Presence Of Heat-inactivated Serum Research Articles

Maitake Beta-Glucan Enhances the Therapeutic Effect of Trastuzumab via Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake β-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.

Elevated Serum Lysophosphatidylcholine in Patients with Systemic Lupus Erythematosus Impairs Phagocytosis of Necrotic Cells In Vitro.

Impaired clearance of dying and dead cells by professional and amateur phagocytes plays a crucial role in the etiology of systemic lupus erythematosus (SLE). While dying, cells expose and release a plethora of eat-me and find-me signals to ensure their timely removal before entering the dangerous stage of secondary necrosis. A well-described chemoattractant for macrophages is dying cell-derived lysophosphatidylcholine (LPC). However, its implications for and/or its association with SLE disease, so far, have not been examined. In the present study, we analyzed the LPC serum concentrations of patients with SLE and rheumatoid arthritis (RA). Subsequently, we examined if and to which extent the measured serum concentrations of LPC and an LPC-rich environment can impact the phagocytosis of necrotic cells. Sera from patients with SLE, RA, and normal healthy donors (NHD) were characterized for several parameters, including LPC concentrations. Phagocytosis of dead cells by human macrophages in the presence of SLE and NHD sera was quantified. Additionally, the impact of exogenously added, purified LPC on phagocytosis was analyzed. Patients with SLE had significantly increased LPC serum levels, and high serum LPC of SLE patients correlated significantly with impaired phagocytosis of dead cells in the presence of heat-inactivated serum. Phagocytosis in the presence of sera from NHD showed no correlation to LPC levels, but exogenous addition of purified LPC in the range as measured in SLE patients' sera led to a concentration-dependent decrease. Our data show that high levels of LPC as observed in the sera of SLE patients have a negative impact on the clearance of dead cells by macrophages. Chemoattraction requires a concentration gradient. The higher the LPC concentration surrounding a dying or dead cell, the smaller the achievable gradient upon LPC release will be. Thus, it is feasible to assume that elevated LPC levels can interfere with the build-up of a local LPC gradient during cell death, and hence might play a role in the establishment and/or perpetuation of SLE disease.

Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatusorganisms

BackgroundAspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response.ResultsThe inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed to A. fumigatus was investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis during A. fumigatus defensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed to A. fumigatus points to the biological significance of described phenomena.ConclusionOur findings provide evidence that respiratory epithelium might play an important role in the immune response during Aspergillus infection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.

Intracellular survival of Leishmania major in neutrophil granulocytes after uptake in the absence of heat-labile serum factors.

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of Leishmania. However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of Leishmania. Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.

Ln vitro effects of platelet derived growth factor on human keratocyte response to excimer laser ablation

SUMMARY Purpose: To evaluate the in vitro effects of platelet derived growth factor (PDGF) on the response ofhuman keratocytes to excimer laser ablation. Methods: Keratocytes were cultured to confluence in 6-well tissue culture plates and exposed to an excimer laser beam. After the laser they were mantained in one of the following media: Group I -Dulbecco's Modified Eagle Medium supplemented with heat-inactivated fetal bovine serum (FBS); Group II - DMEM + fetal bovine serum (FBS); Group III - DMEM + heat-inactivated FBS plus PDGF at a concentration of 1 ng/ml and Group IV - DMEM + heat-inactivated FBS plus PDGF at a concentration of 10 ng/ml. To quantitate the rate of wound closure micrographs were taken at O, 24, 48, 72 and 96 hours. Results: Ninety-six hours after the excimer laser ablation only keratocytes cultured in FBS (Group II) resurface completely. At evaluations after 48, 72 and 96 hours, the residual area in Groups I, III and IV was bigger than in Group II (p < 0.05). There was no significant difference between groups treated with different concentrations of PDGF. Conclusion: Cultured human keratocytes proliferate and migrate to the wounded area after excimer laser ablation and PDGF actually does not stimulate these cells in the presence ofheat-inactivated serum.

Involvement of C3a and C5a in interleukin-8 secretion by human polymorphonuclear cells in response to capsular material of Cryptococcus neoformans.

