Search

Intravenous (iv) injection of secretin activates neurons in brain areas controlling autonomic function and emotion. Peripheral administration of secretin inhibits gastric functions through a central mechanism that is mediated by vagal dependant pathways. We investigated whether the vagus nerve is involved in intraperitoneal (ip) injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 microgram/kg, ip, -90 min) induced a linear dose-related increase in the number of Fos positive neurons in the central nucleus of the amygdala (CeA), and a plateau Fos response in the area postrema (AP), nucleus tractus solitarii (NTS), locus coeruleus (LC), Barrington's nucleus (Bar), external lateral subnucleus of parabrachial nucleus (PBel) and arcuate nucleus (Arc), and at the highest dose, in the dorsal motor nucleus of the vagus (DMV) compared with ip injection of vehicle. Double immunohistochemistry in the NTS showed that a substantial number of neurons activated by secretin (40 microgram/kg, ip) are tyrosine hydroxylase positive. Subdiaphragmatic vagotomy (-7 days) abolished Fos expression-induced by ip secretin (40 microgram/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, capsaicin (-10 days) did not influence the Fos induction in the above nuclei. Quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared to the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in pontine and limbic nuclei regulating autonomic functions and emotion through vagal dependent capsaicin resistant pathways. Secretin injected ip may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood brain barrier.

Examines the role of epithelial gamma/delta T cells in tissue repair. Ontogeny and tissue distribution of intraepithelial gamma/delta T cells; Antigen recognition by intraepithelial gamma/delta T cells; Signal transduction via gamma/delta T cell receptor and CD3 complex; Cytokines, chemokines and growth factors.

BIVV003 is a gene-edited autologous cell therapy in clinical development for the potential treatment of sickle cell disease (SCD). Hematopoietic stem cells (HSC) are genetically modified with mRNA encoding zinc finger nucleases (ZFN) that target and disrupt a specific regulatory GATAA motif in the BCL11A erythroid enhancer to reactivate fetal hemoglobin (HbF). We characterized ZFN-edited HSC from healthy donors and donors with SCD. Results of preclinical studies show that ZFN-mediated editing is highly efficient, with enriched biallelic editing and high frequency of on-target indels, producing HSC capable of long-term multilineage engraftment in vivo, and express HbF in erythroid progeny. Interim results from the Phase 1/2 PRECIZN-1 study demonstrated that BIVV003 was well-tolerated in seven participants with SCD, of whom five of the six with more than 3 months of follow-up displayed increased total hemoglobin and HbF, and no severe vaso-occlusive crises. Our data suggest BIVV003 represents a compelling and novel cell therapy for the potential treatment of SCD., (© 2024. The Author(s).)

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoβ (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects., Competing Interests: Competing interest statement: All authors, except for L.M., were employees of Genentech, Inc., a member of the Roche group, at the time this work was performed and own/owned Roche stock and/or options at the time of their employment. L.M. is an employee and shareholder of ProSciento, Inc.; ProSciento, Inc. received research funding from Genentech, Inc. A.B. is currently an employee of Calico Life Sciences. J.S. is currently an employee of Regeneron Pharmaceuticals. R.B. is currently an employee of IgGenix. P.S.A. and L.W.C. are currently employees of Principia Biopharma., (Copyright © 2020 the Author(s). Published by PNAS.)

Secretin infused into rats activates neurons located in brain areas controlling autonomic function and emotion. The brain activity of secretin is mediated, at least in part, through vagal pathways. It is known that afferent stimulation of the vagus nerve results in considerable antiepileptic effects. Whether or not secretin has an effect on seizures is unknown. In this study, we evaluated the efficacy and safety of secretin as an antiepileptogenic agent in electrical kindling and as an anticonvulsant in fully kindled seizures. To assess antiepileptogenic effects, we administered secretin (10, 30, or 100 microg/kg/dose) or normal saline intravenously 5 min before twice-daily kindling stimulation. To assess the anticonvulsant effect of secretin, we administered either normal saline or secretin (100 microg/kg/dose) 5 min before the electrical stimulation to fully kindled rats. We observed no effect on kindling rate or afterdischarge duration. In fully kindled rats, secretin administration had no effect on kindling stage or afterdischarge duration. Thus, in the dose range used in this preliminary acute treatment study, secretin had no discernible antiepileptogenic or anticonvulsant effects. Secretin was very well tolerated in this multidose protocol.

