Modulation Of Cell Surface Receptors Research Articles

Multiplex interrogation of the NK cell signalome reveals global downregulation of CD16 signaling during lentivirus infection through an IL-18/ADAM17-dependent mechanism.

Despite their importance, natural killer (NK) cell responses are frequently dysfunctional during human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) infections, even irrespective of antiretroviral therapies, with poorly understood underlying mechanisms. NK cell surface receptor modulation in lentivirus infection has been extensively studied, but a deeper interrogation of complex cell signaling is mostly absent, largely due to the absence of any comprehensive NK cell signaling assay. To fill this knowledge gap, we developed a novel multiplex signaling analysis to broadly assess NK cell signaling. Using this assay, we elucidated that NK cells exhibit global signaling reduction from CD16 both in people living with HIV-1 (PLWH) and SIV-infected rhesus macaques. Intriguingly, antiretroviral treatment did not fully restore diminished CD16 signaling in NK cells from PLWH. As a putative mechanism, we demonstrated that NK cells increased surface ADAM17 expression via elevated plasma IL-18 levels during HIV-1 infection, which in turn reduced surface CD16 downregulation. We also illustrated that CD16 expression and signaling can be restored by ADAM17 perturbation. In summary, our multiplex NK cell signaling analysis delineated unique NK cell signaling perturbations specific to lentiviral infections, resulting in their dysfunction. Our analysis also provides mechanisms that will inform the restoration of dysregulated NK cell functions, offering potential insights for the development of new NK cell-based immunotherapeutics for HIV-1 disease.

Development of an Antibody-Based Platform for the Analysis of Immune Cell-Specific N-linked Glycosylation.

N-linked glycosylation plays an important role in both the innate and adaptive immune response through the modulation of cell surface receptors as well as general cell-to-cell interactions. The study of immune cell N-glycosylation is gaining interest but is hindered by the complexity of cell-type-specific N-glycan analysis. Analytical techniques such as chromatography, LC-MS/MS, and the use of lectins are all currently used to analyze cellular glycosylation. Issues with these analytical techniques include poor throughput, which is often limited to a single sample at a time, lack of structural information, the need for a large amount of starting materials, and the requirement for cell purification, thereby reducing their feasibility for N-glycan study. Here, we report the development of a rapid antibody array-based approach for the capture of specific nonadherent immune cells coupled with MALDI-IMS to analyze cellular N-glycosylation. This workflow is adaptable to multiple N-glycan imaging approaches such as the removal or stabilization and derivatization of terminal sialic acid residues providing unique avenues of analysis that have otherwise not been explored in immune cell populations. The reproducibility, sensitivity, and versatility of this assay provide an invaluable tool for researchers and clinical applications, significantly expanding the field of glycoimmunology.

Water-Soluble BODIPY Photocages with Tunable Cellular Localization.

Photoactivation of bioactive molecules allows manipulation of cellular processes with high spatiotemporal precision. The recent emergence of visible-light excitable photoprotecting groups has the potential to further expand the established utility of the photoactivation strategy in biological applications by offering higher tissue penetration, diminished phototoxicity, and compatibility with other light-dependent techniques. Nevertheless, a critical barrier to such applications remains the significant hydrophobicity of most visible-light excitable photocaging groups. Here, we find that applying the conventional 2,6-sulfonation to meso-methyl BODIPY photocages is incompatible with their photoreaction due to an increase in the excited state barrier for photorelease. We present a simple, remote sulfonation solution to BODIPY photocages that imparts water solubility and provides control over cellular permeability while retaining their favorable spectroscopic and photoreaction properties. Peripherally disulfonated BODIPY photocages are cell impermeable, making them useful for modulation of cell-surface receptors, while monosulfonated BODIPY retains the ability to cross the cellular membrane and can modulate intracellular targets. This new approach is generalizable for controlling BODIPY localization and was validated by sensitization of mammalian cells and neurons by visible-light photoactivation of signaling molecules.

The Potential for Cancer Immunotherapy in Targeting Surgery-Induced Natural Killer Cell Dysfunction.

