ABSTRACT
OBJECTIVE: while smell training appears to be effective for post viral smell loss, its effectiveness in COVID-19 induced smell loss is currently not well known. Therefore, we aim to investigate the potential effect of smell training on patients with COVID-19 induced smell loss. METHODS: we conducted a case-control study with two comparable cohorts. One of which (n=111) was instructed to perform smell training twice daily for 12 weeks, therapeutical adherence was monitored on a daily schedule, while the other cohort (n=50) did not perform smell training. The Sniffin' Sticks Test (SST) was used to objectify participants' sense of smell at baseline and after 12 weeks, reported as a Threshold, Discrimination, and Identification (TDI) score. We also determined the association between therapeutical adherence and the TDI scores. RESULTS: we found a significant difference in psychophysical smell function between patients with COVID-19 induced smell disorders who performed 12 weeks of smell training and those who did not. Median TDI difference between groups was 2.00 However, there was no association between the therapeutical adherence and olfactory function. CONCLUSION: we discovered a significant moderate difference in psychophysical smell function between patients with COVID-19-induced smell disorders who performed smell training and those who did not, implying a possible advantage of training. However, no relationship was found between therapeutical adherence of smell training and olfactory function.
Subject(s)
COVID-19 , Olfaction Disorders , SARS-CoV-2 , Humans , COVID-19/complications , Olfaction Disorders/etiology , Olfaction Disorders/virology , Olfaction Disorders/rehabilitation , Olfaction Disorders/therapy , Case-Control Studies , Male , Female , Middle Aged , Adult , Smell/physiology , Anosmia/etiology , Olfactory TrainingABSTRACT
Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.
Subject(s)
Bacterial Proteins/metabolism , Immune Evasion , Neutrophils/enzymology , Serine Proteases/metabolism , Serine Proteinase Inhibitors/metabolism , Staphylococcus aureus/immunology , Virulence Factors/metabolism , Cells, Cultured , Humans , Neutrophils/immunology , Proteolysis , Staphylococcus aureus/pathogenicity , Staphylococcus aureus/physiologyABSTRACT
UNLABELLED: Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF', exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study, the molecular basis of the host specificity of LukMF' is elucidated by identification of chemokine receptor CCR1 as its target. Bovine neutrophils, the major effector cells in the defense against staphylococci, express significant cell surface levels of CCR1, whereas human neutrophils do not. This causes the particular susceptibility of bovine neutrophils to pore formation induced by LukMF'. Bovine S. aureus strains produce high levels of LukMF' in vitro. In culture supernatant of the mastitis field isolate S1444, LukMF' was the most important cytotoxic agent for bovine neutrophils. In a fibrin gel matrix, the effects of the in situ secreted toxins on neutrophils migrating toward S. aureus were visualized. Under these physiological ex vivo conditions, bovine S. aureus S1444 efficiently killed approaching neutrophils at a distance through secretion of LukMF'. Altogether, our findings illustrate the coevolution of pathogen and host, provide new targets for therapeutic and vaccine approaches to treat staphylococcal diseases in the cow, and emphasize the importance of staphylococcal toxins in general. IMPORTANCE: This study explains the mechanism of action of LukMF', a bicomponent toxin found in bovine lineages of S. aureus that is associated with mastitis in cattle. At a molecular level, we describe how LukMF' can specifically kill bovine neutrophils. Here, we demonstrate the contribution of toxins in the determination of host specificity and contribute to the understanding of mechanisms of coevolution of pathogen and host. Our study provides new targets that can be used in therapeutic and vaccine approaches to treat staphylococcal diseases in the cow. We also demonstrate the importance of toxins in specific elimination of immune cells, which has broader implications, especially in human infections.