Background: Secretin is a "gut-brain" peptide whose neural function is as yet poorly understood. Several clinical studies have reported modestly increased social interaction in autistic children following intravenous secretin administration. Very recently secretin also was administered to schizophrenic patients and found to increase social interaction in some individuals., Methods: In light of this finding, we assessed the ability of secretin to reverse phencyclidine- (PCP) induced impairment in prepulse inhibition (PPI), a leading animal model of sensorimotor gating deficits in schizophrenia., Results: Similar to atypical antipsychotics, secretin (1, 3, 10, 30, and 100 microg/kg) partially and dose-dependently reversed the PCP-induced deficit in PPI without significantly affecting baseline startle when administered intraperitoneally (IP) 10 minutes following IP administration of PCP (3 mg/kg)., Conclusions: This finding may be relevant to observations of antipsychotic efficacy of secretin in schizophrenic patients as well as our previous report that systemically administered secretin is capable of modulating conditioned fear, even at quite low doses.

In the present study expression levels of secretin and secretin receptor mRNAs in several brain regions of rats ranging in age from postnatal days 7 to 60 were investigated by quantitative real-time PCR. Expression of secretin and secretin receptor was detected in the central amygdala, hippocampus, area postrema, nucleus of the tractus solitary and cerebellum. The cerebellum expressed secretin receptor at significantly higher levels than that found in other brain regions within all the ages examined. In contrast, secretin mRNA was significantly higher in the nucleus of the tractus solitary than in the other four brain regions examined in postnatal day-21, -30 and -60 rats. Within most brain regions, both secretin and secretin receptor mRNAs were more abundant in postnatal day-7 and -14 rats as compared to postnatal day-21, -30 and -60 rats. Thus, secretin and its receptor are widely expressed in rat brain and the expression of both genes is developmentally regulated during the first few weeks following birth.

The recent suggestion that secretin may be useful in treating autism and schizophrenia has begun to focus attention on the mechanisms underlying this gut-brain peptide's actions in the central nervous system (CNS). In vitro autoradiographic localization of (125)I-secretin binding sites in rat brain shows the highest binding density in the nucleus tractus solitarius (NTS). Recent evidence suggests that intravenous infusion of secretin causes fos activation in NTS, a relay station playing important roles in the central regulation of autonomic functions. In this study, whole cell patch-clamp recordings were obtained from 127 NTS neurons in rat medullary slices. The mean resting membrane potential of these neurons was -54.7 +/- 0.3 mV, the mean input resistance was 3.7 +/- 0.2 GOmega, and the action potential amplitude of these neurons was always >70 mV. Current-clamp studies showed that bath application of secretin depolarized the majority (80.8%; 42/52) of NTS neurons tested, whereas the remaining cells were either unaffected (17.3%; 9/52) or hyperpolarized (1.9%; 1/52). These depolarizing effects were maintained in the presence of 5 microM TTX and found to be concentration dependent from 10(-12) to 10(-7) M. Using voltage-clamp techniques, we also identified modulatory actions of secretin on specific ion channels. Our results demonstrate that while secretin is without effect on net whole cell potassium currents, it activates a nonselective cationic conductance (NSCC). These results show that NTS neurons are activated by secretin as a consequence of activation of a NSCC and support the emerging view that secretin can act as a neuropeptide within the CNS.

Rationale: Previous results indicate that peripheral administration of secretin leads to robust Fos protein expression in the central nucleus of the rat amygdala. The implications of this observation on rat brain function, if any, remain unclear., Objectives: We examined the effect of systemic secretin administration on the expression of fear-potentiated startle in rats, a behavioral response known to require an intact, functional central nucleus of the amygdala., Methods: Rats were trained to associate a neutral light conditioned stimulus (CS) with footshock, a fear-inducing unconditioned stimulus (US). Twenty-four hours later, rats were administered secretin or vehicle and were tested immediately for their startle response to a loud noise in the presence or absence of the light., Results: Within a dose range relevant to its clinical use in autistic children, secretin dose-dependently decreased the magnitude of fear-potentiated startle in rats., Conclusions: This investigation provides additional evidence that systemically administered secretin can influence a neural network implicated in the acquisition and expression of emotional behaviors, including fear and anxiety.

gammadelta intraepithelial T lymphocytes (IEL) represent a major T cell population within the intestine of unclear functional relevance. The role of intestinal gammadelta IEL was evaluated in the dextran sodium sulfate (DSS) induced mouse colitis model system. Large numbers of gammadelta T cells, but not alphabeta T cells, were localized at sites of DSS-induced epithelial cell damage. gammadelta IEL in DSS treated mice expressed keratinocyte growth factor (KGF), a potent intestinal epithelial cell mitogen. gammadelta cell-deficient mice (TCRdelta(-/-)) and KGF-deficient mice (KGF(-/-)), but not alphabeta cell-deficient mice (TCRalpha(-/-)), were more prone than wild-type mice to DSS-induced mucosal injury and demonstrated delayed tissue repair after termination of DSS treatment. Termination of DSS treatment resulted in vigorous epithelial cell proliferation in wild-type mice but not in TCRdelta(-/-) mice or KGF(-/-) mice. These results suggest that gammadelta IEL help preserve the integrity of damaged epithelial surfaces by providing the localized delivery of an epithelial cell growth factor.

