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Circulating T-lymphocyte subpopulations were enumerated in 65 patients with histoplasmosis and correlated with the different clinical manifestations of the disease. Acute pulmonary histoplasmosis, rheumatologic, disseminated, and chronic inflammatory manifestations of histoplasmosis were all associated with a significant elevation above normal of OKT8+ (suppressor-cytotoxic) lymphocytes and a significantly lower than normal OKT4+ (helper-inducer)-lymphocyte to OKT8+-lymphocyte ratio. In contrast, cavitary disease was associated with an increase in OKT4+ lymphocytes, a decrease in OKT8+ lymphocytes, and a higher than normal OKT4/OKT8 ratio. Clinical recovery was associated with normalization of these values. Functional activity determined by coculture techniques correlated closely with T-lymphocyte subset measurements. These distinct subset abnormalities may help monitor immunological aspects of disease activity.

The neuropeptide substance P, a known mitogen for human blood T-lymphocytes, now is shown to stimulate proliferation of embryonic rat aortic smooth muscle cells of the A7r5 line, at concentrations of 10 −9 M to 10 −6 M. Neurotensin (NT), that has vascular and smooth muscle activity similar to SP, failed to induce proliferation of A7r5 cells. At proliferation-enhancing concentrations, SP increased the concentration of cytosolic Ca 2+ in A7r5 cells, suggesting activation of the phosphatidylinositol pathway. Binding of [ 125 I]-substance P to A7r5 cells reached equilibrium rapidly at 4°C, and was saturable, implying that the activation of smooth muscle cells by SP is a receptor-mediated process.

Histamine is an important mediator of immediate hypersensitivity for both animals and humans. The action of histamine on target tissues is believed to be mediated by specific cell surface receptors, especially H1 and H2 receptors for hypersensitivity and inflammatory reactions, which involve stimulation of smooth muscle contractility, alterations in vascular permeability, and modifications in the activities of macrophages and lymphocytes. Although the nature of histamine receptors in the brain and peripheral tissues has been studied extensively by many laboratories, the molecular mechanism of histamine receptor-mediated reactions is not fully understood, mainly because histamine receptors are incompletely characterized from the biochemical point of view. In previous studies, we have found that the cultured smooth muscle cell line DDT1MF-2, derived from hamster vas deferens, expresses low-affinity histamine H1 receptors and responds biochemically and functionally to H1-specific stimulation (Mitsuhashi and Payan, J Cell Physiol 134:367, 1988). This cell line provides a model for analyzing the biochemical responses of H1 receptor-mediated reactions in peripheral tissues. In this review, we summarized our recent progress in the study of low-affinity H1 receptors on DDT1MF-2 cells.

Cultured cells of the smooth muscle line DDT1MF-2, which was derived from a hamster vas deferens tumor, expressed histamine H1-type receptors and responded biochemically and functionally to H1-specific stimulation. The H1-receptor antagonist [3H]-pyrilamine bound specifically to 9.7 x 10(6) sites/DDT1MF-2 cell with a dissociation constant (Kd) of 219 nM. The addition of histamine to suspensions of fura-2-loaded DDT1MF-2 cells elicited a rapid, transient, and stimulus concentration-dependent increase in the intracellular concentration of Ca2+ with an EC50 of 3 x 10(-5) M, which demonstrated H1 receptor specificity. Moreover, in order to evaluate in vitro contractile response of individual DDT1MF-2 cells, the degree of intracellular actin polymerization was quantified by a DNase inhibition assay. The percentage of nonpolymerized or G-actin in DDT1MF-2 cells was reduced in a histamine concentration-dependent manner with an EC50 of 1 x 10(-5) M and H1 receptor specificity. Histamine-induced actin polymerization was accompanied by changes in cell shape that were consistent with cellular contraction, as assessed by flow cytometry. The H1-type receptors of cultured DDT1MF-2 cells thus couple histamine stimulation to a variety of functional responses of smooth muscle cells.

