other neurons showed short burst patterns of activity or were silent. The majority of ACN neurons were activated by bath application of angiotensin II, and a substantial number of them showed inhibitory or excitatory responses to hypertonic bathing medium. These results indicate that magnocellular neurosecretory neurons in the ACN may participate in the regulation of water balance.

Angiotensin II↗

PubMed1992

The interleukin 2 receptor (IL-2R): the IL-2R alpha subunit alters the function of the IL-2R beta subunit to enhance IL-2 binding and signaling by mechanisms that do not require binding of IL-2 to IL-2R alpha subunit.

Interleukin 2 (IL-2)-mediated signaling through its high-affinity receptor involves a complex interrelationship between IL-2 and two IL-2-binding chains, IL-2R alpha and beta chains. Previously with the reagents available it was difficult to define functional interactions between these two IL-2R subunits involved in IL-2 binding and signal transduction. To extend our understanding of the interplay between the two binding subunits we have done studies with the monoclonal antibody HIEI, which interferes with interaction of IL-2R alpha and beta chains (IL-2R alpha and IL-2R beta, respectively). Furthermore, we used two forms of IL-2, recombinant native IL-2 and F42A, an IL-2 analog (Phe-42----Ala substitution) that binds only to IL-2R beta. Analog F42A manifested 75-100% of the bioactivity of wild-type IL-2. This observation is inconsistent with the strict hierarchical IL-2-binding affinity conversion model previously proposed by Saito and coworkers [Saito Y., Sabe, H., Suzuki, N., Kondo, S., Ogura, T., Shimizu, A. & Honjo, T. (1988) J. Exp. Med. 168, 1563-1572] that predicted an ordered sequence of events in which IL-2 must first bind to IL-2R alpha before its interaction with IL-2R beta. Previous investigations using IL-2 variants were interpreted to show that IL-2R alpha merely acts to concentrate IL-2 to the cell surface and that no other meaningful interaction occurred between IL-2R alpha and IL-2R beta. However, our data are inconsistent with this view. We draw this conclusion on the basis of our observation that antibody HIEI, which reacts with an epitope of IL-2R alpha and interferes with interaction of this chain and IL-2R beta, inhibits the IL-2-dependent proliferative effects mediated by analog F42A. Furthermore, by blocking interaction of IL-2R alpha and IL-2R beta with the antibody HIEI, a decrease in the affinity of radiolabeled analog F42A for IL-2R beta was seen. In our proposed model IL-2R alpha contributes several functions to IL-2-mediated signaling through the high-affinity IL-2R. These functions include concentration of IL-2 within the two-dimensional surface of the plasma membrane as well as alteration of the functional capacity of IL-2R beta, an effect that does not require prior binding of IL-2R to IL-2R alpha. The IL-2R alpha-mediated augmentation of IL-2R beta functions involves affinity conversion of IL-2R beta, increasing its affinity for IL-2, and may involve facilitation of Il-2-mediated signaling after binding of IL-2 to this IL-2R beta.

Amino Acid Sequence↗

PubMed1992

Third ventricular subependymal oxytocin-like immunoreactive neuronal plexus in the rat.

In a previous retrograde tracing study, a dense subependymal neuronal plexus was found along the anterior ventral third ventricle that projects to the posterior pituitary. In the work reported here, the oxytocin-like immunoreactive neurons of this plexus were studied in detail. It has a population of about 650 cells with a great wealth of dendrites. The neurons are of magnocellular neurosecretory type with long straight dendrites running parallel to the ependyma. The plexus is composed of a dorsal and a ventral part. The dorsal part consists of about 75% of the whole population and is most dense at the levels of the anterior and medial magnocellular paraventricular nuclei. Their dendrites appear vacuous in immunohistochemically stained sections and have a tendency to form fascicles. The ventral part is more sparse. The dendrites of the subependymal plexus are well organized so the anteriorly located ones tend to be directed rostrally and the posteriorly ones caudally. The functional significance of the plexus is discussed.

Animals↗

PubMed1991

Identification of the discontinuous binding site in human interleukin 1 beta for the type I interleukin 1 receptor.

Human interleukin 1 beta (IL-1 beta) exerts its diverse biological effects by binding to specific receptors on target cells. Two types of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68). Using site-specific mutagenesis, we have identified the binding site on IL-1 beta for the murine type I IL-1R. Analogs of the IL-1 beta protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs. Substitutions of the amino acids at seven positions resulted in analogs that had greater than or equal to 100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R. These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) are clustered in the IL-1 beta molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-1 beta. The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.

