Lack of augmenting effect of interferon-γ on dengue virus multiplication in human peripheral blood monocytes

The effect of interferon—γ (IFN-γ) on dengue virus multiplication in human peripheral blood mono-cytes was investigated. Enriched monocytes were treated with IFN-γ and then infected with dengue virus type 2 either directly or in the presence of optimal infection-enhancing levels of antibodies. Pretreatment of monocytes from dengue-immune donors with 100 IU/ml of IFN-γ caused 12- to 97-fold and 13- to 137-fold reduction of virus yields at 24 hr after infection in the absence and presence of an anti-flavivirus monoclonal antibody, respectively. IFN-γ also diminished virus yields when infection of monocytes from a donor who lacked anti-dengue antibody was enhanced 40-fold. The percentage of infected monocytes in IFN-γ-pretreated cultures was similarly reduced. Dominance of the antiviral effect of IFN-γ in monocytes is in contrast to an augmenting effect previously observed in the promonocytic cell line U937. © 1995 Wiley-Liss, Inc.     

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.     

Causes and prevalence of inadequate pulmonary function testing among patients with systemic sclerosis

Introduction

Spirometry is a screening tool for evaluating the degree of restrictive lung disease in systemic sclerosis (SSc). Observations indicated that some patients could not complete the test. The aim of the study was to identify the prevalence, causes and clinical predictors of an inadequate pulmonary function test (PFT) in SSc.

Material and methods

A cross-sectional study was performed among SSc patients over 18 years old followed up at Srinagarind Hospital, Khon Kaen, Thailand, during January 2006–December 2012. The adequacy of the PFT was based on the acceptable blow criteria as set out by the American Thoracic Society and the European Respiratory Society 2005 Standardizations of Spirometry.

Results

Two hundred and forty-nine patients were included (female to male ratio was 2 : 1). The mean age at performing PFT was 51.4 ±11.1 years (range: 19.6–79.5). Median duration of disease at performing PFT was 2 years (IQR: 0.6–4.4). Inadequate PFT occurred in 73 cases (prevalence 29.3%: 95% CI: 23.6–35.0); the majority (60 cases; 82.2%) had an expiration time < 6 s and the others were due to plateau < 1 s (11 cases; 15%), air leak around mouth piece (1 case; 1.4%) and hesitation (1 case; 1.4%). Thirteen of 73 (17.8%) had an unusable graph with the overall prevalence of 5.2% (95% CI: 2.4–8.0). The factor associated with inadequate PFT was docy mass index (BMI) < 18.5 kg/m² (OR = 2.17: 95% CI: 1.49–3.17); the same factor was associated with an unusable graph, which was confirmed by the multivariate analysis (OR = 5.21; 95% CI: 1.60–16.95).

Conclusions

One-third of Thai SSc patients had an inadequate pulmonary function test – the majority because of inadequate time for expiring. Low BMI influenced the effectiveness of the test, leading to an incomplete graph for evaluating lung disease in SSc.     

CD99 ligation induces intercellular cell adhesion molecule-1 expression and secretion in human gingival fibroblasts

ObjectiveTo examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3.BackgroundEngagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells.MethodsTotal mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA.ResultsCD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment.ConclusionOur study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.     

Production of monoclonal antibodies to P-glycoprotein: its application in detection of soluble and surface P-glycoprotein of leukemia patients

Multidrug resistance (MDR) in leukemia is commonly associated with the expression of a transmembrane protein, P-glycoprotein (P-gp). In this study, two monoclonal antibodies (mAbs) specific for the extracellular domain of P-gp were generated. By employing the generated mAbs, a two-color lysed whole blood flow cytometric method for surface P-gp and an efficient sandwich ELISA for soluble P-gp determinations were established. By using the established methods, surface and soluble P-gp were detected in several leukemia patients. The presence of soluble P-gp could be used to identify the P-gp surface expression patients. Detection of soluble P-gp reported provides a new basis that may lead to a better understanding of the MDR mechanism in leukemia.     

Enumeration of interleukin 2-producing cells from rat spleen

A method for the enumeration of IL2-producing cells from rat spleen has been developed. Rat spleen cells were stimulated with concanavalin A (Con A), washed, then mixed with IL2-dependent cells (3 day Con A blasts) and plated in soft agar. Clusters of IL2-dependent cells formed around IL2-producing cells, giving colonies which were easy to count under a dissecting microscope. All experimental factors influencing development of colonies of IL2-producing cells surrounded by IL2-dependent cells were evaluated and set up. Optimum number of effector cells was 2.5 x 10(5) cells/culture, while optimum number of IL2-dependent cells was 6 x 10(6) cells/culture. Optimum concentration of Con A for IL2 stimulation was 40 micrograms/ml with an optimal stimulation time of 10 hours. Optimum incubation time for development of IL2-producing cell colonies was 5 days. The number of IL2-producing cells by this technique had a good correlation with the level of IL2 in the cell culture fluid (r = 0.885). When colonies were aspirated from agar and stained by Wright stain, a big purple stained cell at the center was surrounded by small cells in almost all colonies examined. All cells from colonies were fluoresed with anti-mouse Thy 1.2-fluorescein conjugate. However, they were negative with anti-mouse Ig-fluorescein conjugate. The number of IL2-producing cells was 816-2080 cells/10(6) of rat spleen cells with mean +/- S.E.M. = 1404 +/- 154/10(6) cells.     