Natural Killer (NK) cells are granular lymphocytes of the innate immune system that are able to recognize and kill tumor cells without undergoing clonal selection. Discovered over 40 years ago, they have since been recognized to possess both cytotoxic and cytokine-producing effector functions. Following trauma, NK cells are suppressed and their effector functions are impaired. This is especially important for cancer patients undergoing the removal of solid tumors, as surgery has shown to contribute to the development of metastasis and cancer recurrence postoperatively. We have recently shown that NK cells are critical mediators in the formation of metastasis after surgery. While research into the mechanism(s) responsible for NK cell dysfunction is ongoing, knowledge of these mechanisms will pave the way for perioperative therapeutics with the potential to improve cancer outcomes by reversing NK cell dysfunction. This review will discuss mechanisms of suppression in the postoperative environment, including hypercoagulability, suppressive soluble factors, the expansion of suppressive cell populations, and how this affects NK cell biology, including modulation of cell surface receptors, the potential for anergy, and immunosuppressive NK cell functions. This review will also outline potential immunotherapies to reverse postoperative NK dysfunction, with the goal of preventing surgery-induced metastasis.

Abstract A014: Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients

Abstract Imprime PGG (Imprime), a soluble yeast 1,3/1,6 β-glucan, is being developed as a novel cancer immunotherapy in conjunction with anti-tumor antibodies in several cancers. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. Mechanistic studies have demonstrated that Imprime forms an immune complex with endogenous IgG and IgM anti-β-glucan antibodies (ABA). The immune complex then binds to and primes innate immune cells, including macrophages, monocytes and neutrophils, to kill antibody-targeted cancer cells via a complement receptor 3-dependent mechanism. In this study, we sought to investigate how the formation of this Imprime-ABA immune complex might influence patient selection strategies. We have shown that there is a threshold level of ABA required for binding of Imprime-ABA immune complex to the innate immune effectors. In human healthy volunteers (N=143) IgG ABA concentrations vary from 8 to 1448 μg/ml. Imprime treatment of whole blood from individuals with ABA concentrations above a threshold yields immune complex formation that allows both classical and alternative complement pathways leading to complement opsonization of the complex. This complex then binds innate immune cells, triggering the modulation of cell surface receptors and production of selective chemokines such as IL-8. We now show that the predominant subclass of endogenous IgG ABA specific to Imprime is IgG2. Additionally, we show that binding of Imprime to immune cells critically involves FcgRIIA, the only Fc receptor capable of binding IgG2 immune complexes. Blocking FcgRIIA significantly inhibits binding to both neutrophils and monocytes in whole blood. This observation was further confirmed by binding of Imprime to HEK cells overexpressing FcgRIIA. In order to evaluate the effect that ABA IgG subclass has on Imprime, chimeric ABAs with identical affinity but differing in the human IgG subclass (IgG1 vs. IgG2) were generated using the heavy and light chain variable domains from a mouse ABA hybridoma. IgG1 or IgG2 ABA added to whole blood of healthy donors with low ABA levels was able to increase Imprime binding, consistent with previous data showing that Imprime binding is dependent on ABA levels. However, the relative increase in binding due to the addition of IgG1 or IgG2 ABA varied among donors. While addition of either IgG1 or IgG2 ABA was equivalent in increasing binding in some donors, IgG1 was more efficacious than IgG2 in other donors. These results led us to look at allelic variants of the FCGR2A gene. In humans, there is a SNP in FCGR2A, R131H, that defines two alleles whose protein products differ in affinity for IgG2, with H variant designating the higher affinity allele and the R variant indicating lower affinity. The variable binding of Imprime in donors with addition of chimeric ABA was found to be related to the FCGR2A genotype. Individuals homozygous for the R genotype showed lower binding with IgG2 chimeric ABA than with IgG1 chimeric ABA. The findings were similar with exogenously added naturally occurring ABA purified from serum. The effect of the R131H SNP on Imprime binding in relation to endogenous ABA levels was further confirmed in a survey of 140 healthy volunteers. The prevalence and functional relevance of this SNP on Imprime binding to immune cells in cancer patients is being evaluated. Together, these data implicate both the concentration of ABA in serum and FCGR2A genotype as critical determinants of the ability of an individual to bind to Imprime and suggest that either or both may enable the selection of patients most likely to derive therapeutic benefit from Imprime treatment. Citation Format: Nandita Bose, Anissa Chan, Adria Jonas, Nadine Ottoson, Xiaohong Qiu, Lindsay Wurst, Steven Leonardo, Peter Maimonis, Katie Ertelt, Ben Harrison, Keith Gorden. Endogenous anti-β-glucan antibodies and FcgRIIA (CD32a) single nucleotide polymorphisms (SNP) as potential predictive biomarkers for the efficacy of Imprime PGG immunotherapy in cancer patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A014.

