Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.     

Effects of Peritoneal Fluid from Endometriosis Patients on Interferon-γ-Induced Protein-10 (CXCL10) and Interleukin-8 (CXCL8) Released by Neutrophils and CD4+ T Cells

Problem  Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-γ-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4⁺ T cells, and monocytes.
Method of study  Neutrophils, CD4⁺ T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay.
Results  The addition of ePF to cultures of CD4⁺ T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF ( R  = 0.89, P  =   0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4⁺ T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils ( R  = 0.89, P  <   0.001), CD4⁺ T cells ( R  = 0.93, P  <   0.001), and monocytes ( R  = 0.70, P  =   0.01). Moreover, the addition of ePF significantly enhanced the interferon-γ-induced release of IP-10 by nuetrophils and CD4⁺ T cells.
Conclusion  These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.     

Population Dynamics of CD4+ T Cells Lacking Thy-1 in Murine Retrovirus-Induced Immunodeficiency Syndrome (MAIDS)

Increased numbers of CD4⁺ Thy-1 cells have been described in the spleen (SP) of mice with retrovirusinduced immunodeliciency (MAIDS). Since this phenotypic abnormality might have considerable functional importance, the expansion of the CD4⁺ Thy-1 subset in MAIDS was characterized further. CD4⁺ Thy-1⁻ and Thy-1⁺ T-cell is from infected mice expressed similar densities of CD3 and TCR γ/β. In contrast, the Thy-I⁻ subset was uniformly CD44^hi, even early in the disease when part of Thy-I⁺ cells were still CD44¹⁰. The emergence of CD4⁺ Thy-1⁻cells occurred first in SP and lymph nodes and was observed later in thymus. The important fraction ofCD4⁺ cells lacking Thy-1 normally present in Peyer's patches was only weakly modified. Despite the major expansion of the CD4⁺ Thy-1⁻ phenotype. the proliferating fraction was not higher in this subset than in CD4⁺ Thy-1⁺ cells from infeeted miee. Persistence after hydroxyurea administration was identical in both subsets, indicating similar mean cell lifespans. Taken together, these results show that the major expansion of CD4⁺ Thy-I⁻ T-cells in MAIDS is not ascribable solely to increased proliferation within this subset. Phenotypic analysis suggests that CD4⁺ Thy-I⁻ cells result from the differentiation of Thy-I⁺ cells induced by activation signals related to retroviral infection.     

Glycine-extended gastrin-17 stimulates acid secretion only via CCK-2 receptor-induced histamine release in the totally isolated vascularly perfused rat stomach

The effects of gastrin precursors have been discussed during recent years. However, the mechanism for their action, whether through a novel receptor on the parietal cell or a cholecystokinin-2 (CCK-2) receptor on the enterochromaffin like (ECL) cells, is still not settled. This study examines the effect of glycine-extended gastrin-17 (Gly-G-17), the main non-amidated gastrin precursor, on gastric acid secretion and histamine release in the totally isolated vascularly perfused rat stomach. Glycine-extended gastrin-17 at the concentrations from 0.52 to 520 nmol L(-1) was administered to the totally isolated vascularly perfused rat stomach. Glycine-extended gastrin-17 at 52 or 520 nmol L(-1), and gastrin-17 at 0.52 nmol L(-1)were co-administered to examine whether glycine-extended gastrin augmented maximal gastrin stimulated acid secretion and histamine release. Both Gly-G-17 at 52 nmol L(-1) and gastrin-17 (G-17) at 0.52 nmol L(-1) were administered together with the histamine-2 receptor antagonist ranitidine at 10 micromol L(-1). Gastric acid and venous histamine output were measured. Glycine-extended gastrin-17 at lower concentrations from 0.52 to 5.2 nmol L(-1) did not stimulate gastric acid output or histamine release, whereas higher concentrations from 52 to 520 nmol L(-1) elicited a concentration-dependent increase in acid secretion and histamine release. The outputs of acid and histamine at 520 nmol L(-1) Gly-G-17 were at the same level as those found for G-17 at its maximally effective concentration of 0.52 nmol L(-1). Glycine-extended gastrin-17 at maximally effective concentration of 520 nmol L(-1) did not augment maximal gastrin stimulated acid secretion or histamine release. Ranitidine inhibited G-17 and Gly-G-17 stimulated acid secretion to a similar degree. This study confirms that the stimulatory effect of Gly-G-17 on gastric acid secretion is via a CCK-2 receptor on the ECL cell.     