Immunologic defects in leprosy patients. II. Interleukin 1, interleukin 2, and interferon production in leprosy patients.

The capabilities of monocytes and lymphocytes in peripheral blood mononuclear leukocytes (PBML) to produce interleukin-1 (IL-1), IL-2, and interferon (IFN), respectively, were evaluated in various types and treatments of leprosy patients. IL-1 production in response to lipopolysaccharide was significantly lower in LL, BL, BB, and BT patients than in normal controls. However, there were no differences in IL-1 levels between TT patients and normal controls. The percentages of nonspecific-esterase-positive cells adhering to the plastic surfaces were not different in LL, BB and TT patients when compared to normal controls. However, they were significantly higher in BT and BL patients than in normal controls. When PBML from leprosy patients were stimulated with concanavalin-A (ConA) for IL-2 production, there were no differences in the IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients compared to normal controls. Similar results were obtained when PBML were stimulated with phytohemagglutinin-P (PHA-P). However, when purified protein derivative (PPD) was used as the stimulating agent, there were significantly lower IL-2 levels in treated BL/LL, untreated BL/LL, treated BT/TT, and untreated BT/TT patients when compared to normal controls. There were also lower IL-2 levels in untreated BL/LL and BT/TT patients compared to treated BL/LL and BT/TT patients, respectively. PBML were stimulated with PHA-P or ConA for IFN production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic defects in leprosy patients. I. Evidence of immune aberration of suppressor-T lymphocytes in lepromatous leprosy

Immunoregulation in various types of leprosy patients was evaluated in vitro using peripheral blood mononuclear leukocytes (PBML) stimulated with phytohemagglutinin-P (PHA-P) or concanavalin A (ConA) for a cell-mediated immune (CMI) assay or pokeweed mitogen (PWM) for a humoral-mediated immune (HMI) assay. The immune responses were evaluated by a lymphocyte transformation test (LTT) and lymphocyte-mediated cytotoxicity (LMC) for the immunoregulation of CMI, and a reverse hemolytic plaque assay for measuring the plaque-forming cells (PFC) and a sandwich ELISA for measuring IgG concentrations for the immunoregulation of HMI. In LTT with PHA-P or ConA, the mean of the normal controls was not significantly different from the means of the untreated LL, BL, BB, BT, and TT leprosy patients. However, a wide variation of LTT results from BT to LL patients was noted: the LTT results of TT patients and normal controls were less variable. A similar pattern of immune responses was noted when studied by LMC in untreated LL, BL, BB, BT, and TT leprosy patients and normal controls. When the untreated patients and normal controls were studied for PFC, using PBML stimulated with PWM, a very similar pattern of PFC was obtained with the different types of leprosy patients. The immunoregulatory role of lymphocytes in leprosy patients was further evaluated by cell mixing cultures. ConA-stimulated PBML from lepromatous leprosy patients were mixed with normal PBML and then stimulated with PHA-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

M5, a phosphoinositol-linked human myelomonocytic activation-associated antigen.

When investigating the previously described monoclonal antibody (MoAb) VIM-5, raised against THP1 cells and binding to human monocytes and granulocytes, we found that the antigen detected by this antibody, designated M5, becomes very strongly expressed on monocytes after overnight culture with phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) but not with recombinant human interferon-gamma (rhIFN-gamma). Granulocytes stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) become negative for binding VIM-5. Immature granulocytes from bone marrow do not express M5, thus its expression on granulocytes is differentiation linked. The antigen bound by VIM-5 is sensitive to hydrolysis by phosphoinositol-specific phospholipase C (PI-PLC). The immunoprecipitated M5 antigen on monocytes is a broad band, with a peak of 50 kD (unreduced) and two bands of 53 kD and 44 kD (reduced). We have therefore detected an antigen that is upregulated on stimulated monocytes but, conversely, down-regulated on FMLP-stimulated granulocytes.     

Inhibition of PHA-induced cell proliferation by polyclonal CD4 antibodies generated by DNA immunization.

Although the role of CD4 molecule as associative binding element to MHC class II is well documented, their role in T cell activation is unclear. In the present report we used DNA immunization, which is currently shown to induce potent immune responses, to produce the polyclonal antibodies specific for the CD4 molecule and used the generated antibodies to characterize the CD4 function. A rabbit was pre-treated with bupivacaine hydrochloride for 24 h which was followed by intramuscular injection of DNA encoding CD4 protein (CD4-DNA) at weekly interval. By this procedure, CD4 antibodies were detected in the immunized serum after two DNA inoculations. The CD4 antibodies titer was up to 1:800 after five DNA inoculations. The rabbit polyclonal CD4 antibodies recognized both recombinant CD4 protein expressed on CD4-DNA transfected COS cells and native CD4 protein presented on peripheral lymphocytes and CD4+ cell lines. These generated CD4 antibodies could block the binding of standard CD4 mAb, Leu3a and 13B8.2, to the CD4 molecule. To characterize the function of CD4 molecule, PBMC were cultured in the presence of sub-optimal dose of PHA and the produced polyclonal CD4 antibodies. We found that the polyclonal CD4 antibodies strongly suppressed PHA induced cell proliferation. The inhibitory effect of CD4 antibodies may be due to their steric inhibition of the CD4-TCR/CD3 association or may interfere with the binding of CD4 to its ligand IL-16, resulting in the reduction of signal transduction and subsequent cellular responses. Our results indicate the possibility of utilizing DNA immunization to produce polyclonal antibodies against cell surface molecule.