Immunological properties of Andean starch films are independent of their nanometric roughness and stiffness

Starch is a natural material extracted from roots, seeds, stems and tubers of different plants. It can be processed as a thermoplastic to produce a variety promising products for biomedical applications, including foams, sheets and films. In the present work, we investigated the immunological properties of microfilms prepared with starches extracted from six different types of Andean potatoes and their relationship with the different film-surface features. We confirmed the biocompatibility of all the films using THP-1 human monocytes, noticing only slight decrease in cell viability in two of the tested starches. We also analyzed pro-inflammatory cytokine release and immune cell surface receptor modulation on THP-1 plated onto the films. Our data show differences in the immunological profile of the same cells cultured onto the different starch films. Furthermore, we examined whether the dissimilar stiffness or the nanometric roughness of the films might influence the immune stimulation of the THP-1 monocytes. Our results demonstrate no correlation between cultured THP-1 immune activation and surface film characteristics. We conclude that different Andean native potato starch films have specific ability to interact with cell membranes of immune cells, conceivably due to the different spatial localization of amylose and amylopectin in the diverse starches.

One Protein to Rule them All: Modulation of Cell Surface Receptors and Molecules by HIV Nef

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic.

Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

Lipopolysaccharide (LPS) activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R) and smooth (S) forms signal through Toll-like receptor 4 (TLR4), but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS) and nitric oxide (NO) generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

HSP110 induces “danger signals” upon interaction with antigen presenting cells and mouse mammary carcinoma

HSP110 is a large molecular weight heat shock protein highly capable of chaperoning large proteins. When chaperoning tumour antigens, HSP110 is capable of eliciting effective anti-tumour immune responses. In the present study, we have determined whether such immunoadjuvant properties of HSP110 stem from its ability to induce “danger signals” through interaction with antigen presenting cells (APCs) and with tumour cells. In the previous studies, endotoxin contamination of HSP preparations was always a matter of concern and controversy. Therefore, we prepared recombinant HSP110 with low endotoxin concentration at which LPS did not have any effect on dendritic cells (DCs). We then evaluated the ability of the HSP110 to induce “danger signals” while interacting with APCs or mouse mammary carcinoma cell line (MMC), as evaluated by modulation of cell surface receptors and cytokines involved in innate and adaptive immune responses. We also performed competition studies in order to rule out contribution of endotoxin in HSP110 preparations while interacting with DCs and MMC. We showed that low endotoxin HSP110 induced DCs to up-regulate the expression of MHC class II, CD40 and CD86 molecules, and to secrete pro-inflammatory cytokines IL-6, IL-12 and TNF- α. Importantly, HSP110 induced MMC to secrete IL-12 and elevate secretion of IL-6 and expression of CD40 molecule. These findings demonstrate that HSP110 acts as a “danger signal” through its interaction with DCs and tumour cells, regardless of its endotoxin component. These immunoadjuvant properties of HSP110 suggest that pre-existing immunity in tumour-bearing individuals may be due to the release of HSPs from tumours upon necrosis alerting the immune system against the tumours.

The proteasome and the delicate balance between destruction and rescue.

Inside eukaryotic cells there is a massive protein complex called the proteasome whose raison d'etre is to remove unnecessary proteins by breaking them down into short peptides. The proteasome is thus responsible for an important aspect of cellular regulation because the timely and controlled proteolysis of key cellular factors regulates numerous biological processes such as cell cycle, differentiation, stress response, neuronal morphogenesis, cell surface receptor modulation, secretion, DNA repair, transcriptional regulation, long-term memory, circadian rhythms, immune response, and biogenesis of organelles (Glickman and Ciechanover 2002). With the multitude of substrates targeted and the myriad processes involved, it is not surprising that aberrations in the pathway are implicated in the pathogenesis of many diseases, including cancer.

