Human Interferon-γ Enhances the Expression of Class I and Class II Major Histocompatibility Complex Products in Neoplastic Cells More Effectively than Interferon-α and Interferon-β

Human interferon-γ was more effective than interferon-β or -α in stimulating production of immunoassociated antigens; HLA-A, -B, and -C; and β₂-microglobulin in human M14 and Namalva cells. The comparison was made on the basis of antiviral units, and the stimulation could be abolished by treatment of the interferon-γ preparation with pH 2 or anti-interferon-γ serum.     

Animal cell cultivation for production of biological substances with a novel perfusion culture apparatus

Summary Specific standard methods of a new perfusion culture are described for growth and maintenance of mammalian cells in suspension culture at high density. The new system of perfusion consists of a suspension vessel containing a cone-type cell-sedimentation column as cell separator and serving as well for axis of impeller. In this report, Namalva cells (human lymphoblastoid cells), adapted to a serum-free medium, have been grown in this perfusion system. The cultured cells and the conditioned medium were separated in the cell-sedimentation column; cell-free expended medium was drained from culture vessel to effluent vessel. The cell-sedimentation column device has been used for the propagationin vitro of Namalva cells to densities 7 to 10 × 10⁶ cells/ml in serum-free medium supplemented with insulin, transferrin, sodium pyruvate, selenious acid, and galactose.     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

Antigenic Analysis of Rheumatoid Factor

Papain digestion of human γ globulin yields two main antigenic components, of slow (S) and fast (F) electrophoretic mobility. Recent work has shown that β₂M globulin (γ₁ macroglobulin) contains S but not F antigenic groupings. It also contains specific groupings which may be called X. Rabbit antisera have been prepared which are specific for S, F and X.

Sheep red cells, `sensitized' with rabbit anti-sheep cell serum as for the Rose-Waaler test, were incubated with rheumatoid arthritis sera. Under appropriate conditions these cells failed to agglutinate when resuspended in saline, but could be agglutinated by anti-γ globulin (S+F), anti-S and anti-X, but not by anti-F. Cells incubated with normal serum failed to agglutinate. Thus rheumatoid arthritis sera contain a protein possessing the antigenic characteristics of β₂M globulin which specifically coats sensitized sheep cells. This provides further evidence of the antibody nature of rheumatoid factor.

     

Comparative intermolecular cross-relaxation studies of human hemoglobin in red blood cells and bovine serum albumin in solution

Intermolecular cross‐relaxation rate (CR) spectra [1/T_IS(HDO) or 1/T_IS(H₂O) vs f₂(ppm) profiles] for bovine serum albumin [BSA; molecular weight (MW), 66 kDa] solution, partially hydrolyzed BSA gel (BSA*gel) and packed human red blood cells (RBCs) with normal or unstable hemoglobin (Hb; MW, 65 kDa) were studied using f₂ irradiation ranging from – 100 to 100 ppm at γH₂/2π of 250 Hz. The CR spectra for BSA*gel (pD 4.01, 0.10 M NaCl, 4.83 and 14.39%) exhibited different features in the off‐resonance region (below – 2.00 and above 12.0 ppm) relative to that for BSA solution (pD 7.14, 0.10 M NaCl, 14.39%), indicating the association of BSA* molecules in the gel state. The CR spectrum for packed RBCs was compared with those for BSA*gel and BSA solution (14.39%) by correcting for differences in protein concentration. The corrected CR spectrum for packed normal RBCs in the off‐resonance region was similar to that for BSA solution, indicating that the physical characteristics of Hb in normal RBCs may be in a solution‐like state. Our results on normal RBCs were approximately consistent with the previously reported thermodynamic and hydrodynamic findings that Hb in RBCs and/or in concentrated solution seems to be in a suspension of hard scaled particles. Copyright © 2011 John Wiley & Sons, Ltd.     