Subject(s)
Bacterial Proteins/metabolism , Leukocidins/metabolism , Mastitis, Bovine/microbiology , Neutrophils/drug effects , Neutrophils/physiology , Receptors, CCR1/metabolism , Staphylococcus aureus/pathogenicity , Animals , Cattle , Cell Survival/drug effects , Staphylococcus aureus/metabolismABSTRACT
Staphylococcus aureus community-acquired (CA) MRSA strains are highly virulent and can cause infections in otherwise healthy individuals. The most important mechanism of the host for clearing S. aureus is phagocytosis by neutrophils and subsequent killing of the pathogen. Especially CA-MRSA strains are very efficient in circumventing this neutrophil killing. Interestingly, only a relative small number of virulence factors have been associated with CA-MRSA, one of which are the phenol soluble modulins (PSMs). We have recently shown that the PSMs are functionally inhibited by serum lipoproteins, indicating that PSMs may exert their cytolytic function primarily in the intracellular environment. To further investigate the intracellular role of the PSMs we measured the effect of the α-type and ß-type PSMs on neutrophil killing after phagocytosis. Using fluorescently labelled S. aureus, we measured bacterial survival after phagocytosis in a plate reader, which was employed next to flow cytometry and time-lapse microscopy. Phagocytosis of the CA-MRSA strain MW2 by human neutrophils resulted in rapid host cell death. Using mutant strains of MW2, we demonstrated that in the presence of serum, the intracellular expression of only the psmα operon is both necessary and sufficient for both increased neutrophil cell death and increased survival of S. aureus. Our results identify PSMα peptides as prominent contributors to killing of neutrophils after phagocytosis, a finding with major implications for our understanding of S. aureus pathogenesis and strategies for S. aureus vaccine development.
Subject(s)
Bacterial Toxins/metabolism , Methicillin-Resistant Staphylococcus aureus/metabolism , Neutrophils/pathology , Phagocytosis/physiology , Antibodies, Anti-Idiotypic/immunology , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/immunology , Bacterial Vaccines/immunology , Bacterial Vaccines/therapeutic use , Cell Death/physiology , Cells, Cultured , Humans , Neutrophils/physiologyABSTRACT
BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.
Subject(s)
Astrocytoma/pathology , Astrocytoma/prevention & control , Bacterial Proteins/pharmacology , Brain Neoplasms/prevention & control , Chemotaxis/drug effects , Peptide Fragments/pharmacology , Receptors, Formyl Peptide/antagonists & inhibitors , Animals , Astrocytoma/metabolism , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Calcium/metabolism , Cell Movement/drug effects , Enzyme-Linked Immunosorbent Assay , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Grading , Receptors, Formyl Peptide/metabolism , Tumor Cells, CulturedABSTRACT
Toll-like receptors (TLRs) are crucial for our host defense against microbial infections. TLR2 is especially important to fight bacterial infections, as it specifically recognizes bacterial lipoproteins of both Gram-positive and Gram-negative origin. Present on a variety of immune cells, TLR2 is critical for host protection against several bacterial infections, including those caused by Staphylococcus aureus. This major human pathogen causes increasing health care problems due to its increased resistance to antibiotics. S. aureus secretes a wide variety of proteins that inhibit innate immune responses. Recently, several staphylococcal superantigen-like proteins (SSLs) have been described to mediate immune evasive properties. Here, we describe that SSL3 specifically binds and inhibits TLR2 activation on human and murine neutrophils and monocytes. Through binding of the extracellular TLR2 domain, SSL3 inhibits IL-8 production by HEK cells expressing TLR1/2 and TLR2/6 dimers, stimulated with their specific ligands. The SSL3-TLR2 interaction is partially glycan dependent as binding of SSL3 to TLR2 is affected upon removal of sialic acid residues. Moreover, the SSL3(R308A) mutant lacking glycan-binding properties shows lower TLR2 inhibition. An SSL3 mutant, lacking the N-terminal 126 amino acids, still retains full TLR2 inhibiting activity. Of other SSLs tested, only SSL4, which shares the highest homology with SSL3, blocks TLR2 activation. SSL3 is the first-described bacterial protein that blocks TLR2 activation through direct extracellular interaction with the receptor. This unique function of SSL3 adds to the arsenal of immune evasive molecules that S. aureus can employ to subvert both innate and adaptive immunity.
Subject(s)
Antigens, Bacterial/immunology , Staphylococcus aureus/immunology , Superantigens/immunology , Toll-Like Receptor 2/metabolism , Adaptive Immunity , Animals , Antigens, Bacterial/pharmacology , Antigens, CD/metabolism , Glycosylation , HEK293 Cells , Host-Pathogen Interactions , Humans , Immune Evasion , Immunity, Innate , Interleukin-8/biosynthesis , Interleukin-8/metabolism , Mice , Monocytes/immunology , Monocytes/metabolism , Neutrophils/immunology , Neutrophils/metabolism , Protein Binding , Sialic Acids/metabolism , Staphylococcus aureus/physiology , Superantigens/pharmacology , Toll-Like Receptor 2/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVES: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). METHODS AND RESULTS: When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, complete and irreversible aggregation was observed. Proteolysis of the extracellular part of GPIb alpha or the addition of dRGDW abrogated platelet aggregation. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s(-1), stable platelet aggregates were observed, and platelet deposition was substantially reduced after proteolysis of GPIb or after addition of dRGDW. SSL5 was shown to interact with glycocalicin, a soluble GPIb alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3 zeta. In addition, SSL5 was shown to interact with endothelial cell matrix (ECM) and this interaction enhanced aggregation of platelets from whole blood to this ECM. CONCLUSIONS: SSL5 activates and aggregates platelets in a GPIb alpha-dependent manner, which could be important in colonization of the vascular bed and evasion of the immune system by S. aureus.