Ubiquitin/proteasome-mediated protein degradation and quality control of misfolded proteins in the endoplasmic reticulum

Ubiquitin is a 76-amino acid protein that is covalently linked to target proteins, and the proteasome recognizes and degrades proteins that are tagged with ubiquitin chains. The proteasome consists of a cylindrical 20S catalytic particle, capped by PA700 or PA28 regulatory unit. The ubiquitin/proteasome system plays an important role in the degradation of short-lived and regulatory proteins in a variety of cellular processes, including cell cycle progression, modulation of cell surface receptors and ion channels, and quality control of misfolded proteins in the endoplasmic reticulum (ER). Thus, misfolded proteins currently are postulated to be degraded by ubiquitin/proteasome pathways after association with ER chaperones and retrotranslocation from the ER to the cytosol. We have shown that misfolded secretory proteins such as gamma-carboxylation-deficient protein C (cd-PC), a vitamin K-dependent coagulation protein, and genetic variants of thyroglobulin, antithrombin and factor XII were degraded by the proteasome system. Our studies also showed that specific mannose trimming catalyzed by ER mannosidase I was critical for the ER-associated degradation of these glycoproteins. Notably, the undegraded protein fraction remained microsome-associated. To clarify the retrotranslocation in ER-associated degradation of cd-PC, we constructed and expressed chimeras with GFP which was NH2-terminally fused to a fragment of protein C. Interestingly, LC-GFP, which is a chimera of the light chain of protein C and GFP, time-dependently formed intranuclear inclusions in the presence of a proteasome inhibitor. After removal of the proteasome inhibitor, these inclusions disappeared with chase, suggesting that inhibition of the proteasome activity correlates with the formation of the intranuclear inclusions. Taken together, the data suggest that specific mannose trimming enhances the efficiency of retrotranslocation of misfolded glycoproteins from the ER lumen to the cytosol, and subsequently they form intranuclear inclusions under the inhibition of the proteasome activity that might be related with the cellular basis of several neurodegenerative diseases.

The downregulation of CD4 and MHC-I by primate lentiviruses: a paradigm for the modulation of cell surface receptors.

The human and simian immunodeficiency viruses (HIV and SIV) downregulate the cell surface expression of CD4, their primary receptor, and of class I histocompatibility complex (MHC-I), a critical mediator of immune recognition. While the first of these effects seems important to preserve viral infectivity, the second likely promotes immune evasion. Three HIV-1 proteins, Nef, Env and Vpu, contribute to downregulate CD4, Env forms a complex with CD4 in the endoplasmic reticulum, thereby retaining the receptor in this compartment. Nef and Vpu, on the other hand, act as connectors between CD4 and specific intracellular trafficking pathways, targeting the receptor for degradation in the lysosome and the proteasome, respectively. Some of the downstream partners of the viral proteins in these events have been identified, and include the adaptor complex of clathrin-coated pits, the beta subunit of COP-I coatomer, and the ubiquitin pathway-related h-beta TrCP protein. HIV-induced MHC-I downregulation, mostly the effect of Nef, also reflects a redistribution of this receptor, with its accumulation in the Golgi. The modalities of this process, however, are as yet imperfectly understood. New evidence indicates that the mechanisms employed by primate lentiviruses to downmodulate CD4 and MHC-I are also exploited by a number of cellular regulatory processes.

The ubiquitin-proteasome pathway and pathogenesis of human diseases.

The ubiquitin-proteasome pathway plays a pivotal role in the degradation of short-lived and regulatory proteins important in a variety of basic cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels, and antigen presentation. The pathway involves an enzymatic cascade through which multiple ubiquitin molecules are covalently attached to the protein substrate, which is then degraded by the 26S proteasome complex. The pathway has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases (including cystic fibrosis, Angelman's syndrome, and Liddle syndrome), in immune surveillance/viral pathogenesis, and in the pathology of muscle wasting. The molecular mechanisms that underlie these processes are being unraveled at present.

The ubiquitin-proteasome pathway: on protein death and cell life.