The regulation of prostate cancer cell adhesion to human bone marrow endothelial cell monolayers by androgen dihydrotestosterone and cytokines

A previous study from our laboratory suggested that prostate cancer metastasis to bone may be mediated, in part, by preferential adhesion to human bone marrow endothelial (HBME) cells. Tumor cell adhesion to endothelial cells may be modulated by the effect of cytokines on cell adhesion molecules (CAMs). Tumor necrosis factor-alpha (TNF-α) regulates VCAM expression on the endothelium and this effect is enhanced by dihydrotestosterone (DHT). Transforming growth factor-beta (TGF-β) stimulates the expression of α₂β₁integrin on PC-3 cells. The current study investigated the effects of the above cytokines and DHT (singularly and in various combinations) upon HBME and prostate cancer cell expression of VCAM, α₂ integrin subunit, and β₁ integrin subunit by flow cytometry. We also monitored the effects of the above treatments on PC-3 cell adhesion to HBME monolayers. The data demonstrate that none of the treatments significantly altered the expression of selected CAMs on HBME cell and neoplastic prostate cell lines. The treatment of HBME monolayers with various combinations of cytokines and DHT prior to performing adhesion assays with PC-3 demonstrates that treatments containing TGF-β reduced PC-3 cell adhesion to HBME monolayers by 32% or greater (P<0.05). The reduction in PC-3 cell adhesion to TGF-β-treated HBME monolayers was dose dependent. Interestingly, LNCaP cells but not PC-3 cells treated with TGF-β had a reduced ability to adhere to untreated HBME monolayers. These results suggest that TGF-β may reduce tumor cell adhesion to bone marrow microvascular endothelium, in vivo. The biological significance of this observation is discussed.     

Protective effect of bone marrow mesenchymal stem cells on PC12 cells apoptosis mediated by TAG1

Objective: This study aims to explore the protection effect of bone marrow mesenchymal stem cells (BMSCs) on PC12 cells apoptosis mediated by transient axonal glycoprotein 1 (TAG1). Methods: PC12 cells were divided into control group, Aβ_25-35 group and BMSCs + Aβ_25-35 group. The effects of BMSCs on PC12 cells treated by Aβ_25-35 were detected using MTT, Hoechst 33258 and Annexin V-FITC/PI staining methods. The expression levels of TAG1, β-amyloid precursor protein (APP), AICD and p53 were determined by RT-PCR and Western blotting methods. The expression levels of Bax and Bcl-2 were determined by Western blotting method. The activity of Caspase 3 was detected by spectrophotometric method. Results: MTT results showed that cell activity decreased after the treatment of 20 μM Aβ_25-35 for 48 h (P<0.01) while it increased in BMSCs + Aβ_25-35 group (P<0.01). Hoechst 33258 and Annexin V-FITC/PI staining results showed that Aβ_25-35 could induce the apoptosis of PC12 cells while the apoptosis of PC12 cells was inhibited in BMSCs + Aβ_25-35 group. RT-PCR and Western blotting methods showed that 20 μM Aβ_25-35 could increase the expression levels of TAG1, APP, AICD and p53 (P<0.01) while they decreased in BMSCs + Aβ_25-35 group (P<0.01). 20 μM Aβ_25-35 could increase the expression levels of Bax and decrease the expression levels of Bcl-2 (P<0.01), while the expression levels of Bax decreased and the expression levels of Bcl-2 increase in BMSCs + Aβ_25-35 group (P<0.01). 20 μM Aβ_25-35 could enhance Caspase 3 activity while it decreased in BMSCs + Aβ_25-35 group (P<0.01). Conclusions BMSCs with Aβ_25-35 could inhibit the apoptosis of PC12 cells, which maybe related with TAG1/APP/AICD signal pathway.     

Vitamin C modulates DNA damage induced by hydrogen peroxide in human colorectal adenocarcinoma cell lines (HT29) estimated by comet assay in vitro

Introduction

Cancer cells, compared to normal cells, are under increased oxidative stress associated with oncogenic transformation, alterations in metabolic activity, and increased generation of reactive oxygen species.