Secretin is a gastrointestinal peptide belonging to the vasoactive intestinal peptide (VIP)/glucagon/pituitary adenylate cyclase-activating polypeptide (PACAP) family recently suggested to have therapeutic effects in autism. A direct effect on brain would require secretin to cross the blood-brain barrier (BBB), an ability other members of the VIP/PACAP family have. Herein, we examined whether a secretin analog (SA) radioactively labeled with (131)I (I-SA) could cross the BBB of 4-week-old mice. We found I-SA was rapidly cleared from serum with fragments not precipitating with acid appearing in brain and serum. Levels of radioactivity were corrected to reflect only intact I-SA as estimated by acid precipitation. After i.v. injection, I-SA was taken up by brain at a modest rate of 0.9 to 1.5 microl/g-mm. Capillary depletion, brain perfusion, and high-performance liquid chromatography were used to confirm the passage of intact I-SA across the BBB. I-SA entered every brain region, with the highest uptake into the hypothalamus and cerebrospinal fluid (CSF). Unlabeled SA (10 microg/mouse) did not inhibit uptake by brain but did inhibit clearance from blood and uptake by the CSF, colon, kidney, and liver. The decreased clearance of I-SA from blood increased the percentage of the i.v. injected dose taken up per brain (%Inj/g) from about 0.118 to 0.295%Inj/g. In conclusion, SA crosses the vascular barrier by a nonsaturable process and the choroid plexus by a saturable process in amounts that for other members of its family produce central nervous system (CNS) effects. This passage provides a pathway through which peripherally administered SA could affect the CNS.

Gammadelta T cell receptor-bearing dendritic epidermal T cells (DETCs) found in murine skin recognize antigen expressed by damaged or stressed keratinocytes. Activated DETCs produce keratinocyte growth factors (KGFs) and chemokines, raising the possibility that DETCs play a role in tissue repair. We performed wound healing studies and found defects in keratinocyte proliferation and tissue reepithelialization in the absence of wild-type DETCs. In vitro skin organ culture studies demonstrated that adding DETCs or recombinant KGF restored normal wound healing in gammadelta DETC-deficient skin. We propose that DETCs recognize antigen expressed by injured keratinocytes and produce factors that directly affect wound repair.

Epithelial tissues house gammadelta T cells, which are important for the mucosal immune system and may be involved in controlling malignancies, infections and inflammation. Whole-genome gene-expression analysis provides a new way to study the signals required for the activation of gammadelta T cells, their mode of action and relationships among cells of the mucosal immune system.

We have examined the role of CD81 in the activation of murine splenic alphabeta T cells. Expression of the CD81 molecule on T cells increases following activation, raising the possibility of a role for this molecule in progression of the activation process. Using an in vitro costimulation assay, we show that CD81 can function as a costimulatory molecule on both CD4+ and CD8+ T cells. This costimulation functions independently of CD28, and unlike costimulation through CD28, is susceptible to inhibition by cyclosporin A. Strikingly, the pattern of cytokine production elicited by costimulation via CD81 is unique. IL-2 production was not up-regulated, whereas both IFN-gamma and TNF-alpha expression significantly increased. Together our results demonstrate an alternate pathway for costimulation of T cell activation mediated by CD81.

Emerging studies using mouse models of experimental colitis are defining the nature of the immunological disturbances that initiate inflammation and destruction of the intestine. A better understanding of disease-promoting and -suppressing CD4+ T cells is providing insight into the mechanisms controlling immune responses within the intestinal compartment. Moreover, a role for distinct T cell populations, including intraepithelial gammadelta T cells, in maintaining the physical integrity of the intestine was suggested by recent studies. Cytokine gene-knockout mice and anti-cytokine treatments remain important tools to define the pro- and anti-inflammatory functions of cytokines. These advances are fostering the design and evaluation of new therapeutic approaches that may eventually be applied to treat human inflammatory bowel disease.