Peptide-containing nerve fibers (peptidergic fibers) abundantly innervate the mammalian cornea. We investigated their role in ocular herpes simplex infection in mice by using capsaicin, which causes degeneration and permanent loss of peptidergic neurons in neonates and temporary peptide depletion in adult animals. The corneas of neonatally denervated BALB/c mice were observed for capsaicin-induced keratitis at 11-14 wk of age and were then infected bilaterally with herpes simplex virus 1 (HSV-1); trigeminal (TG) ganglia were cocultivated 6 wk later to establish the rate of latent infection. We also applied capsaicin eye drops to adult BALB/c mice that had been infected with HSV-1 6 wk earlier, and measured viral shedding before, and 3 days and 2 months after, administration of capsaicin drops; TG ganglia of these animals were cocultivated at 3 days and 2 months after capsaicin application. Neurotrophic keratitis was found in 81% of neonatally denervated animals; mortality rate due to HSV-1 infection was reduced from 80% in the controls to 24% in the capsaicin-treated group, and recovery of latent virus by cocultivation was reduced by 50%. Viral shedding could not be produced by capsaicin eye drops in adult animals infected with HSV-1. However, recovery of latent virus was significantly reduced in TG ganglia sampled 3 days and 2 months after capsaicin drops were instilled. Our findings suggest 1) that peptidergic fibers play a crucial role in the establishment of trigeminal HSV-1 latency and 2) that reactivation of latently infected ganglia can be inhibited by topical capsaicin.

Human T lymphocytes in modified Boyden migration chambers exhibited a chemokinetic response, but no detectable chemotaxis, to 5(S),12(R)-dihydroxyeicosa-6, 14-cis-8,10-trans-tetraenoic acid (leukotriene B4) and to 5(S)-hydroxyeicosatetraenoic acid (5-HETE), which are 5-lipoxygenase products of arachidonic acid, and failed to respond to either leukotriene C4 or 11-HETE. Concentrations of lymphocyte-derived 15-HETE and of 15-hydroperoxyeicosatetraenoic acid that inhibited the 5-lipoxygenation of endogenous arachidonic acid suppressed significantly both random migration and the chemokinetic responses of T lymphocytes to concanavalin A and α-thioglycerol. That the suppressive effect of 15-HETE on T lymphocyte chemokinesis was attributable in part to the inhibition of 5-lipoxygenase was further suggested by the ability of exogenous 5-hydroperoxyeicosatetraenoic acid to restore chemokinetic responsiveness to lymphocytes that had been preincubated with 15-HETE.

A high-resolution triple-laser sorter was designed and constructed to provide flexible switchover and high-resolution sorting of cells or chromosomes with any combination of one, two, or three lasers. These features provide a central facility instrument that currently serves multiple users and analyzes different stain combinations with minimal switchover effort between experiments. Improved optics and mounts that focus the three laser beams independently are able to resolve beads and chromosomes better than our previously reported dual-laser sorter. An improved signal collection unit with electronically controlled reference positions can be focused more quickly and precisely for any signal combination. A removable dye laser extends the range of usable fluorochrome labels. A rapid sheath switchover permits sorting of sterile cells and sterile chromosomes sequentially without additional sterilization or reservoir sheath change. Improved dual-laser chromosome resolution is at least as good, analyzing 8,000 chromosomes/s, compared to the previous dual-laser bench at 2,000/s. Stimulated and unstimulated peripheral blood lymphocytes were analyzed according to simultaneous measurements of cell surface receptors labeled with a fluoresceinated neuropeptide and a Texas red-labeled antibody as well as DNA content during the cell cycle. These results demonstrate the broad range of potential applications of this triple-laser system.

The mitogenic effect of corticotropin-releasing factor (CRF) on rat lymphocytes was investigated. When rat splenocytes were cultured for 48 hr with CFR, a dose-dependent increase in incorporation of 3H-thymidine (3H-Tdr) was observed, with a maximal response at 10 nM CRF. Comparison of the proliferative effect of CRF on enriched populations of B lymphocytes, T lymphocytes, or macrophages revealed that only B lymphocytes responded following treatment with CRF. When lymphocytes derived from different lymphoid tissues were compared, CRF had a greater proliferative effect on lymphocytes derived from gut-associated lymphoid tissue (mesenteric lymph nodes and Peyer's patches) than on lymphocytes from spleen or inguinal lymph nodes; CRF had no effect on thymocytes. Synthetic fragments of CRF were used to determine which portions of the peptide are recognized by lymphocytes. The C-terminal fragments alpha-helical CRF9-41 and CRF21-41 were as potent as native CRF in stimulating B-lymphocyte proliferation, whereas CRF1-20 did not stimulate proliferation. The activity of these peptides suggests that CRF stimulates lymphocyte proliferation by cellular recognition of structural determinants in the C-terminal one-half of the peptide.