Material and methods

We investigated the ability of vitamin C to reduce the damage induced by hydrogen peroxide, in human colorectal adenocarcinoma cells in vitro by the comet assay. Additionally, we measured the kinetics and efficacy of the repair of DNA damage after incubation with vitamin C in the presence of H₂O₂.

Results

The obtained results showed that 1 h pre-incubation with vitamin C and exposure to H₂O₂ for the last 10 min of incubation caused a statistically significant (p < 0.05) increase in DNA migration in comet tails in all experimental series. For the 10 µM, 25 µM, 50 µM, 100 µM vitamin C concentrations the levels of DNA damage were as follows: 18.6%, 21.1%, 25.3% and 27.2%, respectively, as compared to the untreated cells (3.26%). However, in comparison with H₂O₂ alone (29.1%), we observed a statistically significant (p < 0.05) decrease of the genotoxic effect in HT29 cells induced by H₂O₂ for the two lowest of concentrations of vitamin C: 10 µM and 25 µM. The HT29 cells were able to achieve effective repair of the damaged DNA within 60 and 120 min after incubation with the tested compounds. All the values obtained in the test were statistically significant (p < 0.05).

Conclusions

Vitamin C caused a weaker DNA damaging effect of hydrogen peroxide and positively influences the level of oxidative DNA damage in HT29 cells (decrease ∼ 30%). We noted that DNA damage was effectively repaired during 120 min postincubation in the tested cells and that oxidative damage was the major type of damage.     

The Separation and Antigenic Characteristics of Some Fractions of Papain Digests of Human Serum Gamma Globulins, and the Antigenic Relationships of Some Human Gamma Globulins with the Gamma Globulins of Some Other Mammalian Sera

1. Papain digests of human γ globulin (7S γ globulin) containing slow (S) and fast (F) immunoelectrophoretic components were fractionated by chromatography on diethylaminoethyl cellulose and by salting out with ammonium sulphate. No direct correlation was found between the antigenic characteristics and the chromatographic and electrophoretic properties of the S component fractionated by these methods.

2. β₂M globulin (γ₁ macroglobulin or 19S γ globulin) contained the S component of γ globulin, but the F component was not detected.

3. Rhesus monkey γ globulin more readily absorbed the anti-F antibodies than the anti-S from anti-human γ-globulin sera. Both rhesus and digested human γ globulins showed a stage of inhibition, when they completely prevented the precipitation of intact human γ globulin by its antiserum. Rhesus γ globulin, however, showed a reaction of partial identity (spur formation) on Ouchterlony analyses with human γ globulin and its antiserum, whereas digested human γ globulin showed a reaction of complete identity. The significance of these observations is discussed.

     

Mycobacterium-Specific γ9δ2 T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity

Numerous pathogens, including Mycobacterium tuberculosis, can activate human γ₉δ₂ T cells to proliferate and express effector mechanisms. γ₉δ₂ T cells can directly inhibit the growth of intracellular mycobacteria and may also act as antigen-presenting cells (APC). Despite evidence for γδ T cells having the capacity to function as APC, the mechanisms involved and importance of these effects on overall tuberculosis (TB) immunity are unknown. We prepared M. tuberculosis-specific γ₉δ₂ T cell lines to study their direct protective effects and APC functions for M. tuberculosis-specific αβ T cells. The direct inhibitory effects on intracellular mycobacteria were measured, and the enhancing effects on proliferative and effector responses of αβ T cells assessed. Furthermore, the importance of cell-to-cell contact and soluble products for γ₉δ₂ T cell effector responses and APC functions were investigated. We demonstrate, in addition to direct inhibitory effects on intracellular mycobacteria, the following: (i) γ₉δ₂ T cells enhance the expansion of M. tuberculosis-specific αβ T cells and increase the ability of αβ T cells to inhibit intracellular mycobacteria; (ii) although soluble mediators are critical for the direct inhibitory effects of γ₉δ₂ T cells, their APC functions do not require soluble mediators; (iii) the APC functions of γ₉δ₂ T cells involve cell-to-cell contact that is dependent on CD40-CD40 ligand (CD40L) interactions; and (iv) fully activated CD4⁺ αβ T cells and γ₉δ₂ T cells provide similar immune enhancing/APC functions for M. tuberculosis-specific T cells. These effector and helper effects of γ₉δ₂ T cells further indicate that these T cells should be considered important new targets for new TB vaccines.     

