Biomedical subjects

John P Atkinson

Publications and source records attributed to John P Atkinson.

At least 19 recordsLinked to original sources

PubMed2005

In vivo correction of complement regulatory protein deficiency with an inhibitor targeting the red blood cell membrane.

Because of the complement system's involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to approximately 2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.

Animals↗

PubMed2005

Emerging roles and new functions of CD46.

In the past 20 years, our understanding of the workings of complement regulatory protein, CD46 (membrane cofactor protein), has grown as has the impressive list of pathogens interacting with this membrane-bound complement inhibitor. Referred to as a "pathogen magnet," CD46 serves as a receptor for seven human pathogens. Initially discovered as a widely expressed C3b- and C4b-binding protein, it was subsequently shown to be a cofactor for the serine protease factor I to inactivate by limited proteolysis these two opsonins and components of the convertases. The involvement of CD46 in reproductive processes continues to be an emerging story. It is a protector of placental tissue, but it may also play a more direct role in reproduction through its expression on the inner acrosomal membrane of spermatozoa. Cross-linking CD46 with antibodies or natural or pathogenic ligands induces rapid turnover and signaling events. In this regard, much attention is currently focused on generating human T lymphocyte regulatory cells by cross-linking CD46. Finally, highlighting its importance in protecting cells against excessive complement activation is the discovery that even a heterozygous deficiency of CD46 predisposes to hemolytic uremic syndrome.

Amino Acid Sequence↗

PubMed2005

Complement-induced regulatory T cells suppress T-cell responses but allow for dendritic-cell maturation.

Concurrent activation of the T-cell receptor (TCR) and complement regulator CD46 on human CD4+ T lymphocytes induces Tr1-like regulatory T cells that suppress through IL-10 secretion bystander T-cell proliferation. Here we show that, despite their IL-10 production, CD46-induced T-regulatory T cells (Tregs) do not suppress the activation/maturation of dendritic cells (DCs). DC maturation by complement/CD46-induced Tregs is mediated through simultaneous secretion of GM-CSF and soluble CD40L, factors favoring DC differentiation and reversing inhibitory effects of IL-10. Thus, CD46-induced Tregs produce a distinct cytokine profile that inhibits T-cell responses but leaves DC activation unimpaired. Such "DC-sparing" Tregs could be desirable at host/environment interfaces such as the gastrointestinal tract where their specific cytokine profile provides a mechanism that ensures unresponsiveness to commensal bacteria while maintaining reactivity to invading pathogens.

Antigens, CD↗

PubMed2005

Synergy between two active sites of human complement receptor type 1 (CD35) in complement regulation: implications for the structure of the classical pathway C3 convertase and generation of more potent inhibitors.

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.

Binding Sites↗

PubMed2005

Virulence differences between monkeypox virus isolates from West Africa and the Congo basin.

Studies indicate that West African and Congo basin isolates of monkeypox virus (MPXV) are genetically distinct. Here, we show Congo basin MPXV-ZAI-V79 is more virulent for cynomolgus monkeys as compared to presumed West African MPXV-COP-58. This finding may explain the lack of case-fatalities in the U.S. 2003 monkeypox outbreak, which was caused by a West African virus. Virulence differences between West African and Congo basin MPXV are further supported by epidemiological analyses that observed a similar prevalence of antibodies in non-vaccinated humans in both regions, while >90% of reported cases occurred in the Congo basin, and no fatal cases were observed outside of this region. To determine the basis for this difference in virulence, we sequenced the genomes of one human West African isolate, and two presumed West African isolates and compared the sequences to Congo basin MPXV-ZAI-96-I-16. The analysis identified D10L, D14L, B10R, B14R, and B19R as possible virulence genes, with D14L (ortholog of vaccinia complement protein) as a leading candidate.

Africa, Western↗

PubMed2005

Induction of a regulatory phenotype in human CD4+ T cells by streptococcal M protein.

