Peripheral secretin-induced Fos expression in the rat brain is largely vagal dependent.

I.v. 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 dependent pathways. We investigated whether the vagus nerve is involved in i.p. injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 microg/kg, i.p., 90 min) induced a 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, and at 100 microg/kg, in the dorsal motor nucleus of the vagus (DMV) compared with i.p. injection of vehicle. Double immunohistochemistry showed that secretin (40 microg/kg, i.p.) activates tyrosine hydroxylase neurons in the NTS. Subdiaphragmatic vagotomy (7 days) abolished Fos expression-induced by i.p. secretin (40 microg/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, s.c. capsaicin (10 days) did not influence the Fos induction in the above nuclei. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared with the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in medullary, pontine and limbic nuclei regulating autonomic functions and emotion through vagal-dependent capsaicin-resistant pathways. Secretin injected i.p. may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood-brain barrier.

A secretin i.v. infusion activates gene expression in the central amygdala of rats.

For the last 100 years secretin has been extensively studied for its hormonal effects on digestion. Recent observations that the deficits in social reciprocity skills seen in young (3-4-year-old) autistic children are improved after secretin infusions suggest an additional influence on neuronal activity. We show here that i.v. administration of secretin in rats induces Fos protein expression in the neurons of the central amygdala as well as the area postrema, bed nucleus of the stria terminalis, external lateral parabrachial nucleus and supraoptic nucleus. However, secretin infusion did not induce Fos expression in the solitary tract nucleus or paraventricular nucleus, regions normally activated by related peptides such as cholecystokinin. The peak blood levels of secretin that induce Fos protein expression in rat brain are similar to the peak blood levels observed during i.v. treatment with secretin in humans. The amygdala is known to be critical for developing reciprocal social interaction skills and abnormalities in this brain region have been demonstrated in autistic children.

Orally administered RDP58 reduces the severity of dextran sodium sulphate induced colitis.

Oral administration of the novel anti-inflammatory peptide RDP58 markedly reduced the severity of dextran sulphate sodium (DSS) colitis as determined by clinical and quantitative histological criteria. The architecture of the colonic epithelium in DSS treated mice receiving RDP58 remained relatively normal compared with that of control DSS treated animals. 5-Bromo-2'-deoxyuridine (BrdU) labelling studies showed a pronounced inhibition of colonic epithelial cell proliferation during DSS treatment, which was partially reversed by RDP58 therapy. Remarkably, RDP58 almost completely prevented colonic epithelial cell death induced by DSS treatment. RDP58 therapy also inhibited the accumulation of neutrophils in the colon of DSS treated mice and effectively down regulated tumour necrosis factor (TNF) expression. Preservation of the intestinal mucosa by RDP58 may thus derive from its influence on TNF expression as well as additional anti-inflammatory properties. These findings indicate that RDP58 represents a new, orally available agent potentially useful in the treatment of inflammatory bowel disease.

Intraepithelial gamma delta T cells exposed by functional genomics.

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.

CD81 and CD28 costimulate T cells through distinct pathways.

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.

Function of intestinal gammadelta T cells.

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).

Insights from mouse models of colitis.

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.

Gammadelta T cells in host defense and epithelial cell biology.

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.

Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli.

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.

An innate view of gamma delta T cells.

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.

Molecular and cellular biology of dendritic epidermal T cells.

The function and specificity of gamma delta T cells remain enigmatic. Dendritic epidermal T cells (DETC) expressing an invariant gamma delta TCR represent a well established model system to investigate these key issues. Accumulating evidence supports the initial observation that recognition of a keratinocyte self-antigen by DETC proceeds without a requirement for MHC gene products. In addition, recent data have identified bioactive polypeptides expressed by DETC but not by most other T cells. For example, keratinocyte growth factor appears to be exclusively produced by activated DETC and intestinal intraepithelial gamma delta cells. These findings suggest that DETC may recognize antigen in novel ways as well as perform specialized functions complementary to those normally attributed to T cells.

Chemokine expression by intraepithelial gamma delta T cells. Implications for the recruitment of inflammatory cells to damaged epithelia.

T cells expressing gamma delta TCR may have evolved to recognize Ag in a different manner as well as perform a broader set of functions than T cells with alpha beta TCR. In this study, we tested the hypothesis that dendritic epidermal T cells (DETC) bearing the invariant V gamma 3V delta 1 TCR may be able to signal the migration of peripheral alpha beta T cells to the epidermis by secreting specific chemokines. Expression of macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES, and lymphotactin was inducible in DETC 7-17 cells, whereas mRNA for monocyte chemoattractant protein (MCP)-1 could not be detected. Strikingly, lymphotactin was the most abundant chemokine produced by activated DETC 7-17 cells. Activated primary DETC cultures also produced copious amounts of lymphotactin mRNA. Similarly, freshly isolated and activated intestinal intraepithelial T cells (i-IEL) with gamma delta TCR expressed high levels of lymphotactin mRNA. In contrast, lymphotactin mRNA was present in activated spleen gamma delta T cells at low basal levels. Migration of CD8+ T cells induced by culture supernatants from stimulated DETC 7-17 cells was strongly reduced in the presence of a neutralizing anti-lymphotactin antiserum and to a lesser extent by neutralizing anti-MIP-1 alpha, anti-MIP-1 beta, or anti-RANTES antiserum. The presence of lymphotactin in supernatants from activated DETC 7-17 cultures was directly demonstrated by Western blot analysis. These observations are consistent with a model in which gamma delta IEL play an active multi-faceted role in the maintenance of epithelia homeostasis.

A role for CD81 in early T cell development.

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.

T-cell development. T-cell lineage commitment revisited.

Years of controversy about the lineage relationship between alpha beta and gamma delta T cells may at last have been resolved: it now appears that most T cells derive from an identical T-lineage committed precursor.

Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule.

Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.

Modulation of epithelial cell growth by intraepithelial gamma delta T cells.

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.

Activation and function of gamma delta T cells.

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.