Charge and size of the conversion products of serum β1C-globulin

Using gel filtration on Sephadex G-200 combined with antigen—antibody crossed electrophoresis of the fractions, serum β_1C-globulin and the conversion products present in stored native serum and stored serum to which EDTA had been added, respectively, were analysed. The conversion products in the inter-β- and γ₁-zone demonstrated in EDTA serum after 1 days storage and after hydrazine treatment, respectively, could not be characterized by gel filtration, probably because of their lability. Sephadex filtration did not induce any conversion of β_1C-globulin in serum. It was found that β_1C- and β_1A-globulin left the column in homogeneous peaks in the `7S' peak, the former slightly before the latter. The conversion product obtained in stored EDTA sera and on electrophoresis migrating in the α₂-zone was found in the right extension of the macroglobulin peak. The precipitation line produced by this component was diffuse and differed distinctly from the precipitation lines of the other components. The possibility of some kind of complex is discussed.     

A surrogate method for assessment of β2-integrin-dependent adhesion of human eosinophils to ICAM-1

We have developed and validated an inexpensive and equivalent method for measuring eosinophil adhesion by β₂-integrin to endothelial ICAM-1 using bovine serum albumin (BSA) as a surrogate for the immunoglobulin supergene. The number of adherent eosinophils on BSA or ICAM-1 coated microplates was quantified by residual eosinophil peroxidase activity. Non-stimulated eosinophils did not adhere to either BSA or ICAM-1. However, after IL-5 stimulation, either BSA or ICAM-1 caused comparable and concentration-dependent adhesion of eosinophils. Eosinophil adhesion was rapid and occurred within 15 to 30 min of incubation for either BSA or ICAM-1. Preincubation of cells with CD11b or CD18 antibody specifically decreased adhesion to either BSA or ICAM-1. IL-5, PAF and fMLP all induced adhesion of eosinophils to either BSA or ICAM-1 in a concentration-dependent manner, and the optimal IL-5, fMLP and PAF concentrations for adhesion to BSA were the same as for adhesion to ICAM-1. BSA-binding was specific for β₂-integrin; neither α-CD49d mAb directed against the α₄-chain or α-CD29 directed against the common β₁-chain of VLA-4 blocked adhesion to BSA or ICAM-1 controls. The protein tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor, wortmanin, and mitogen-activated protein kinase kinase (MEK) inhibitor, U0126, all inhibited IL-5-induced eosinophil adhesion to either BSA or ICAM-1 comparably. These results indicate that BSA is a reliable and economical surrogate ligand for ICAM-1 adhesion to β₂-integrin-dependent adhesion to ICAM-1. Ligation characteristics of BSA are identical to those for soluble ICAM-1, and the assay is suitable for assessment of signal transduction pathways mediating adhesion.     

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [³H]thymidine incorporation. The responses of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the μ or δ chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.     

A kinetic and quantitive analysis of antibody and protein synthesis during the primary response to keyhole limpet haemocyanin

Rabbits were injected with alum-precipitated keyhole limpet haemocyanin. On various days after immunization popliteal lymph node cells were prepared for synthesis of antibody as well as non-antibody proteins by incorporation of ¹⁴C-amino acids. Antibody and proteins were characterized by gel filtration, radio-immunoelectrophoretic and zone electrophoretic analyses of the culture media. Some `natural' antibody appeared to be synthesized by lymph node cells from uninjected animals. The first definite increase in numbers of nucleated lymph node cells and in antibody synthesis occurred on the third day after immunization. The peak of cell numbers and antibody synthesis was attained on the sixth day after antigenic stimulation. The antigen-stimulated cells also synthesized increased amounts of non-antibody proteins, including IgM and IgG, and proteins tentatively identified as α₂ macroglobulin, α₁ globulin and a microglobulin. Most of the radioactive protein which was synthesized following injection of antigen was demonstrably not antibody. Both IgM and IgG antibody were synthesized at all times, but relatively more radioactivity was incorporated into IgM antibody on day 3 and into IgG antibody on day 6 after immunization.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

In Vitro Inhibition of Cytopathic Effect of Influenza Virus and Human Immunodeficiency Virus by Bamboo Leaf Extract Solution and Sodium Copper Chlorophyllin

Background

Although the link between oral and oropharyngeal health status and susceptibility to infection has long been recognized, there is a limit to the selection of antiseptics for oral care.