The mas oncogene/angiotensin II receptor was subcloned into a mammalian expression vector pCDM8 and used to transiently transfect monkey kidney derived COS-7 cells. As a result, the mas transfected COS-7 cells expressed a functional angiotensin II receptor capable of transducing an increase in intracellular Ca2+ following stimulation with angiotensin II. The angiotensin II stimulated changes in Ca2+ could be measured 24 hours after transfection in both a fluorimeter and a fluorescence activated cell sorter. These results describe a rapid method for the functional analysis of the 7-transmembrane domain receptor genes.

Peptide mediators of sensory nerves that are released in tissues by noxious stimuli or inflammatory reactions rapidly elicit local and systemic responses similar to those of immediate hypersensitivity. These sensory neuropeptides affect functions of smooth muscles, blood vessels, leukocytes, and epithelial glands both directly and indirectly, through the actions of mediators released from mast cells stimulated by the peptides. Stereospecific receptors transduce the effects of neuropeptides of the peripheral nervous system (PNS) and central nervous system (CNS) on diverse functions of human, murine and guinea pig mononuclear and polymorphonuclear leukocytes, mast cells, and basophils in vitro and in vivo. Stimulatory and inhibitory effects of neuropeptides on leukocytes are attained in vitro at concentrations which are similar to those in the circulation and in tissues. The dissociation constant (KD) for the binding of a neuropeptide to its leukocyte receptor is within the range of concentrations that evoke cellular responses critical to immunity and hypersensitivity. Neuropeptides exhibit both cellular and stimulus specificities, as exemplified by the greater potency of substance P in activating mucosal than connective tissue mast cells and the capacity of somatostatin to inhibit the release of mediators from basophils challenged by IgE-dependent mechanisms, but not by basic peptides or ionophores. The selective release of distinct neuropeptides from different subsets of sensory nerve endings, the specificity of neuropeptide recognition by mast cells, basophils, lymphocytes, and other target cells, and the diversity of relevant activities of the neuropeptides suggest that the nervous system may initiate and modulate immediate and delayed hypersensitivity by unique mechanisms.

Persistent lymph node syndrome, epidemiologically related to acquired immune deficiency syndrome, is characterized by reactive lymphadenopathy and an increased incidence of localized bacterial, viral, and fungal infections. Seven typical patients were found to have a localized infection every 4.5 +/- 2.0 months (mean +/- SD) since onset of adenopathy. Because recurrent bacterial infections may be associated with defective polymorphonuclear leukocyte (PMNL) function, studies of PMNL function in these patients were undertaken. The patients' PMNLs had diminished chemotactic responses to high concentrations of two structurally distinct chemotactic factors, leukotriene B4 and N-formylmethionylleucylphenylalanine. The patients' PMNLs also had deficient degranulating responses to the former but not to the latter. Diminished PMNL function may contribute to the observed increased incidence of localized infections, which are a major source of morbidity, in these patients.

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.

Background: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. Results: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 μM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. Conclusions: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.

Leukotriene B4, but not (12S)-leukotriene B4, coupled to fluorescein-labeled human serum albumin interacts specifically with human T lymphocytes, as assessed by fluorescence-activated flow cytometry. Approximately 11% of blood T lymphocytes bind the fluorescent conjugate of leukotriene B4. The leukotriene B4-reactive T lymphocytes are distributed between the suppressor-cytotoxic (14%) and helper-inducer (8%) subsets, which were identified concurrently by phycoerythrin-labeled monoclonal antibodies to subset-specific antigens. Leukotriene B4, but not (12S)-leukotriene B4 or leukotriene C4, enhances significantly the proliferation of mitogen-stimulated purified suppressor-cytotoxic T lymphocytes, at concentrations of 10(-12) M to 10(-6) M, and inhibits significantly the proliferation of mitogen-stimulated purified helper-inducer T lymphocytes, at concentrations of 10(-8) M to 10(-6) M, as assessed by the uptake of [3H]thymidine. The net effect of leukotriene B4 on blood T lymphocytes is immunosuppression, which may be expressed in some human hypersensitivity and inflammatory diseases.