The discovery of the ubiquitin pathway and its many substrates and functions has revolutionized our concept of intracellular protein degradation. From an unregulated, non‐specific terminal scavenger process, it has become clear that proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process which plays important roles in a broad array of basic cellular processes. It is carried out by a complex cascade of enzymes and displays a high degree of specificity towards its numerous substrates. Among these are cell cycle and growth regulators, components of signal transduction pathways, enzymes of house keeping and cell‐specific metabolic pathways, and mutated or post‐translationally damaged proteins. The system is also involved in processing major histocompatibility complex (MHC) class I antigens. For many years it has been thought that activity of the system is limited to the cytosol and probably to the nucleus. However, recent experimental evidence has demonstrated that membrane‐anchored and even secretory pathway‐compartmentalized proteins are also targeted by the system. These proteins must be first translocated in a retrograde manner into the cytosol, as components of the pathway have not been identified in the endoplasmic reticulum (ER) lumen. With the multiple cellular targets, it is not surprising that the system is involved in the regulation of many basic cellular processes such as cell cycle and division, differentiation and development, the response to stress and extracellular modulators, morphogenesis of neuronal networks, modulation of cell surface receptors, ion channels and the secretory pathway, DNA repair, regulation of the immune and inflammatory responses, biogenesis of organelles and apoptosis. One would also predict that aberrations in such a complex system may be implicated in the pathogenesis of many diseases, both inherited and acquired. Recent evidence shows that this is indeed the case. Degradation of a protein by the ubiquitin system involves two distinct …

Cell surface receptor modulation on monocytes and granulocytes during clinical and experimental hemodialysis.

We studied the modulation of cell surface receptors related to cell adhesion (L-selectin and Mac-1) on monocytes and granulocytes during clinical (7 patients treated with cuprophan, Cu, and polysulfone, PS, membranes, n = 14) and experimental Cu and PS hemodialysis (n = 14). The objective was to compare cell surface receptor modulation in vivo when large subpopulations of cells are withdrawn from the circulating pool with the experimental model when cells are not sequestrated. The expression of Mac-1 and L-selectin on monocytes increased during clinical Cu dialysis (p = 0.024 and p = 0.0096, respectively) but remained stable during PS dialysis. On granulocytes, an inverse receptor modulation of Mac-1 and L-selectin was observed during clinical Cu dialysis but not during PS dialysis. Mac-1 was significantly higher and L-selectin lower on granulocytes after 15 min of clinical Cu as compared to PS dialysis (p = 0.001 and p = 0.0093, respectively). During experimental Cu dialysis, Mac-1 expression increased and L-selectin decreased markedly and continuously on both monocytes and granulocytes. The L-selectin/Mac-1 ratio on monocytes and granulocytes may be used as an index of the ability of leukocytes to adhere and to be recruited to an inflammatory focus. This ratio was significantly lower during clinical Cu as compared to PS dialysis (p = 0.0008 and p = 0.0015 respectively) indicating that the recruitment of leukocytes to infection foci may be precluded in patients on Cu membranes. Both monocytes and granulocytes showed significantly lower L-selectin/Mac-1 ratio during and after experimental Cu as compared to PS dialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitogenic Response of Osteoblast Cells to Prostate-Specific Antigen Suggests an Activation of Latent TGF-β and a Proteolytic Modulation of Cell Adhesion Receptors

During studies of mitogens in prostate, PSA quantities as low as 2.5 ng/mL caused cultured osteoblast cells to proliferate beyond controls ( p=<0.05). Investigation of this novel mitogenicity suggested the use of several mechanisms by PSA, namely: 1) the activation of latent hTGF-β in PC-3 conditioned medium, PSA treated conditioned medium stimulated DNA uptake in UMR-106 cells to 78% of acid treated conditioned medium, while DNA incorporation was less than controls with anti-hTGF-β neutralizing IgG; and 2) the proteolytic modulation of cell surface receptors with temporary contact inhibition, PSA significantly stimulated cell detachment while hTGF-β enhanced cell attachment of confluent Saos-2 cells above controls. Clinically, these results suggest that PSA may provide a mechanism for both tumor spread and the osteoblastic metastasis so common to prostate cancer.