Methods

Madin-Darby canine kidney (MDCK) cells were exposed to influenza virus and cultured in the presence or absence of test reagents: bamboo leaf extract solution and sodium copper chrolophyllin. MDCK cells were pre-incubated with the reagents to assess the inhibitory activity at adsorption (viral attachment). Similarly, anti-HIV activity and the inhibitory mechanism at adsorption were assessed by MT-2 cell culture system. Mixture of HIV and bamboo leaf extract solution was fixed and examined by transmission electron microscopy.

Results

The 50% inhibitory concentration (IC₅₀) of bamboo leaf extract solution against influenza virus and the 50% cytotoxic concentration (CC₅₀) in MDCK cells of the solution lay between 0.0313–0.0625% and 0.5–1.0%. The solution inhibited the influenza virus adsorption at the concentration of 0.5% (P < 0.05). The values of IC₅₀ and CC₅₀ of sodium copper chlorophyllin lay between 50–100 µM and 200–400 µM, respectively. This inhibited the virus adsorption at 200 µM (P < 0.05). The bamboo leaf extract solution showed values of IC₅₀ against HIV and CC₅₀ in MT-2 cells at around 0.0313% and between 0.25–0.5%, respectively. This solution inhibited HIV adsorption at 1.25% (P < 0.05). The IC₅₀ and CC₅₀ of sodium copper chlorophyllin lay between 50–100 µM and 200–400 µM, respectively. Sodium copper chlorophyllin inhibited HIV adsorption at 2.5 mM (P < 0.05). HIV particles survived after the exposure to 0.5% bamboo leaf extract solution.

Conclusion

Sodium copper chlorophyllin exerted antiviral activities against influenza virus and HIV as the major ingredient of bamboo leaf extract solution by blocking adsorption. This mechanism of action is different completely from the one of povidone-iodine.     

Characterization of an Experimental Vaccine for Bovine Respiratory Syncytial Virus

Bovine respiratory syncytial virus (BRSV) and human respiratory syncytial virus (HRSV) are major causes of respiratory disease in calves and children, respectively, and are priorities for vaccine development. We previously demonstrated that an experimental vaccine, BRSV-immunostimulating complex (ISCOM), is effective in calves with maternal antibodies. The present study focuses on the antigenic characterization of this vaccine for the design of new-generation subunit vaccines. The results of our study confirmed the presence of membrane glycoprotein (G), fusion glycoprotein (F), and nucleoprotein (N) proteins in the ISCOMs, and this knowledge was extended by the identification of matrix (M), M2-1, phosphoprotein (P), small hydrophobic protein (SH) and of cellular membrane proteins, such as the integrins α_Vβ₁, α_Vβ₃, and α₃β₁. The quantity of the major protein F was 4- to 5-fold greater than that of N (∼77 μg versus ∼17 μg/calf dose), whereas G, M, M2-1, P, and SH were likely present in smaller amounts. The polymerase (L), M2-2, nonstructural 1 (NS1), and NS2 proteins were not detected, suggesting that they are not essential for protection. Sera from the BRSV-ISCOM-immunized calves contained high titers of IgG antibody specific for F, G, N, and SH. Antibody responses against M and P were not detected; however, this does not exclude their role in protective T-cell responses. The absence of immunopathological effects of the cellular proteins, such as integrins, needs to be further confirmed, and their possible contribution to adjuvant functions requires elucidation. This work suggests that a combination of several surface and internal proteins should be included in subunit RSV vaccines and identifies absent proteins as potential candidates for differentiating infected from vaccinated animals.     