Regulatory T cells (Tregs) participate in the control of the immune response. In the human system, an IL-10-secreting, T regulatory type 1 cell (Tr1)-like subset of Tregs can be induced by concurrent cross-linking of the TCR and CD46 on naive CD4(+) T cells. Because many viral and bacterial pathogens, including the major human pathogen Streptococcus pyogenes, bind to CD46, we asked whether this bacterium can directly induce Tr1-like cells through the streptococcal ligand for CD46, the M protein. The M5 and M22 proteins were found to induce T cells to develop into the IL-10-producing Tr1-like phenotype. Moreover, whole M5-expressing bacteria, but not isogenic M-negative bacteria, led to proliferation and IL-10 secretion by T cells. The interaction between the M5 protein and T cells was dependent on CD46 and the conserved C repeat region of M5. Supernatants derived from T cells stimulated with M proteins or M protein-expressing bacteria suppressed bystander T cell proliferation through IL-10 secretion. In addition, activation of CD46 through streptococcal M protein induced the expression of granzyme B, providing a second means for these cells to regulate an immune response. These findings suggest that binding to CD46 and exploiting its signaling pathway may represent a strategy employed by a number of important human pathogens to induce directly an immunosuppressive/regulatory phenotype in T cells.

Antigens, Bacterial↗

PubMed2005

Targeted and restricted complement activation on acrosome-reacted spermatozoa.

A specific hypoglycosylated isoform of the complement regulator membrane cofactor protein (MCP; CD46) is expressed on the inner acrosomal membrane (IAM) of spermatozoa. This membrane is exposed after the acrosome reaction, an exocytosis event that occurs upon contact with the zona pellucida. We initiated this investigation to assess MCP's regulatory function in situ on spermatozoa. Upon exposure of human spermatozoa to autologous serum or follicular fluid, we unexpectedly observed that acrosome-reacted spermatozoa activated the complement cascade efficiently through C3 but not beyond. Using FACS to simultaneously evaluate viability, acrosomal status, and complement deposition, we found that complement activation was initiated by C-reactive protein (CRP) and was C1q, C2, and factor B dependent. This pattern is consistent with engagement of the classical pathway followed by amplification through the alternative pathway. C3b deposition was targeted to the IAM, where it was cleaved to C3bi. Factor H, and not MCP, was the cofactor responsible for C3b cleavage. We propose that this localized deposition of complement fragments aids in the fusion process between the spermatozoa and egg, in a role akin to that of complement in immune adherence. In addition, we speculate that this "targeted and restricted" form of complement activation on host cells is a common strategy to handle modified self.

Acrosome↗

PubMed2005

Polychromatic selective population inversion for TROSY experiments with large proteins.

This paper presents polychromatic selective polarization inversion (PC-SPI) as an alternative to the polarization transfer methods recently developed for the application of NMR to large biological molecules. Theoretical and numerical considerations indicate that PC-SPI has the potential for more efficient polarization transfer under conditions of rapid transverse relaxation compared to J coupling- and cross-correlated relaxation-based transfers. The main advantage offered by the method presented here is the maintenance of near-optimal trajectories of inversion of the individual components of the spin magnetization while using broadband optimized pulses. A 2D experiment was implemented combining PC-SPI with TROSY-based chemical shift correlation. The experiment was applied to detect (15)N-(1)H chemical shift correlation spectra of a 200 kDa complex consisting of an 80% (2)H- and uniformly (15)N,(13)C-labeled 22 kDa portion of complement receptor type 1 and unlabeled C3b of complement (180 kDa).

Binding Sites↗

PubMed2005

Engagement of CD46 and alpha5beta1 integrin by group A streptococci is required for efficient invasion of epithelial cells.