In a previous paper we demonstrated that human polymorphonuclear cells (PMN) in the presence of normal human serum (NHS) secrete proinflammatory cytokines in response to Cryptococcus neoformans or its major capsular component, glucuronoxylomannan (GXM). The hypothesis that activation of the complement system could be responsible for the observed phenomenon is supported by the fact that encapsulated and acapsular C. neoformans isolates are activators of the complement system and, in particular, large encapsulated isolates are powerful activators. In the present study we demonstrate that (i) interleukin-8 (IL-8) release in response to acapsular or encapsulated strains of C. neoformans is significantly reduced in the presence of heat-inactivated serum rather than NHS and is completely abrogated in the absence of human serum; (ii) GXM-induced IL-8 release is strictly dependent on the presence of NHS, is inhibited by specific antibodies to either C3a and C5 complement components, and is completely abrogated by the combined use of these antibodies; (iii) the addition of purified C3a and C5a directly stimulates IL-8 release by PMN; and (iv) monoclonal antibody to GXM in combination with GXM or encapsulated C. neoformans potentiates IL-8 release by PMN. These data shed light on the mechanism involved in GXM-induced IL-8 secretion by PMN, provide an additional potential role for complement in the control of C. neoformans infections, and suggest a complex interplay between the complement system, humoral immunity, and cytokine regulation.

Involvement of C3a and C5a in Interleukin-8 Secretion by Human Polymorphonuclear Cells in Response to Capsular Material of Cryptococcus neoformans

In a previous paper we demonstrated that human polymorphonuclear cells (PMN) in the presence of normal human serum (NHS) secrete proinflammatory cytokines in response to Cryptococcus neoformans or its major capsular component, glucuronoxylomannan (GXM). The hypothesis that activation of the complement system could be responsible for the observed phenomenon is supported by the fact that encapsulated and acapsular C. neoformans isolates are activators of the complement system and, in particular, large encapsulated isolates are powerful activators. In the present study we demonstrate that (i) interleukin-8 (IL-8) release in response to acapsular or encapsulated strains of C. neoformans is significantly reduced in the presence of heat-inactivated serum rather than NHS and is completely abrogated in the absence of human serum; (ii) GXM-induced IL-8 release is strictly dependent on the presence of NHS, is inhibited by specific antibodies to either C3a and C5 complement components, and is completely abrogated by the combined use of these antibodies; (iii) the addition of purified C3a and C5a directly stimulates IL-8 release by PMN; and (iv) monoclonal antibody to GXM in combination with GXM or encapsulated C. neoformans potentiates IL-8 release by PMN. These data shed light on the mechanism involved in GXM-induced IL-8 secretion by PMN, provide an additional potential role for complement in the control of C. neoformans infections, and suggest a complex interplay between the complement system, humoral immunity, and cytokine regulation.

Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.

Complement receptor-mediated uptake and tumor necrosis factor-alpha-mediated growth inhibition of Mycobacterium tuberculosis by human alveolar macrophages.

The relative phagocytosis and intracellular fate of Mycobacterium tuberculosis (MTB) (H37Ra) in human alveolar macrophages (AM) and their precursors blood monocytes (MN) was investigated. Uptake of MTB by MN and AM was confirmed by electron microscopy. At an infection ratio of 100:1 (MTB:target cell), the percentage of infected AM and the number of MTB per AM was > MN (p < 0.001, p < 0.0001, respectively). Uptake of MTB was increased by increasing concentrations of serum and decreased in the presence of heat-inactivated serum. Among complement receptors (CR) CR1, CR3, and CR4, the major CR mediating uptake of MTB by MN were CR1 and CR3, whereas for AM, CR4 was the major CR. When MN and AM were infected with MTB and cultured for up to 7 days, AM limited intracellular growth of MTB more effectively than MN as determined by a CFU assay. MTB stimulated production of TNF-alpha by mononuclear phagocytes and by AM > MN (p < 0.007). Pentoxifylline inhibited TNF-alpha production by mononuclear phagocytes and concurrently increased MTB growth (AM > MN). A polyclonal neutralizing antibody to TNF-alpha also increased MTB growth in AM. Thus, AM are more efficient in phagocytosis of MTB than MN, and uptake is mediated through CR4 to a greater extent than CR1 or CR3. The slowed replication of MTB in AM is associated with an increase in TNF-alpha production, and intracellular growth is promoted by pentoxifylline and neutralizing antibody to TNF-alpha. These data suggest that AM may play a prominent and efficient role in the primary defense of the lung in tuberculosis through CR-mediated uptake, predominantly CR4, and TNF-alpha-mediated killing of MTB.