Anti-inflammatory effect of β2-agonists: Inhibition of TNF-α release from human mast cells

β₂-Agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D₂ from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-α (TNF-α), there is no information about their regulation by β₂-agonists. Thus given the importance of TNF-α in inflammation and the widespread use of β₂-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) β₂-agonists on the secretion of TNF-α from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-α (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-α–sensitive cell line, WEHI-164, with an IC₅₀ of 71, 50, and 29 nmol/L, respectively. Specificity for β-adrenergic receptors was shown with propranolol. The inhibitory effect of β₂-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-α release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, β₂-agonists did not show tachyphylaxis for the inhibition of TNF-α release. Thus selective β₂-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-α from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of β-agonists may be important in their mode of action in the treatment of allergic diseases. (J Allergy Clin Immunol 1997;100:825-31.)     

Quantitative T cell subsets profile in peripheral blood from patients with idiopathic inflammatory myopathies: tilting the balance towards proinflammatory and pro-apoptotic subsets

The role of T cells in idiopathic inflammatory myopathies (IIM) is not yet clear. Some alterations in certain subsets have been reported in inflamed muscle cells. However, a broad quantitative assessment of peripheral T cell subsets has not been evaluated. The aim of this study was to address the quantitative profile of potential pathogenic T cell subsets, namely follicular helper T cells (Tfh), T helper type 17 (Th17), CD28^null and regulatory T cells (T_regs) in peripheral blood from IIM patients. Thirty IIM patients and 30 age- and gender-matched healthy donors were included. Peripheral blood mononuclear cells were isolated. T cell subsets were evaluated by flow cytometry, as follows: Tfh (CD4⁺CXCR5⁺) and its subsets Tfh1 (CXCR3⁺CCR6⁻), Tfh2 (CXCR3⁻CCR6⁻), Tfh17 (CXCR3⁻CCR6⁺), Th17 (CD4⁺IL17A⁺), CD28^null (CD4⁺CD28⁻CD244⁺) and T_regs (CD4⁺CD25^highforkhead box protein 3 (FoxP3⁺); CD8⁺CD25^highFoxP3⁺). Percentage, absolute numbers and mean fluorescence intensity were analysed. We found increased numbers of total Tfh cells (28 ± 8·16 versus 6·64 ± 1·29, P = 0·031) in IIM patients when compared to healthy controls. Moreover, this increment was dependent upon Tfh2 and Tfh17 (Tfh2:9·49 ± 2·19 versus 1·66 ± 0·46, P = 0·005; Tfh17 9·48 ± 2·83 versus 1·18 ± 0·21, P = 0·014). Also, IIM patients showed higher numbers of Th17 cells (30·25 ± 6·49 versus 13·46 ± 2·95, P = 0·031) as well as decreased number of T_regs (5·98 ± 1·61 versus 30·82 ± 8·38, P = 0·009). We also found an expansion of CD28^null cells (162·88 ± 32·29 versus 64 ± 17·35, P = 0·015). Our data suggest that IIM patients are characterized by an expansion of peripheral proinflammatory T cells, such as Tfh and Th17, as well as pro-apoptotic CD28 null cells and a deficiency of suppressor populations of T_regs (CD4⁺ and CD8⁺).     

Identification of the Components of Complement Participating in the Antiglobulin Reaction

Red cells sensitized with a complement-binding antibody and then incubated with fresh serum have been shown to be coated with β_1C and β_1E globulin, two components of the complement system that have been isolated recently. Red cells presumably in the state EAC′_1,4 reacted with anti-β_1E, and cells presumably in the state EAC′_1,4,2,3a reacted with anti β_1E and with anti-β_1C. Agglutination of complement-coated cells by `broad spectrum' antiglobulin sera was effectively inhibited by purified β_1E and β_1C globulin. Red cells from certain patients with acquired haemolytic anaemia were found to be coated in vivo with β_1E and β_1C globulin. The function and significance of the two serum components have been discussed.