In order to investigate the relationship between the biochemical pathways that characterize contraction and cell growth, we have studied both contraction, mitogenesis and protein synthesis induced by the vasoactive neuropeptides, substance P (SP), calcitonin gene related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) on four different established vascular and non-vascular smooth muscle cell lines. Contraction in vitro was evaluated by light microscopy and recorded photographically. Mitogenesis and protein synthesis were evaluated by [3H]-thymidine incorporation into cells and [3H]-amino acid incorporation into trichloroacetic acid precipitated materials, respectively. SP stimulated mitogenesis of A7r5 cells (embryonic rat aorta), but failed to induce significant contraction of these cells, whereas, SP induced contraction of cultured adult rat vascular smooth muscle cells (VSMC), but failed to stimulate mitogenesis. CGRP and VIP stimulated mitogenesis and protein synthesis, respectively, of DDT1MF-2 cells (hamster vas deferens), but neither induced contraction of this cell line. All three neuropeptides showed no effect on BC3Hl (mouse smooth muscle-like) cells. These results suggest that neuropeptides with vasoactive properties modulate different stages of cellular mitogenic responses which may be regulated by the degree of maturation of smooth muscle cell.

Two groups of six adults with persistent asthma, who were identical clinically, received 0.1 or 4 g of purified eicosapentaenoic acid ethyl ester (EPA) daily for 8 weeks. Both doses increased significantly the generation of leukotriene B5 (LTB5) from EPA by polymorphonuclear (PMN) and mononuclear leukocytes, while only the high dose decreased leukocyte arachidonic acid (AA) and the generation of LTB4 and prostaglandin E2 from AA. Only the high dose led to inhibition of PMN leukocyte chemotaxis to multiple stimuli by a mean of 57-70% (P less than 0.01), without altering monocyte chemotaxis, the production of platelet-activating factor by mononuclear leukocytes, or the IgE-dependent release of histamine from basophils. Both doses of EPA increased the responses of T lymphocytes to phytohemagglutinin by a mean of 73% or more (P less than 0.01) without modifying the numbers of helper and suppressor T lymphocytes. EPA affects the functions of several types of leukocytes critical to inflammation and immunity.

Receptors for somatostatin (SOM) were identified on mouse bone marrow derived mast cells (MBMMC) and shown to vary in expression with the state of proliferation and differentiation of the MBMMC. Flow cytometric studies of the binding of fluorescein-labeled SOM and concurrent analyses of the binding of [125I]SOM demonstrated that the population of MBMMC capable of recognizing SOM specifically is that exhibiting a proliferative response to interleukin-3. The MBMMC that bound SOM reached a maximal number at 72 hr following the addition of interleukin-3, and were distributed principally in the G2/M phase of the cell cycle. SOM did not influence directly the proliferative responses of MBMMC to interleukin-3. The level of expression of SOM receptors thus may reflect the state of differentiation of mast cells, as well as determining the funcitonal sensitivity to the inhibitory effects of SOM.

The effect of 1 μ and 5 μ aspirin on oxygenation of arachidonic acid in vitro by basophils stimulated with anti-IgE was assessed with basophil-enriched suspensions of mononuclear leukocytes from five patients with asthma and hypertrophic allergic rhinitis and one patient with rhinitis alone who had recent adverse pulmonary reactions to aspirin, four aspirin-tolerant patients with asthma receiving therapy similar to that of the aspirin-sensitive patients, and eight normal subjects who resembled the patients with respect to sex and age. As quantified by the combined application of high-performance liquid chromatography and radioimmunoassays, the generation of the principal products prostaglandin E 2 (PGEZ 2 ) and leukotriene D 4 was modified differently by aspirin but not sodium salicylate in the aspirin-sensitive patients. The alterations of PGE 2 generation and of the ratio of leukotriene D 4 to PGE 2 generation by aspirin were much greater for basophil-containing leukocytes of aspirin-sensitive patients than those of the nvo control groups. Although the basic mechanism and relevance to pulmonary reactions 9f the observed effects of aspirin were not elucidated, aspirin clearly modifies the leukocyte oxygenation of arachidonic acid differently in aspirin-sensitive patients.

The concentrations of the neuropeptides substance P, somatostatin, and calcitonin gene-related peptide in human nasal secretions were quantified by radioimmunoassays, concurrently with that of histamine, in the course of nasal challenge of allergic and control subjects with ryegrass antigen to examine contributions of neuromediation of the tissue response. Each of the neuropeptides and histamine were detected in nasal lavage fluid prior to challenge. In allergic patients, but not normal controls, antigen evoked significant increases of 3-fold in histamine at 15-60 min, 1.5- to 4-fold in calcitonin gene-related peptide at 15 min-24 hr, and more than 2-fold in somatostatin at 6 hr, without altering the concentration of substance P in nasal lavage fluid. The identity of the neuropeptides was confirmed chromatographically. Thus calcitonin gene-related peptide may mediate nasal congestion directly and somatostatin may be one of the factors regulating the late involvement of basophils and mast cells in allergic rhinitis.