Membrane cofactor protein (MCP or CD46), a widely distributed complement regulatory human protein, is a cell surface receptor for many pathogens including group A streptococci (GAS). The surface M protein of GAS binds CD46 and mediates GAS adherence to keratinocytes. In the present study, we studied the role of CD46 in GAS invasion of human lung epithelial cells, A549. Anti-CD46 antibody which specifically blocks the domain to which M protein binds inhibited adherence to and invasion of A549 cells by GAS. Moreover, downregulation of CD46 expression on A549 by RNA interference resulted in reduced invasion of these cells by GAS. A mutant form of CD46 with a deletion in the cytoplasmic domain was overexpressed in A549 cells, which resulted in partial inhibition of invasion. This indicates that the cytoplasmic tail is required for CD46 to promote invasion by GAS. Invasion assays with Lactococcus lactis that express M protein demonstrated the dependence of CD46-promoted invasion on interaction with M protein. In addition, CD46-mediated invasion was also found to be dependent on the extracellular matrix protein fibronectin.

Antigens, Bacterial↗

PubMed2005

Release of host-derived membrane vesicles following pilus-mediated adhesion of Neisseria gonorrhoeae.

Following attachment of Neisseria gonorrhoeae to human epithelial cell lines, the cellular pilus receptor CD46 is shed from the cell and accumulates in the media. In this report, we assess Neisseria-induced alterations in CD46 surface distribution and characterize this complement regulatory protein following its release from the infected cell. Within 3 h of attachment of gonococci to human epithelial cell lines, CD46 is enriched beneath sites of microcolony adhesion. By 6 h post infection, differential ultracentrifugation of culture media from ME-180 monolayers resulted in sedimentation of structurally and functionally intact CD46. Electron microscopy of these 100,000 g pellets revealed 30-200 nm vesicles. These vesicles likely originated from the host cell as they contained additional host cell surface proteins including CD55 and the epidermal growth factor receptor. Further, these vesicles were visualized by quick-freeze, deep-etch electron microscopy in association with the surface of infected ME-180 cells and with pili of adherent gonococci. Like CD46 shedding, CD46 redistribution and vesicle release were insensitive to colchicine and cytochalasin-D but dependent on expression of the pilus retraction protein PilT. This vesiculation may represent a host cell defence response in which surface proteins that are commonly exploited by pathogens, such as CD46, are removed from the cell.

Adenosine Triphosphatases↗

PubMed2005

Localization of regions in CD46 that interact with adenovirus.

A variety of pathogens use CD46, a ubiquitously expressed membrane protein that regulates complement activation, as a cellular attachment receptor. While the CD46 binding sites of several pathogens, including measles virus, Neisseria gonorrhea, and human herpesvirus 6, have been described, the region of CD46 responsible for adenovirus binding has not been determined. In this study, we used competition experiments with known CD46 ligands, CD46-specific antibodies, and a set of CD46 mutants to localize the binding domain for the group B adenovirus serotype 35 (Ad35). Our results show that Ad35 competes with measles virus for binding to CD46 but not with complement protein C3b. We further show that this interaction is a protein-protein interaction and that N glycosylations do not critically contribute to infection with Ad35 fiber-containing Ad vectors. Our data demonstrate that the native conformation of the CCP2 domain is crucial for Ad35 binding and that the substitution of amino acids at positions 130 to 135 or 152 to 156 completely abolishes the receptor function of CD46. These regions localize to the same planar face of CD46 and likely form an extended adenovirus binding surface, since no single amino acid substitution within these areas eliminates virus binding. Finally, we demonstrate that the infection with a virus possessing human group B serotype Ad11 fibers is also mediated by the CCP2 domain. This information is important to better characterize the mechanisms of the receptor recognition by adenovirus relative to other pathogens that interact with CD46, and it may help in the design of antiviral therapeutics against adenovirus serotypes that use CD46 as a primary cellular attachment receptor.

Adenoviruses, Human↗

PubMed2005

CD46 is a cellular receptor for all species B adenoviruses except types 3 and 7.