ATP release from human airway epithelial cells studied using a capillary cell culture system

Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study. We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells. The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re-circulating medium pump. Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (≈250 μg (10⁸ cells)⁻¹ day⁻¹). Elevating apical flow rate 5-fold increased ATP secretion from ≈200 to 6618 fmol min⁻¹. Reducing apical osmolarity by 25–43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained. Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis. Lowering apical [Ca²⁺] to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca²⁺ solution, and therefore another secretory pathway may operate with physiological (i.e. m m ) calcium. Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.     

Incorporation of Human Chorionic Gonadotropin Alpha-Subunit into Different Types of Granule in Stomach Endocrine Cells

In order to demonstrate the ultrastructural localization of the α subunit of human chorionic gonadotropin (hCG) in the stomach mucosa, an immunoelectron microscopic study was performed using formalin-fixed specimens. In pyloric glands, α hCG-positive granules were irregular in outline, with a mean area and maximum diameter of 2.857 × 10⁴ nm² and 218.4 nm, respectively. In fundic glands, the granules had a smoother outline and were larger in both area and maximum diameter (3.943 × 10⁴ nm², 246.8 nm) than those of pyloric glands (p <0.001). In the atrophic fundic glands of non antral gastritis, the αhCG granules showed a differece in shape; more elliptical granules appeared to be increased, as indicated by the higher value of the axial ratio (1.452) and lower value of D (1.862) (log₁₀ area ∞ D log₁₀ perimeter) compared with those in pyloric (1.231, 1.968) and fundic glands (1.148, 1.979) (p< 0.001). The a-subunit of hCG is thus Incorporated into different types of endocrine cell in pyloric and fundic glands, and the granule morphology appears to differ in hyperplastic αhCG cells of non-antral gastritis. Acta Pathol Jpn 39: 737-742, 1989.     

Methacholine induces wheal-and-flare reactions in human skin but does not release histamine in vivo as assessed by the skin microdialysis technique

A number of investigations have indicated that cholinergic agonists release histamine from isolated mast cells and suggested that cholinergic stimulation releases histamine in vivo. The purpose of this study was to investigate whether the cutaneous wheal-and-flare reaction induced by methacholine challenge in human skin involves histamine release as measured by the skin microdialysis technique. Five hollow dialysis fibers were inserted intradermally in forearm skin in eight healthy subjects. Each fiber was perfused with Kreb's-Ringer bicarbonate at a rate of 3 μl/min. Dialysates were collected in 2-min fractions before skin challenge and for 20 min after intradermal injection of methacholine 10–^-3 -10^-1 M, the vehicle, and a positive control, codeine phosphate 0.3 mg/ml. Histamine was assayed spectrofluorometrically. Methacholine caused a statistically significant dose-related wheal-and-flare reaction, the flare reaction to methacholine 10^-1 M being comparable with that seen with codeine 0.3 mg/ml. No significant histamine release was observed with methacholine, cumulative histamine release of 16 ± 8nM by methacholine 10^-1 M being similar to vehicle responses of 15 ± 9 nM. Histamine release by codeine was 2524 ± 435 nM. In conclusion, methacholine-induced wheal-and-flare reactions in human skin appeared not to involve histamine release from skin mast cells.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

Culture of Regulatory T-Cell Lines from Bronchial Mucosa

T lymphocytes play a major role in many immune responses. In the last decade, special focus has been on the function of Th1 and Th2 effector cells. Now the importance of regulatory CD4⁺CD25⁺ T cells in maintenance of the immunological homeostasis emerges. Sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. The typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly CD4⁺ T cells of Th1 phenotype. We have cultured T cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (IL-2) and IL-4 and demonstrate spontaneously arising CD4⁺ CD25⁺ populations and high concentrations of IL-10 in these cultures. The main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines IL-6 and TNF-α in cultures of sarcoid origin.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.     

Interleukin-4 and Interferon-Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-γ was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105–1748pg/ml) in 13 and seven cultures, and IFN-γ production (1.7- > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-γ production was abrogated by depletion of CD2⁺ or CD4⁺ but not CD8⁺ cells. CD2⁺ or CD4⁺ but not CD8⁺ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4⁺ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-γ and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-γ and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.     

Rate, extent and concentration dependence of histamine-evoked Weibel–Palade body exocytosis determined from individual fusion events in human endothelial cells

The rate, concentration dependence and extent of histamine-evoked Weibel–Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 ± 0.26 ( s.d. , n = 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s ( n = 9 cells) at 0.3 μ m histamine and 1.57 ± 0.21 s ( n = 15 cells) at 100 μ m histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s⁻¹ ( n = 9) at 0.3 μ m and 3.66 ± 0.45 WPB s⁻¹ at 100 μ m histamine ( n = 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca²⁺ indicating that the initial burst of secretion is driven by Ca²⁺ release from internal stores, but sustained exocytosis required external Ca²⁺. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μ m ), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s ( n = 6), and a peak fusion rate of ∼10 WPB s⁻¹.     