The clinical outcome of patients with the various manifestations of collagen vascular disease (CVD) has significantly improved over the past two decades with the increased use of diuretics, cytotoxic agents, dialysis, transplantation, and the judicious use of corticosteroids and other antiinflammatory agents directed at altering or delaying end-organ damage by the underlying immunopathologic process.1–4 A more rigorous definition of the various CVD clinical syndromes and a greater sophistication in the serologic, radiologic, and pathologic diagnostic methods have meant that patients with these diseases are now coming to clinical attention earlier in the course of their illness. Consequently, the physician is now encountering a greater number of clinical problems over a longer time span for each individual patient, rather than just the well-known complications of their end-stage disease.

The neuropeptide substance P modulates the activities of a number of different leukocytes that characterize both acute and delayed inflammatory responses. Substance P may play a role in the pathogenesis of such diverse diseases as arthritis, asthma, and inflammatory bowel disease.

Recent evidence suggests that the neuropeptide somatostatin (SOM) plays an immunoregulatory role. We demonstrated previously that SOM inhibits concanavalin A-induced cell proliferation and immunoglobulin synthesis by murine Peyer's patch and splenic lymphocytes. Available data suggest that these effects are in part mediated by specific SOM receptors expressed by lymphocytes, but as yet these receptors have not been characterized. Using cytofluorimetry we investigated the distribution and specificity of binding of fluorescent SOM (SOM*) to murine Peyer's patch and splenic T- and B-lymphocyte subpopulations. The specificity of binding was confirmed by radioassay. T and B cells from both organs showed specific binding of SOM*. In Peyer's patches, approximately 50% of all cell populations studied (whole, T- and B-cell-enriched) bound SOM specifically and this was significantly higher than the corresponding splenic lymphocyte populations. Eighty to eighty-four percent of Peyer's patch Thy1.2+, Lyt1+, or L3T4+ cells and 94% of Lyt2+ cells bound SOM. Greater than 80% of B cells from this organ bound SOM (sIgA+ = sIgM+ greater than sIgG+ cells). In spleen, approximately 30% of Thy1.2+, Lyt1+, or L3T4+ cells bound SOM and this was significantly less than the proportion of Lyt2+ cells (53%) which did so. More sIgA+ (89%) than sIgG+ (66%) than sIgM+ (55%) B cells bound SOM*. Although we have previously shown that the effect of SOM on immunoglobulin synthesis was relatively isotype-specific (IgA synthesis was predominantly affected, especially in Peyer's patches) this cannot be explained solely on the basis of preferential expression of SOM receptors by distinct lymphocyte subsets. Instead, it is probably the result of the specific immunological microenvironment in which the lymphocytes reside.

Pulmonary immunologic responses may be mediated and modulated in part by neuropeptides released from the endings of nerve fibers that innervate the airways. Bronchoconstriction, mucosal edema, and increased airway secretions may be mediated by multiple locally generated neuropeptides. The neuropeptides affect directly and indirectly through other mediators the functions of endothelial cells, smooth muscle cells, blood vessels, and leukocytes. The high content of diverse potent neuropeptides in the ganglionated plexuses close to pulmonary smooth muscle bundles, along blood vessels, and in neural projections into the bronchial epithelium and regions containing acini of mucus glands provide tissue concentrations well within the range required to attain such biologic effects. The airway responses mediated principally by neuropeptides exhibit unique mechanisms of specificity attributable to the selective distribution of each neuropeptide in a distinct subset of nerve fibers that react differently to each stimulus and to the target cell specificity of the neuropeptides. At physiologically relevant in vitro concentrations, many of the neuropeptides have different effects on mast cells, basophils, monocytes, and lymphocytes. The direct actions of substance P (SP) and the capacity to initiate and modulate leukocyte responses are described in relation to the physiologic and pathopharmacologic consequences of pulmonary nerve stimulation. Selected cellular and molecular characteristics of the interactions of SP with mast cells, basophils, lymphocytes, endothelial cells, and smooth muscle cells also will be presented in order to define the functionally critical properties of receptors for the neuropeptide and to expose possible means of specifically modifying the interactions.