膜のホスフォリピドによる細胞表層レセプターの調節

膜のホスフォリピドによる細胞表層レセプターの調節

Transforming growth factor-β and the immune system

It is now apparent that the transforming growth factor beta (TGF-beta) family of proteins has potent immunoregulatory properties ranging from effects on the growth and differentiation of primitive stem cells to the differentiated functions of immune effector cells. Although most reports have described the immunosuppressive activities of TGF-beta, recent evidence supports the concept that TGF-beta can have both inhibitory and stimulatory actions on these systems. Recently, it has been found that TGF-beta can have autocrine as well as paracrine effects on the immune system, indicating that immune cells can activate the inactive secreted form of TGF-beta. Furthermore, TGF-beta has differential intracellular effects on cell surface receptor modulation, tyrosine phosphorylation, and cytokine gene transcription as well as cell-mediated cytotoxicity. Importantly, the administration of TGF-beta has proven beneficial in several animal disease models such as septic shock, allograft rejection, and autoimmunity. Moreover, the increased levels of TGF-beta found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases, and AIDS implicate the involvement of TGF-beta in the pathogenesis of some diseases. Ultimately, well designed clinical trials will determine whether the exciting potential of TGF-beta can be used to treat or prevent disease.

Effects of glucocorticoids on deoxyribonucleic acid(DNA) synthesis stimulated by growth factors in cultured rat skin fibroblasts.

The effects of glucocorticoids on deoxyribonucleic acid (DNA) synthesis were studied by using confluent cultured rat skin fibroblasts prepared by enzymatic dispersion and expanded up to passage 3. Dexamethasone caused the inhibition of the DNA synthesis stimulated by 10% fetal calf serum (FCS) in a dose dependent manner. Maximum inhibition (90%-100%) was achieved by the concentration of 10(-7)M. A similar dose dependent inhibition was also obtained in the experiment using epidermal growth factor (EGF) (1 ng/ml) as a stimulant. Dexamethasone (10(-7)M) also inhibited the DNA synthesis stimulated by somatomedin C (100 ng/ml) or platelet derived growth factor (1 half-maximum unit/ml) almost to control levels. Binding studies with 125I-labeled EGF suggested that dexamethasone caused this inhibitory action without modulation of cell surface receptors for EGF. Furthermore, the effects of a variety of glucocorticoids on the DNA synthesis were studied to clarify the structural requirement of glucocorticoids for the inhibition of the DNA synthesis. The results showed that 11 beta-hydroxyl and 21-hydroxyl groups on the steroid nucleus were necessary for the inhibition of the growth factor-stimulated DNA synthesis. Meanwhile, the inhibitory action on the DNA synthesis was markedly diminished by the replacement of a 16 alpha-methyl group by a 16 beta-methyl group in the presence of a bulky group at C-17 (e.g. 17 alpha-valerate). For further elucidation of mechanisms of action of glucocorticoids on the inhibition of the growth factor-stimulated DNA synthesis, the relationships between the structural features of glucocorticoids and their binding ability to the glucocorticoid receptor ([3H]-dexamethasone-binding receptor) were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The modulation of polymorphonuclear leukocyte function by Bacteroides gingivalis.

B acteroides gingivalis has been recognized as a pathogen causing adult periodontal disease. In this study, we examined the culture supernatants of B. gingival is for their ability to alter the function of polymorphonuclear leukocytes (PMNs). After a 60-minute incubation with the culture supernatants of B. gingivalis, it was found that there was a significant reduction of f-methionyl-leucyl-phenylalanine (FMLP) receptor in human and guinea pig PMNs. Superoxide anion (O- 2) production of PMNs stimulated by FMLP was also suppressed when cells were pre-incubated with the culture supernatants of B. gingivalis. A similar suppression was observed when these cells were stimulated by phorbol myristate acetate. An exohemagglutinin fraction of the culture supernatants showed suppressive activity at a low dose (10 μg/mL). The data presented in this paper suggest that B. gingivalis releases substances which suppress the functions of PMNs via the modulation of cell surface receptors and internal cellular events. These suppressive effects may be important in the pathogenesis of this micro-organism.