Cytokine and chemokine expression in the skin from patients with maculopapular exanthema to drugs

Background:  Maculopapular exanthema (MPE) is the most frequent clinical manifestation of nonimmediate allergic reactions to drugs and T helper 1 (Th1) cytokines and CD4⁺ T cells have been shown to play an important role in its pathogenesis. We assessed the role of cytokines and chemokines and their receptors in the pathogenesis of MPE.
Methods:  We evaluated skin biopsies and peripheral CD4⁺ and CD8⁺ T cells from 27 patients during the acute phase of the reaction and 26 exposed controls. Semiquantitative real-time PCR was performed to determine the expression of cytokines and chemokines and their receptors and immunohistochemistry was used to determine the same chemokines and their receptor proteins in skin.
Results:  There was a high expression of the Th1 cytokines interferon-γ ( P  = 0.006) and tumor necrosis factor-α ( P  = 0.022) in skin and CD4⁺ T cells ( P  = 0.007 and P  = 0.005, respectively); and of the Th1 chemokines CXCL9 ( P  = 0.005) and CXCL10 ( P  = 0.028) in the skin, while their receptor CXCR3 was increased in skin ( P  = 0.006) and CD4⁺ T cells ( P  = 0.03). Homing chemokine receptors were also increased: CCR6 in skin ( P  = 0.026) and CD4⁺ T cells ( P  = 0.016), and CCR10 only in CD4⁺ T cells ( P  = 0.016), as well as their ligands, CCL20 and CCL27, in skin alone. Immunohistochemistry confirmed these results.
Conclusions:  These data show significant differences in the expression of chemokines and chemokine receptors, related with a Th1 profile, in both skin biopsies and peripheral CD4⁺ T cells in patients with drug-induced MPE.     

Expression of CZ-1: a CD45RB Epitope on Progenitors of Natural Killer and Other Haematopoietic Cells

CZ-1 is a novel sialic acid-dependent epitope of the murine CD45RB molecule which is expressed on cells that proliferate when cultured in IL-2. Because IL-2 appears to be Important in the differentiation of NK cells, the authors examined the expression of CZ-1 on immature NK-Iineage cells within the bone marrow. All mature NK1.1⁺ cells as well as their NK1. l⁻ IL-2 responsive precursors were CZ-1⁺. Furthermore, IL-2 unresponsive transplantable NK progenitor cells expressed CZ-1 also. To examine expression of CZ-1 on other immature lymphoid progenitor cells. CZ-1⁺ and CZ-1 marrow cells were tratisplanted Into lightly irradiated scid mice. Transfer of CZ-1⁺ cells resulted iti rapid and sustained generation of thymocytes and splenic B cells, whereas CZ-1⁻ cells caused delayed repopulation. This suggested that the slowly repoptilating pluripotent stem cells lacked CZ-1. Therefore, expression of CZ-1 on Ly6⁶ Lin⁻ c-kit⁺ cells, highly enriched for pluripotent stem cells, was examined. This population appeared lo be homogeneously CZ-1^dull. Thus, it appears that expression of CZ-1 is developmentally regulated, with diflerentiation associated with increased expression. Since CZ-1 is expressed on a protein tyrosine phosphatase, it is likely that this molecule regulates differentiation of NK and other lymphoid cells.     

ASRI2005-88 Comparable levels of CD4+CD25+ CTLA4+ Treg cells in peripheral blood of pre-eclampsia and normal pregnant patients

The acceptance of the semiallogeneic fetus within the maternal environment requires tolerance mechanisms not fully characterized yet. Normal pregnancy is known to be associated with a Th2 profile. Furthermore, T-regulatory cells were proposed to regulate the Th2/Th1 balance at early stages of pregnancy. Treg may avoid the shift to a Th1 profile preventing miscarriage. Accordingly, spontaneous abortion is characterized by a Th1 dominance and diminished levels of Tregulatory cells (Treg). The major aim of the present work was to investigate if pre-eclampsia, a late immunological complication of pregnancy, is characterized by similar hallmarks. Therefore, we measured the surface antigens CD4, CD25, CD8, CTLA4 (as well as the secretion of IL-10) in peripheral blood from patients suffering from pre-eclampsia (n = 8) and age-matched patients undergoing normal pregnancies (n = 9) by 4-colour flow-cytometry. We were not able to find any significant differences in the levels of CD4⁺, CD25⁺, CD8⁺, CTLA4, CD4⁺/CD25⁺, CD4⁺/CD25^bright, CD4⁺/CTLA4, CD25⁺/CTLA4, CD4⁺/CD25⁺/CTLA4, CD8⁺/CD25⁺, CD8⁺/CTLA4 or CD8⁺/CD25⁺/CTLA4 cell subsets. Our data suggest that Treg may not participate in the onset of pre-eclampsia and suggest other regulatory mechanisms during late pregnancy.     