Subject(s)
Blood Platelets/microbiology , Platelet Activation/immunology , Platelet Adhesiveness/immunology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Staphylococcus/immunology , Superantigens/physiology , Blood Platelets/cytology , Cells, Cultured , Erythrocytes/cytology , Perfusion , Platelet Activation/drug effects , Platelet Adhesiveness/drug effects , Platelet Aggregation/drug effects , Platelet Aggregation/immunology , Superantigens/pharmacologyABSTRACT
Lipopolysaccharide (LPS)-binding components in serum play an important role in modifying LPS toxicity. We analyzed the binding characteristics of LPS in the presence of serum using gel filtration of FITC-labeled LPS (FITC-LPS) with on line detection of optical density and fluorescence. FITC-LPS separately behaves as an aggregate resulting in a low, dequenched, fluorescence. Binding of single LPS molecules, segregated from the aggregate, to serum components results in an increase in the fluorescence due to dequenching, and a comigration of fluorescence and optical density signals using gel filtration. This method, in combination with the use of specific antibodies inducing additional shifts, demonstrated that in serum high-density lipoproteins (HDL), albumin and low-density lipoproteins (LDL) were able to monomerize LPS. An ELISA on collected fractions of the gel filtration revealed binding of the recently identified LPS-binding protein, serum amyloid P component (SAP), to the high molecular weight LPS aggregate. In serum, binding of soluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detected. However, this was probably due to an overshadowing effect of albumin, as an extra addition of recombinant sCD14 to serum clearly monomerized FITC-LPS. Biosensor technology revealed that, of all LPS-binding components tested, only SAP clearly bound to the LPS-coated sensor chip. These results show that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components.
Subject(s)
Acute-Phase Proteins , Blood Proteins/metabolism , Fluorescein-5-isothiocyanate , Lipopolysaccharides/metabolism , Membrane Glycoproteins , Carrier Proteins/analysis , Chromatography, Gel/methods , Chromatography, Gel/standards , Fluorescence , Fluorescent Dyes , Humans , Lipopolysaccharide Receptors/analysis , Lipoproteins, HDL/metabolism , Serum Albumin/metabolism , Serum Amyloid P-Component/metabolismABSTRACT
Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.
Subject(s)
Lipopolysaccharides/metabolism , Lipoproteins, HDL/metabolism , Serum Amyloid P-Component/pharmacology , Apolipoprotein A-I/metabolism , Fluorescein-5-isothiocyanate , Humans , Luminescent Measurements , Serum Amyloid P-Component/metabolismABSTRACT
Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.
Subject(s)
Complement Activation , Gram-Negative Bacteria/immunology , Lipopolysaccharides/immunology , Serum Amyloid P-Component/immunology , Serum Amyloid P-Component/physiology , Animals , Antibodies, Monoclonal/immunology , Dose-Response Relationship, Drug , Escherichia coli/immunology , Gram-Negative Bacteria/growth & development , Humans , Hybridomas/immunology , Mice , Neutrophils/microbiology , Phagocytosis , Salmonella typhimurium/immunology , Serum Amyloid P-Component/pharmacologyABSTRACT
Serum amyloid P component (SAP) is a highly preserved plasma protein named for its ubiquitous presence in amyloid deposits. Although SAP is described to bind many ligands, no clear biological function has been ascribed to it as yet. This review summarizes the current knowledge about the protein SAP, its ligands and functional properties. Finally, the author focuses on the recent finding of the binding of SAP to lipopolysaccharide (LPS) and Gram-negative bacteria and the possible functional consequences of these interactions.
Subject(s)
Lipopolysaccharides/metabolism , Serum Amyloid P-Component/physiology , Animals , Gram-Negative Bacteria/metabolism , HumansABSTRACT
Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans.