The possibility that cyclooxygenase products of arachidonic acid might contribute to the defective T lymphocyte function of homosexual men with the reactive lymph node syndrome was examined in vitro. T lymphocyte proliferation, assessed by the uptake of [3H]thymidine after the addition of phytohemagglutin, was 72,870 +/- 66,816 counts per minute (mean +/- SD) for eight patients and 119,589 +/- 64,913 counts per minute for 30 controls (P less than 0.05, Student's t test). Treatment with the cyclooxygenase inhibitor indomethacin increased the phytohemagglutin-induced proliferation of the T lymphocytes from five of eight patients, but none of 12 healthy homosexual and heterosexual control subjects. The production of prostaglandin E2 by T lymphocytes from six patients was 16.1 +/- 10.5 pg/5 X 10(6) cells/hr, as contrasted with that of 4.9 +/- 1.3 and 4.3 +/- 2.1 pg/5 X 10(6) cells for four healthy homosexual and six healthy heterosexual control subjects, respectively (P less than 0.01, Student's t test). The production of prostaglandin E2 by the patients' monocytes was normal. Abnormalities of the cyclooxygenase pathway of T lymphocytes of patients with the reactive lymph node syndrome may reflect an immunoregulatory defect, which predisposes to infections and may evolve into the more severe abnormalities of the acquired immune deficiency syndrome.

Substance P (SP) is an undecapeptide neuromediator that stimulates human T-lymphocyte function by binding to stereospecific membrane receptors. Human IM-9 cultured B-lymphoblasts express approximately 20,000 receptors per cell for [125I]SP with a Kd of 0.3 nM. [125I]SP was specifically crosslinked by disuccinimidyl suberate to IM-9 cell membrane proteins of 78, 58, and 33 kDa. An indirect immunoaffinity purification procedure has now been developed based on immunoabsorption of detergent-solubilized [125I]SP-labeled IM-9 cell membrane proteins to anti-SP antibody that was bound to an epoxide ultraffinity high-performance liquid chromatography column, followed by elution in acidic 8 m urea. The 58- and 33-kDa SP-receptor complexes were purified to apparent homogeneity by immunoaffinity chromatography and identified by autoradiography and silver staining of sodium dodecyl sulfate-polyacrylamide gels.

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10 −12 to 10 −10 M and 10 −13 to 10 −9 M somatostatin 14 , as assessed by the uptake of [ 3 H]thymidine and [ 3 H]leucine, respectively. The inhibitory effect of somatostatin 14 was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 °C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.

The interaction of substance P with human blood T-lymphocytes, which stimulates T-lymphocyte proliferation, was quantified by both flow cytometric and direct binding assays. Fluorescence-detection flow cytometry recorded the binding of dichlorotriazinylamino-fluorescein-labeled substance P to 21 +/- 10% (mean +/- SD, n = 6) and 35 +/- 8% (n = 2) of human blood T-lymphocytes before and after stimulation with 10 micrograms/ml of phytohemagglutinin, respectively. The suppressor-cytotoxic (leu 2a) and helper-inducer (leu 3a) subsets identified by phycoerythrin-labeled monoclonal antibodies contained substance P-reactive T-lymphocytes at respective mean frequencies of 10 and 18%. [3H]substance P bound rapidly and reversibly to a mean of 7035 +/- 2850 sites/T-lymphocyte, which exhibited a dissociation constant (KD) of 1.85 +/- 0.70 X 10(-7) M (mean +/- SD, n = 5). [D-Pro2,D-Phe7,D-Trp9]substance P inhibited the binding of dichlorotriazinylamino-fluorescein-labeled substance P and [3H]substance P to T-lymphocytes at concentrations that suppressed the proliferative response to substance P. Substance P, eledoisin, and substance K (alpha-neurokinin), which all share with substance P the carboxy-terminal substituent -Gly-Leu-Met-NH2, were more potent than substance P in inhibiting the binding of [3H]substance P to T-lymphocytes, suggesting the importance of this sequence in the interaction. Purified human blood B-lymphocytes, monocytes, polymorphonuclear leukocytes, and platelets, and cultured Hut 78 cutaneous lymphoma T-cells, Jurkat cells, Molt-4 lymphoblasts, and HL-60 and U-937 monocyte-like cells all showed only minimal specific binding of [3H]substance P. The recognition of substance P by T-lymphocytes provides one mechanism for selective modulation of immunity by sensory nerves.