Amino Acid Sequence↗

PubMed1991

Convergence of somatosensory and baroreceptive inputs onto parabrachio-subfornical organ neurons in the rat: an electrophysiological study.

Electrophysiological characteristics were described for neurons of the parabrachial nucleus (PBN) which receive baroreceptive and somatosensory inputs in the rat. Following focal electrical stimulation in the ipsilateral caudal nucleus of the tractus solitarii (NTS), the firing rates of these neurons were increased in 94 (55.6%), and decreased in 38 (22.5%). Fifty-three (54.5%) of 97 PBN neurons tested were excited, and 11 (11.3%) inhibited in response to contralateral common peroneal nerve (CPN) stimulation. Of these neurons, 52 were found to respond to both caudal NTS and CPN stimulation. Effects of subfornical organ (SFO) stimulation were also examined in 151 of 169 parabrachial neurons. 13 (7.7%) were activated antidromically and were located in the lateral division of the PBN, while 34 (22.5%) were affected orthodromically. Stimulation of the caudal NTS resulted in both a fall in the heart rate and changes of PBN neuronal firing rates. Similar effects were elicited by activating peripheral baroreceptors by the administration of phenylephrine, an alpha-adrenergic agonist. These results strongly indicate that: (1) the lateral PBN is involved in central cardiovascular control; (2) somatosensory and baroreceptive messages may converge onto some PBN neurons; and (3) some PBN neurons may relay baroreceptive information from the caudal NTS to the SFO.

Action Potentials↗

PubMed1991

The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain.

The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.

Antibodies, Monoclonal↗

PubMed1991

Activation of corticotropin-releasing factor-containing neurons in the paraventricular nucleus of the hypothalamus by interleukin-1 in the rat.

Interleukin-1 (IL-1) alpha was injected into the lateral ventricle of the rat. An antiserum against Fos oncoprotein was used to detect the activated neurons immunohistochemically. A large number of neurons in the parvocellular paraventricular nucleus of the hypothalamus, in an area corresponding to the location of the corticotropin-releasing factor (CRF)-containing neurons, were strongly Fos-like immunoreactive (LI). Double immunostaining for Fos and CRF revealed that many of the Fos-LI cells were CRF-LI. Furthermore, the CRF-like immunoreactivity was greatly enhanced in the rats injected with IL-1 alpha, indicating an increase in CRF synthesis.

Animals↗

PubMed1991

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.

Amino Acid Sequence↗

PubMed1991

Conversion of the interleukin 1 receptor antagonist into an agonist by site-specific mutagenesis.

Interleukin 1 (IL-1) receptor antagonist (IL-1ra) is a naturally occurring protein that binds to the IL-1 receptor present on T cells, fibroblasts, and other cell types and acts to block IL-1-induced responses. IL-1ra is a pure antagonist and has no agonist activity in in vitro or in vivo systems. By site-specific mutagenesis, an analog of IL-1ra was created that contained a substitution of a single amino acid, Lys-145----Asp. This analog, IL-1ra K145D, exhibited partial agonist activity in the D10.G4.1 cell proliferation assay. The newly acquired agonist activity could not be neutralized by antisera to IL-1 alpha or IL-1 beta, but it could be blocked by a monoclonal antibody to the T-cell IL-1 receptor. The analog also showed agonist activity as assayed by increased prostaglandin E2 synthesis from CHO cells expressing recombinant mouse IL-1 receptor. These results with IL-1ra K145D demonstrate the importance of the region surrounding the corresponding Asp-145 residue in IL-1 beta for triggering the biological response to IL-1.

Animals↗

PubMed1991

Calcitonin gene-related peptide-like immunoreactivity and its relation with neurotensin- and corticotropin-releasing hormone-like immunoreactive neurons in the bed nuclei of the stria terminalis in the rat.

The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactive (LI) nerve cells and terminals (BST) was studied in the rat. Only the fusiform nucleus was found to consistently contain CGRP-LI neurons. The CRGP-LI terminals had a wide distribution in the BST, being most numerous in the oval and the fusiform nuclei. In the oval nucleus the CGRP-LI terminals formed characteristic "baskets." Since the oval nucleus was known to be studded with neurotensin (NT)- and corticotropin-releasing hormone (CRH)-LI neurons, the relationship of the CGRP-LI nerves to NT- and CRH-LI neurons was investigated by means of a double immunostaining technique. A part of the CGRP-LI baskets were found to contain NT- or CGRP-LI neurons. The possible involvement of the CGRP/NT or CRH relationship in reciprocal connection between the BST and the parabrachial nucleus and in cardiovascular regulation is discussed.

Animals↗