Ultrastructure of IL2-stimulated Tumor-infiltrating Lymphocytes Showing Cytolytic Activity Against Tumor Cells

Tumor-infiltrating lymphocytes (TIL) obtained from tumor tissue and pleural effusion of breast carcinoma were cultured with interleukin-2 (IL2) and thus activated. The ultrastructure of TIL stimulated by IL2 to kill various breast carcinoma cells was then investigated. Freshly isolated TIL cultured with autologous tumor cells for 48 h without IL2 were small, round and showed neither binding to nor killing of tumor cells. TIL stimulated to proliferate by IL2 became effector cells and showed cytotoxicity against tumor cells. Ultrastructurally, the effector TIL resembled large granular lymphocytes, and adhered to tumor cells through interdigitation or close apposition of the two plasma membranes accompanied by spot-like close membrane contacts. At the site of each spot-like contact, there was a 5-nm intercellular space. The morphology of the TIL processes did not differ from those of LAK and other CTL or NK cell processes during contact, invagination or the killing of target cells. The granules in TIL were considered to participate in the cytotoxic effect. Phenotypically heterogeneous TIL, CD87CD57- and CD8⁺/CD57⁺, adhered to autologous tumor cells and MCF7 (human breast carcinoma cell line). However, it was unclear which cell or cells acted as the effector for tumor-cell killing. Acta Pathol Jpn 41: 94-105, 1991.     

Control of T-cell activation by CD4+ CD25+ suppressor T cells

Summary: Depletion of the minor (∼10%) subpopulation of CD4⁺ T cells that co-expresses CD25 (interleukin (IL)-2 receptor α-chain) by thymectomy of neonates on the third day of life or by treatment of adult CD4⁺ T cells with anti-CD25 and complement results in the development of organ-specific autoimmunity. Autoimmune disease can be prevented by reconstitution of the animals with CD4⁺ CD25⁺ cells. CD4⁺ CD25⁺-mediated protection of autoimmune gastritis does not require the suppressor cytokines IL-4, IL-10, or transforming growth factor (TGF)-β. Mice that express a transgenic T-cell receptor (TCR) derived from a thymectomized newborn that recognizes the gastric parietal cell antigen H/K ATPase all develop severe autoimmune gastritis very early in life. CD4⁺ CD25⁺ T cells are also powerful suppressors of the activation of both CD4⁺ and CD8⁺ T cells in vitro . Suppression is mediated by a cell contact-dependent, cytokine-independent T–T interaction. Activation of CD4⁺ CD25⁺ via their TCR generates suppressor effector cells that are capable of non-specifically suppressing the activation of any CD4⁺ or CD8⁺ T cell. Activation of suppressor effector function is independent of co-stimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We propose that CD4⁺ CD25⁺ T cells recognize organ-specific antigens, are recruited to sites of autoimmune damage where they are activated by their target antigen, and then physically interact with autoreactive CD4⁺ or CD8⁺ effector cells to suppress the development of autoimmune disease.     

Determination of in vitro postantibiotic effects in Staphylococcus aureus and Escherichia coli by [3H]thymidine incorporation

Postantibiotic effects (PAE) and control-related effective regrowth time (CERT) of dicloxacillin, vancomycin, rifampin and gentamicin in Staphylococcus aureus and imipenem, gentamicin, tobramycin, doxycycline and rifampin in Escherichia coli were measured by standard viability counting and [³H]thymidine incorporation. For PAE determination, the two methods correlated well; r ² = 0.821 for S. aureus and r ² = 0.939 for E. coli . For viable counts below the detection limits of 10⁵ to 10⁶ log₁₀ CFU/mL, the PAE was overestimated by the [³H]thymidine method. Quantitation of CERT by both methods showed a good correlation, r ² = 0.867 for S. aureus and r ² = 0.997 for E. coli . Measuring [³H]thymidine incorporation in bacteria is a novel alternative method for the determination of PAE and CERT.