Effects of plasminogen and interleukin-1β on bone resorption in vitro

Inflammatory bone resorption, a characteristic feature of periodontal disease and rheumatoid arthritis, appears to be mediated by interleukin-1β (IL-1β). IL-1β has been shown to stimulate a wide range of proteolytic enzymes, including collagenases and plasminogen activators, in particular chondrocytes, synovial cells, and isolated osteoblasts. In this study, we have examined the hypothesis that IL-1β may stimulate bone loss by inducing the activity of plasminogen activators (PAs) in bone cultures. The latter would convert plasminogen to plasmin, which in turn can activate precursor procollagenase to collagenase. Active collagenase would then break down the bone collagen matrix. In the present study, release of 45Ca from fetal rat long bones in culture was studied in the presence of plasminogen and IL-1β. Plasminogen and IL-1β separately enhance resorption of fetal rat long bones in vitro. When plasminogen and IL-1β are added together at suboptimal levels, mainly additive effects are observed. The presence of heat-inactivated serum does not alter these results. These data tend to indicate that IL-1β is stimulating bone resorption through both PA-dependent and PA-independent pathways.

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva.

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a.) and Haemophilus aphrophilus (H.a.) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission--chemiluminescence (CL)--from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a. strains and two out of five A.a. strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a. strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a. strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.

Role of complements C3 and C5 in the phagocytosis of liposomes by human neutrophils.

During the course of a previous investigation, we noticed that the uptake of liposomes by human polymorphonuclear neutrophils (PMNs) was significantly lower in the presence of heat-inactivated serum compared to that in intact whole serum (Scieszka et al., Pharm. Res. 5:352, 1988). This observation suggested the participation of heat-labile complement components in the phagocytic process. In this report we conclude that complement C3bi is the component responsible for opsonization of the liposome surface. Phagocytosis was not supported by C3-deficient serum, and phagocytosis in whole serum was blocked by the antibody to the receptor for C3bi (CR3) but not by the antibody to the receptor for C3b (CR1). We also found that with C5-deficient serum the level of uptake was minimal but slightly higher than without any serum. When exogenous C5a was added along with C5-deficient serum, uptake levels similar in magnitude to those observed with intact serum were obtained. We conclude that C5a enhances phagocytosis of opsonized liposomes by activating the phagocytic capacity of CR3 on the PMN.

Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3.

We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes. mAb against complement receptors (CR) inhibit adherence and phagocytosis of M. tuberculosis in fresh nonimmune serum. A mAb against the type 1 CR (CR1) inhibits adherence of M. tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4%. A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7%. Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2%. mAb against other monocyte surface Ag do not significantly influence adherence. In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M. tuberculosis in the presence of heat-inactivated serum. By electron microscopy, monocytes ingest all M. tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion. In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M. tuberculosis adherence or ingestion. Adherence of M. tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence. Heat inactivation of serum markedly reduces adherence of M. tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold. Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively. Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M. tuberculosis (71 +/- 1%). C component C3 is fixed to M. tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA. Human monocytes ingest M. tuberculosis by conventional phagocytosis as viewed by electron microscopy. This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M. tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand.

Aggregated human colostral sIgA stimulates delayed, non-complement-dependent, NBT reduction by human neutrophils.

Antibodies often alert polymorphonuclear neutrophils (PMNs) to the presence of pathogens. In a study to learn if secretory immunoglobulins can carry out this function, we observed that as little as 4 micrograms/ml of secreted human immunoglobulin A from colostrum (sIgA), in the absence of antigen, stimulated human PMNs to reduce nitroblue tetrazolium (NBT). NBT reduction was inhibited 71% by superoxide dismutase. Active complement pathways were not required since comparable activity was obtained in the presence of heat-inactivated serum. Aggregated forms of sIgA were much more stimulatory than nonaggregated dimeric sIgA. Such interaction between PMNs and sIgA could act in situ to enhance protection against infections of exposed body sites or could initiate inflammatory tissue damage.