Subject(s)
Lipopolysaccharides/immunology , Peptides/immunology , Serum Amyloid P-Component/immunology , Amino Acid Sequence , Animals , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Heparin/metabolism , Humans , Lipopolysaccharides/toxicity , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Monocytes/metabolism , Neutralization Tests , Peptides/chemical synthesis , Respiratory Burst , Serum Amyloid P-Component/chemical synthesis , Shock, Septic/prevention & controlABSTRACT
Biosensor technology was employed to study the specific interactions of different lipopolysaccharide (LPS)-binding proteins and peptides with LPS, using an LPS-coated surface. Two methods to immobilize biotinylated LPS to streptavidin-coated sensor chips (SA-chips) were evaluated. Biotinylated LPS in PBS or biotinylated LPS, pretreated with EDTA and sodium-desoxycholate, were injected across an SA-chip, resulting in a 'high-' and 'low- mass' LPS chip, respectively. While the 'high mass' LPS chip appeared to be unstable, the 'low mass' LPS chip resulted in reproducible binding curves for bactericidal/permeability-increasing protein (rBPI21) with a binding affinity corresponding to the literature (Kd: 3.75 nM). New Kd values were obtained for serum amyloid P component (SAP, Kd: 3.9 nM), a recently discovered new LPS-binding protein, and cationic protein 18 (CAP18, Kd: 0.58 nM). Moreover, binding affinities of bioactive BPI- and SAP-derived peptides could be determined. This study shows for the first time the applicability of biosensor technology to study interactions of proteins and peptides with LPS, using an LPS-coated sensor chip.
Subject(s)
Acute-Phase Proteins , Biosensing Techniques , Lipopolysaccharides/metabolism , Membrane Glycoproteins , Membrane Proteins , Peptides/metabolism , Proteins/metabolism , Amino Acid Sequence , Antimicrobial Cationic Peptides , Blood Proteins/genetics , Blood Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Humans , In Vitro Techniques , Kinetics , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptides/genetics , Protein Binding , Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serum Amyloid P-Component/genetics , Serum Amyloid P-Component/metabolism , StreptavidinABSTRACT
LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27-39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27-39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27-39. The ability of pep27-39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27-39 may be a novel therapeutic agent in the treatment of gram-negative sepsis.
Subject(s)
Acute-Phase Proteins , Carrier Proteins/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Membrane Glycoproteins , Peptide Fragments/pharmacology , Serum Amyloid P-Component/pharmacology , Amino Acid Sequence , Binding, Competitive , Carrier Proteins/blood , Carrier Proteins/chemical synthesis , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Humans , Immunization , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/blood , Lipopolysaccharides/immunology , Molecular Sequence Data , Monocytes/metabolism , Neutrophils/immunology , Peptide Fragments/blood , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Binding/drug effects , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Serum Amyloid P-Component/antagonists & inhibitors , Serum Amyloid P-Component/immunology , Serum Amyloid P-Component/metabolismABSTRACT
To investigate the effects of Mycobacterium tuberculosis on HIV-1 replication, peripheral blood mononuclear cells (PBMC) of bacille Calmette-Guerin (BCG)-vaccinated donors and non-BCG-vaccinated donors were infected in vitro with a lymphotropic isolate of HIV-1 and cultured in the presence of purified protein derivative (PPD). Addition of PPD resulted in enhanced HIV-1 replication and lymphoproliferation in BCG-vaccinated donor PBMC, while PPD had no such effects in control PBMC. HIV-1 replication increased even more when monocytes were removed from PBMC, while lymphoproliferation was decreased. High percentages of monocytes were associated with a decreased HIV-1 replication and proliferation that could not be reversed by addition of antibodies against the cytokines IL-1, transforming growth factor-beta (TGF-beta) or indomethacin. PPD stimulates PBMC to release IL-10, a cytokine known to down-regulate proliferation and HIV-1 replication. PPD-induced effects on proliferation as well as HIV-1 replication could be partially blocked by adding a monoclonal antibody against MHC class II molecules, suggesting that part of the mechanism of PPD-induced enhancement is T memory cell activation.