IgA has been recognized as the predominant class of antibody in mucosal secretions even though it constitutes a relatively small percentage of serum immunoglobulin. There are several mechanisms that contribute to IgA making up such a large proportion of the antibody in intestinal secretions (reviewed in ref. 1) including: a high frequency of IgA B cell precursors in Peyer’s patches (2); T cells which facilitate an isotype switch from IgM to IgA (so called switch T cell) (3); IgA-specific helper T cells (4); a propensity of these cells to traffic selectively to mucosal surfaces (1); and the existence of a specific mechanism to facilitate the transport of IgA across the epithelium into secretions (5). In this paper, we report our data concerning two other mechanisms that may be capable of enhancing IgA production, namely neuropeptides and the antisuppressor regulatory circuit.

The role of arachidonic acid metabolites in the pathogenesis of airway inflammation and clinical asthma is currently unknown. The addition of eicosapentaenoic acid (EPA) to the diet of humans has been shown to generate metabolites that are less potent than their arachidonic acid counterparts. The substitution of EPA for arachidonic acid metabolites in patients might cause a decrease in airway inflammation and an improvement in clinical asthma. We studied the effect of addition of EPA to the diet of twelve asthmatic patients. Standard clinical evaluations and pulmonary function tests were done on weeks 0, 3, 6, 10, 12 and 14. Patients ingested either low-dose EPA (0.1 g/day) or high-dose EPA (4.0 g/day) from weeks 6-14 (total of 8 weeks). There was no difference in clinical status or pulmonary function between groups at the start of the study. There was no change in clinical status or pulmonary function between or within groups at the end of 8 weeks of EPA ingestion.

Neurogenic inflammation is mediated by release of tachykinins from sensory nerves, which stimulate plasma extravasation from postcapillary venules. Because there are conflicting results regarding the importance of neurogenic inflammation in the gastrointestinal tract, we quantified plasma extravasation using Evans blue and identified sites of the leak using Monastral blue in the mouse. Substance P and bradykinin stimulated extravasation from postcapillary venules in the stomach, small and large intestine, pancreas, urinary bladder, trachea, and skin by two- to sevenfold by interacting with NK1 and B2 receptors, respectively. Stimulation of sensory nerves with capsaicin also induced extravasation. Capsaicin- and bradykinin-stimulated extravasation was attenuated by an NK1-receptor antagonist and is thus mediated by release of tachykinins and activation of the NK1 receptor. We conclude that 1) substance P stimulates extravasation in the gastrointestinal tract and pancreas of mice by interacting with the NK1 receptors, and 2) capsaicin and bradykinin induce plasma extravasation by stimulating tachykinin release from sensory nerves. Thus neurogenic mechanisms mediate inflammation in the gastrointestinal tract and pancreas of the mouse.

Background The novel small molecule R118 and the biguanide metformin, a first-line therapy for type 2 diabetes (T2D), both activate the critical cellular energy sensor 5′-AMP-activated protein kinase (AMPK) via modulation of mitochondrial complex I activity. Activation of AMPK results in both acute responses and chronic adaptations, which serve to restore energy homeostasis. Metformin is thought to elicit its beneficial effects on maintenance of glucose homeostasis primarily though impacting glucose and fat metabolism in the liver. Given the commonalities in their mechanisms of action and that R118 also improves glucose homeostasis in a murine model of T2D, the effects of both R118 and metformin on metabolic pathways in vivo were compared in order to determine whether R118 elicits its beneficial effects through similar mechanisms. Results Global metabolite profiling of tissues and plasma from mice with diet-induced obesity chronically treated with either R118 or metformin revealed tissue-selective effects of each compound. Whereas metformin treatment resulted in stronger reductions in glucose and lipid metabolites in the liver compared to R118, upregulation of skeletal muscle glycolysis and lipolysis was apparent only in skeletal muscle from R118-treated animals. Both compounds increased β-hydroxybutyrate levels, but this effect was lost after compound washout. Metformin, but not R118, increased plasma levels of metabolites involved in purine metabolism. Conclusions R118 treatment but not metformin resulted in increased glycolysis and lipolysis in skeletal muscle. In contrast, metformin had a greater impact than R118 on glucose and fat metabolism in liver tissue. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-674) contains supplementary material, which is available to authorized users.