Ultrastructure of changes at the surface during the early development phases of Taenia ovis cysticerci in vitro

Harris A., Heath D. D., Lawrence S. B. and Shaw R. J. 1987. Ultrastructure of changes at the surface during the early development phases of Taenia ovis cysticerci in vitro. International Journal for Parasitology 17: 903–910. Transmission and scanning electron microscopy and conventional, interference, and u.v. light microscopy were used to examine the epithelial membrane and underlying cytoplasm during the early development of Taenia ovis cysticerci in vitro. For 2 h after activation of the oncosphere, blebs of plasma membrane containing cytoplasm and secretory granules were released. Two h later the surface area of the plasma membrane had increased by the elaboration of microvilli. Microvilli increased in length and number for 48 h. Synthesis of granules was observed in two of the four lobes of the penetration glands for at least 4 h in vitro. Granules were present in the surface epithelium 4 h after activation. At 24 h granules remaining in the epithelium were electron lucid. In the presence of heat inactivated serum from sheep immunised against T. ovis oncospheres, an electron dense material was precipitated on and between microvilli within 24–48 h of culture. Material in this position reacted positively with fluorescein-labelled antisheep IgG. When metacestodes became motile after 4–5 days, microvilli were sloughed and replaced by a form of microtriche.

The Glucocorticosteroid, Budesonide, Partially Blocks Histamine Release from Human Lung Tissue in Vitro

IgE-receptor dependent, but not A23187-induced, histamine release from passively sensitized chopped human lung tissue, or from a dispersed lung cell population obtained from the tissue after enzymatic digestion, is reduced after incubation overnight of cells/tissue with the glucocorticosteroid budesonide (10(-7) M). Since the inhibitory effect of budesonide is reduced in the continuous presence of diluted reagin-rich serum used for passive sensitization, but not in the presence of heat-inactivated serum, it is suggested that the glucocorticosteroid acts by reducing the binding of IgE-antibody to the target cell(s).

Activation of polymorphonuclear leukocytes by spermatozoa.

Human spermatozoa activated human polymorphonuclear leukocytes (PMN) in the presence of serum. Activation of PMN was studied by measuring the emission of chemiluminescence by the PMN. The amount of chemiluminescence emitted depended on the number of spermatozoa and the serum concentration, and the presence of antibody or complement. Spermatozoa were able to activate PMN in the presence of heat-inactivated serum (only antibody, no complement), serum obtained from a patient with agammaglobulinemia (without antibodies and only complement as opsonin), and in MgEGTA agammaglobulinemic serum (only the alternative pathway of complement intact). In the presence of heat-inactivated agammaglobulinemic serum no significant chemiluminescence was observed. It was concluded that spermatozoa activate the alternative pathway of complement. Dead spermatozoa were more able to activate PMN than viable spermatozoa.

Interaction of human polymorphonuclear leukocytes with Pseudomonas pseudomallei.

The interaction of Pseudomonas pseudomallei with human polymorphonuclear leukocytes (PMNs) has been eamined. Human PMNs ingested P. pseudomallei after incubation for 60min in the presence of normal serum. The bacteria were however relatively resistant to phagocytosis by human PMNs in the absence of serum or in the presence of heat-inactivated serum (56° for 30min). Rapid intracellular killing of P. pseudomallei was observed in the presence of 10% normal human serum; but killing efficiency was reduced in the presence of heat-inactivated serum. Killing in the leukocyte bactericidal assay was shown to be predominantly due to the PMN bactericidal effect; not to the 10% normal serum contained in the incubation mixture. Ingestion of the bacteria was accompanied by a 2-fold stimulation of the hexose monophosphate pathway (HMP). In the absence of phagocytosis, however, the bacteria failed to show a marked enhancement in the HMP-shunt activity. Electron microscopic evidence showed that degranulation and bacterial morphologic changes occurred within 1hr after the organism was ingested. Our data suggest that human PMNs kill P. pseudomallei efficiently after ingestion of the organism in the presence of thermolabile serum opsonins. The occasional chronic nature of the disease caused by P. pseudomallei, therefore, could not be explained simply by the ability of the organism to survive within PMNs.