Mucosal tissues including the intestine, lung, reproductive tract, and skin form the major interfaces between the outside and internal milieus. Facing the outside is an epithelial cell layer, the epithelium, built on a vascular connective surface. In addition to performing specialized functions, mucosal tissues are sites where immune, epithelial, and neuronal cell types act in concert to maintain tissue integrity and fight invading pathogens. This article presents the latest findings from my laboratory describing a novel protective function for the intestinal intraepithelial gammadelta T cells (gammadelta intraepithelial lymphocytes).

Recent studies have demonstrated increased numbers of gammadelta T cells in a variety of human infectious as well as noninfectious diseases. In some cases gammadelta T cells could be shown to destroy infected or transformed cells. Advances in the identification of ligands recognized by gammadelta T cells and the development of animal model systems to study these cells in vivo should overcome some of the major obstacles currently preventing a better understanding of gammadelta T cell function in immune responses. As we gain this knowledge it may become possible to design therapeutic strategies exploiting unique properties of gammadelta T cells to promote more effective immunity., (Copyright 1998 Academic Press.)

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification. Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies. Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography. Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA. Similar N-terminal deletions are observed with AFP isolates originating from natural sources. A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed. The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein. The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

Findings made during the past few years demonstrate that gamma delta T cells apparently share with macrophages a propensity to recognize nonpeptidic molecules of the kind most commonly associated with microorganisms and stressed cells. In general, recognition of these antigens by gamma delta T cells involves the antigen receptor but does not require antigen presenting cells to express MHC gene products or to have a functional antigen processing machinery. Other recent advances continue to support the notion that gamma delta T cells can perform specialized functions related to the repair of tissue damage.

Early stages of T cell development are thought to include a series of coordinated interactions between thymocytes and other cells of the thymus. A monoclonal antibody specific for mouse CD81 was identified that blocked the appearance of alpha beta but not gamma delta T cells in fetal organ cultures initiated with day 14.5 thymus lobes. In reaggregation cultures with CD81-transfected fibroblasts, CD4-CD8- thymocytes differentiated into CD4+CD8+ T cells. Thus, interactions between immature thymocytes and stromal cells expressing CD81 are required and may be sufficient to induce early events associated with T cell development.

The role played in immune surveillance by gamma delta T cells residing in various epithelia has not been clear. It is shown here that activated gamma delta T cells obtained from skin and intestine express the epithelial cell mitogen keratinocyte growth factor (KGF). In contrast, intraepithelial alpha beta T cells, as well as all lymphoid alpha beta and gamma delta T cell populations tested, did not produce KGF or promote the growth of cultured epithelial cells. These results suggest that intraepithelial gamma delta T cells function in surveillance and in repair of damaged epithelial tissues.

T-cell receptor gene rearrangements in gamma delta T cells have been the subject of intense molecular investigations. This year, much has been learned about the mechanisms controlling this process. However, the specificity and function of gamma delta T cells still remains enigmatic. The application of molecular technology including the availability of mutant mice lacking defined T-cell populations and immunologically relevant surface proteins is beginning to provide answers as well as some surprises.

A rapid and reliable purification procedure is described that is useful for both analytical detection and quantitative recovery of milligram amounts of individual molecular variants of mouse alpha-fetoprotein (AFP). The appropriate separation conditions were developed with an analytical-size Mono Q anion-exchange column linked to an automated Fast Protein Liquid Chromatography system. Effective separations of fetal-derived AFP variants was accomplished within 20 min under mild conditions with an L-histidine buffer. Employing the optimal separation conditions established on the Mono Q HR 5/5 column we upscaled the procedure by using a preparative Mono Q HR 16/10 column in order to obtain milligram quantities of each molecular variant of AFP. Seven distinct isomeric forms of AFP could be recovered on the preparative anion exchanger in a highly reproducible manner. Each of the seven protein peaks eluted from the Mono Q column were confirmed to be distinct isoforms of AFP by isoelectric focusing and Western blotting developed with monospecific anti-AFP antisera. This method in its scaled up version offers the benefit of providing milligram quantities of immunochemically pure AFP isomers for structure and function studies.

A mouse alpha-fetoprotein complementary DNA containing the coding region for amino acids 256 to 548 was fused to the lac transcriptional and translational control elements contained on the expression vector pOP203-28. The expression of a 35 kD hybrid lac Z'- alpha-fetoprotein polypeptide in Escherichia coli was demonstrated by the chloramphenicol release assay and by immunoprecipitation using rabbit anti-mouse alpha-fetoprotein antibodies as probe.