The 51 human adenovirus serotypes are divided into six species (A to F). Adenovirus serotypes from all species except species B utilize the coxsackie-adenovirus receptor for attachment to host cells in vitro. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes are also associated with renal disease. We have previously demonstrated that adenovirus type 11 (species B) uses CD46 (membrane cofactor protein) as a cellular receptor instead of the coxsackie-adenovirus receptor (A. Segerman et al., J. Virol. 77:9183-9191, 2003). In the present study, we found that transfection with human CD46 cDNA rendered poorly permissive Chinese hamster ovary cells more permissive to infection by all species B adenovirus serotypes except adenovirus types 3 and 7. Moreover, rabbit antiserum against human CD46 blocked or efficiently inhibited all species B serotypes except adenovirus types 3 and 7 from infecting human A549 cells. We also sequenced the gene encoding the fiber protein of adenovirus type 50 (species B) and compared it with the corresponding amino acid sequences from selected serotypes, including all other serotypes of species B. From the results obtained, we conclude that CD46 is a major cellular receptor on A549 cells for all species B adenoviruses except types 3 and 7.

Adenoviruses, Human↗

PubMed2005

T-cell stimulation and regulation: with complements from CD46.

Crosslinking of CD46 and CD3 on naïve human CD4+ T-lymphocytes induces interleukin-10 secretion and granzyme B expression. These highly proliferative T-regulatory type 1-like T-regulatory T-cells (Tregs) can suppress an immune response. We propose that this process is important in the prevention of chronic inflammation such as at epithelial borders and in deactivation of a successful immune response. Relative to the latter, once a complement-fixing polyclonal antibody response has been mounted, in most cases, the pathogen will be rapidly destroyed. At this time, the C3b/C4b-bearing immune complexes could initiate the deactivation arm of an immune response by shutting down immunocompetent cells through CD46-generated T-cells. Herein, we review this pathway for the induction of Tregs, focusing on a role for the complement system and especially signaling through CD46 on human T-cells.

Amino Acid Sequence↗

PubMed2004

Differential expression of granzymes A and B in human cytotoxic lymphocyte subsets and T regulatory cells.

Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells use the perforin/granzyme pathway as a major mechanism to kill pathogen-containing cells and tumor cells.(1,2) Dysregulation of this pathway results in several human diseases, such as hemophagocytic lymphohistiocytosis. Here we characterize the single-cell expression pattern of granzymes A and B in human lymphocytes using a flow cytometry-based assay. We demonstrate that most circulating CD56(+)8(-) NK cells, and approximately half of circulating CD8(+) T lymphocytes, coexpressed both granzymes A and B. In contrast, few circulating CD4(+) T lymphocytes expressed granzymes A or B. Activation of CD8(+) T lymphocytes with concanavalin A (ConA)/interleukin-2 (IL-2), and activation of CD4(+) T lymphocytes with antibodies to CD3/CD28 or CD3/CD46 (to generate T regulatory [Tr1] cells), induced substantial expression of granzyme B, but not granzyme A. Naive CD4(+)CD45RA(+) cells stimulated with antibodies to CD3/CD46 strongly expressed granzyme B, while CD3/CD28 stimulation was ineffective. Finally, we show that granzyme B-expressing CD4(+) Tr1 cells are capable of killing target cells in a perforin-dependent, but major histocompatibility complex (MHC)/T-cell receptor (TCR)-independent, manner. Our results demonstrate discordant expression of granzymes A and B in human lymphocyte subsets and T regulatory cells, which suggests that different granzymes may play unique roles in immune system responses and regulation.

Animals↗

PubMed2004

Human T regulatory cells can use the perforin pathway to cause autologous target cell death.