Subject(s)
HIV-1/physiology , Leukocytes, Mononuclear/virology , Mycobacterium tuberculosis/virology , Virus Replication/physiology , Antibodies, Monoclonal/pharmacology , BCG Vaccine/immunology , BCG Vaccine/pharmacology , Bacterial Proteins/immunology , Bacterial Proteins/pharmacology , Cell Division/drug effects , Histocompatibility Antigens Class II/immunology , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Lymphocytes/immunology , Lymphocytes/virology , Tuberculin/immunology , Tuberculin/pharmacology , Virus Replication/drug effectsABSTRACT
Unlike the selection of HIV-1 variants resistant to anti-retroviral drugs in human peripheral blood mononuclear cells and T cell lines, induction of resistance in monocyte-derived macrophages has not been widely studied. Since macrophages serve as a potential HIV-1 reservoir in humans, knowledge of the effect of anti-retroviral drugs on macrophage-tropic HIV-1 isolates may help in the design of a strategy for prolonged suppression of viral replication. In-vitro selection and drug susceptibility testing of macrophage-tropic HIV-1 variants with reduced sensitivity to two non-nucleoside reverse transcriptase inhibitors, atevirdine and delavirdine (both bis-heteroarylpiperazines), is described here. The atevirdine-resistant isolate was cross-resistant to delavirdine, and the delavirdine-resistant isolate was cross-resistant to atevirdine. Interestingly, the atevirdine-resistant isolate, but not the delavirdine-resistant isolate, was also cross-resistant to nevirapin while the inhibition of viral replication of both isolates in macrophages by zidovudine was the same as that in the parental HIV-1 strain. Nucleotide sequence analysis of the resistant macrophage-tropic HIV-1 isolates showed that the atevirdine-induced resistance was due to a single amino acid change at codon 106 and that the delavirdine-induced resistance could be attributed to an amino acid change at codon 236. This study demonstrates that monocyte-derived macrophages can be used to investigate the phenotypic and genotypic acquisition of anti-retroviral drug resistance of macrophage-tropic HIV-1.
Subject(s)
Drug Resistance, Microbial/genetics , HIV-1/genetics , Macrophages/virology , Monocytes , Reverse Transcriptase Inhibitors/pharmacology , Amino Acid Sequence , Delavirdine/pharmacology , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Humans , Molecular Sequence Data , Piperazines/pharmacology , Sequence Homology, Amino Acid , Zidovudine/pharmacologyABSTRACT
The objective of the study was to evaluate a rapid screening technique for the presence of mutations in the viral reverse transcriptase gene of HIV following prolonged therapy with zidovudine in patients with AIDS. Peripheral blood mononuclear cells (PBMCs) of 14 HIV-infected patients were analyzed by micro-titer point mutation assay (PMA) before therapy with zidovudine and after at least 10 months of treatment. In addition, five of these were analyzed longitudinally. Three nontreated HIV-seropositive individuals were tested as controls. To confirm the validity of the PMA, patients' material was also analyzed with the single strand conformational polymorphism (SSCP) assay. After 10-55 months of treatment, at codons 41, 70, and 215 a shift from predominantly wild type strains to a mixture of wild type and mutant strains (21%-100% mutant sequences) appeared in the majority of patients' PBMCs. At codons 67 and 219, the wild type strain persisted after therapy in all but 3 patients. Most mutations were detected by SSCP as well as by PMA, except for one mutation at codon 41 and one at codon 70. However, when the two mutations were both present, SSCP and PMA were both able to detect these mutations. In conclusion, both PMA and SSCP are rapid and simple methods for screening for mutations causing drug resistance in zidovudine-treated HIV-infected patients. Although PMA is more labor-extensive than SSCP, the advantage of PMA over SSCP is that it permits the quantitative detection of point mutations coding for zidovudine resistance. The application of these assays may improve procedures of monitoring and modifying antiretroviral therapy on an individual basis.
ABSTRACT
Insulin-like growth factor-I and -II (IGF-I and IGF-II), both of which bind to type I IGF receptors, can modulate certain functions of the immune system. We, therefore, studied the expression of type I IGF receptors on purified subpopulations of peripheral blood mononuclear cells. Using two-color flow cytometry, specific binding of a monoclonal antibody directed against the type I IGF receptor (alpha IR3) was found in every subpopulation. Relatively high numbers of receptors were detected on monocytes, natural killer cells, and CD4+ T-helper cells, an intermediate number of receptors on CD8+ suppressor/cytotoxic T-cells, and a relatively low number of receptors on B-cells. The presence of these receptors was confirmed by specific binding of [125I] IGF-I to purified subpopulations. alpha IR3 inhibited the binding of [125I] IGF-I. The specific binding of [125I]IGF-I to monocytes could be completely inhibited by IGF-II and insulin, but higher doses of these peptides were needed than of IGF-I. Scatchard analysis revealed the presence of 734 +/- 426 receptors/monocyte, with a Kd of 0.23 +/- 0.05 nM. A lower number of receptors (230 +/- 52), but with a higher affinity (Kd = 0.05 +/- 0.02 nM), was found on purified T-cells. The positive effect of IGF-I on natural killer cell cytotoxicity indicates that the type I IGF receptors on this cell type are functional. The possibility that IGF-I mediates hormonal effects on the immune system is discussed.