Cytotoxic T lymphocytes and natural killer cells use the perforin/granzyme pathway to kill virally infected cells and tumor cells. Mutations in genes important for this pathway are associated with several human diseases. CD4(+) T regulatory (Treg) cells have emerged as important in the control of immunopathological processes. We have previously shown that human adaptive Treg cells preferentially express granzyme B and can kill allogeneic target cells in a perforin-dependent manner. Here, we demonstrate that activated human CD4(+)CD25(+) natural Treg cells express granzyme A but very little granzyme B. Furthermore, both Treg subtypes display perforin-dependent cytotoxicity against autologous target cells, including activated CD4(+) and CD8(+) T cells, CD14(+) monocytes, and both immature and mature dendritic cells. This cytotoxicity is dependent on CD18 adhesive interactions but is independent of Fas/FasL. Our findings suggest that the perforin/granzyme pathway is one of the mechanisms that Treg cells can use to control immune responses.

CD18 Antigens↗

PubMed2004

CD46: expanding beyond complement regulation.

During the 1980s CD46 was discovered in a search for C3b binding proteins of human peripheral blood cells. Its role as an inactivator of C3b and C4b deposited on self-tissue is highlighted by the observation that partial deficiency of CD46 is a predisposing factor to hemolytic uremic syndrome. This discovery has an impact on the treatment options for these patients. Other new findings have expanded the role of CD46 in immunity and disease. For example, signaling through CD46 on human T lymphocytes drives them to become regulatory cells, indicating a novel link between the complement system and cellular immunity. Also, CD46 interacts with at least seven human pathogens and participates in reproduction/fertilization, further suggesting that dissecting its multi-faceted activities will have important clinical implications.

Anemia, Hemolytic↗

PubMed2004

Clinical and laboratory evaluation of complement deficiency.

The complement system provides innate defense against microbial pathogens and is a "complement" to humoral (antibody-mediated) immunity. Consisting of plasma and membrane proteins, this proinflammatory system works in part by a cascade involving limited proteolysis whereby one component activates the next, resulting in a dramatic amplification. The overall goal is deposition of complement fragments on pathologic targets for the purposes of opsonization, lysis, and liberation of peptides that promote the inflammatory response. Deficiencies of complement components predispose to infections and autoimmune syndromes. Even though total deficiency of a complement component is rare, patients presenting with certain bacterial infections and autoimmune syndromes, especially SLE, have a much greater incidence of deficiency. This review will summarize the clinical manifestations and pathophysiology of congenital and acquired complement deficiency diseases. We will also present an algorithm for laboratory diagnosis of complement deficiency and discuss current and future therapeutic options.

Complement System Proteins↗

PubMed2004

ScFv-mediated in vivo targeting of DAF to erythrocytes inhibits lysis by complement.

Antibodies or antibody fragments represent a powerful class of targeting moieties to specifically attach proteins to the surface of a given cell or tissue. Since the presence of constant antibody domains in these targeted fusion proteins may have certain disadvantages, we report here the reduction of the targeting epitope to the variable regions of an Ab by the generation of a single chain antibody fragment (scFv). As an effector molecule, we attached the human complement regulatory protein (CRP) decay-accelerating factor (DAF) at its amino-terminus with a scFv specific for TER-119, a red blood cell (RBC) restricted surface antigen of the mouse. This heterologous system enabled us to study (a) the applicability of a scFv as a targeting domain, (b) the functionality of the effector molecule with respect to regulation of the complement cascade in vitro, and (c) the in vivo biodistribution characteristics of a scFv-DAF fusion protein attached to a clinically relevant target cell type. RBCs from C57BL/6 mice loaded in vitro or in vivo with this fusion protein were significantly protected against lysis by human complement. After intravenous injection, a homogeneous population of in vivo tagged RBCs was maintained throughout a 6-day follow-up. This result and in vitro mixing experiments indicated that there was an equilibration of the fusion protein between tagged and non-tagged RBCs. Thus, scFv-mediated targeting of proteins to a selected cell or tissue surface has promise as a means to supplement absent or defective plasma membrane constituents. This approach should therefore be applicable for diseases caused by a membrane protein deficiency such as paroxysmal nocturnal hemoglobinuria (PNH).

Animals↗