Thomas C. S. Keller

Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under six years of age

BackgroundIn Plasmodium falciparum-infected children, the relationships between blood cell histopathology, blood plasma components, development of immunocompetence and disease severity remain poorly understood. Blood from Nigerian children with uncomplicated malaria was analysed to gain insight into these relationships. This investigation presents evidence for circulating neutrophil extracellular traps (NETs) and antinuclear IgG antibodies (ANA). The presence of NETs and ANA to double-stranded DNA along with the cytokine profiles found suggests autoimmune mechanisms that could produce pathogenesis in children, but immunoprotection in adults.MethodsPeripheral blood smear slides and blood samples obtained from 21 Nigerian children under six years of age, presenting with uncomplicated malaria before and seven days after initiation of sulphadoxine-pyrimethamine (SP) treatment were analysed. The slides were stained with Giemsa and with DAPI. Levels of the pro-inflammatory cytokines IFN-γ, IL-2, TNF, CRP, and IL-6, select anti-inflammatory cytokines TGF-β and IL-10, and ANA were determined by immunoassay.ResultsThe children exhibited circulating NETs with adherent parasites and erythrocytes, elevated ANA levels, a Th2 dominated cytokine profile, and left-shifted leukocyte differential counts. Nonspecific ANA levels were significant in 86% of the children pretreatment and in 100% of the children seven days after SP treatment, but in only 33% of age-matched control samples collected during the season of low parasite transmission. Levels of ANA specific for dsDNA were significant in 81% of the children both pre-treatment and post treatment.ConclusionThe results of this investigation suggest that NET formation and ANA to dsDNA may induce pathology in falciparum-infected children, but activate a protective mechanism against falciparum malaria in adults. The significance of in vivo circulating chromatin in NETs and dsDNA ANA as a causative factor in the hyporesponsiveness of CpG oligonucleotide-based malaria vaccines is discussed.

STRUCTURE AND FUNCTION OF TITIN AND NEBULIN

Recent investigations of titin anchorage and elasticity have been supplemented with in vitro expression studies on isolated domains of titin and nebulin. These have yielded new insights into the molecular basis of the functions of these proteins in muscle. The characterization of a cellular (non-muscle) isoform of titin has extended the functional relevance of this family of proteins beyond the realm of muscle.

Smooth muscle cell phenotype modulation and contraction on native and cross-linked polyelectrolyte multilayers.

Smooth muscle cells convert between a motile, proliferative “synthetic” phenotype and a sessile, “contractile” phenotype. The ability to manipulate the phenotype of aortic smooth muscle cells with thin biocompatible polyelectrolyte multilayers (PEMUs) with common surface chemical characteristics but varying stiffness was investigated. The stiffness of (PAH/PAA) PEMUs was varied by heating to form covalent amide bond cross-links between the layers. Atomic force microscopy (AFM) showed that cross-linked PEMUs were thinner than those that were not cross-linked. AFM nanoindentation demonstrated that the Young’s modulus ranged from 6 MPa for hydrated native PEMUs to more than 8 GPa for maximally cross-linked PEMUs. Rat aortic A7r5 smooth muscle cells cultured on native PEMUs exhibited morphology and motility of synthetic cells and expression of the synthetic phenotype markers vimentin, tropomyosin 4, and nonmuscle myosin heavy chain IIB (nmMHCIIB). In comparison, cells cultured on maximally cross-linked PEMUs exhibited the phenotype markers calponin, smooth muscle myosin heavy chain (smMHC), myocardin, transgelin, and smooth muscle α-actin (smActin) that are characteristic of the smooth muscle “contractile” phenotype. Consistent with those cells being “contractile”, A7r5 cells grown on cross-linked PEMUs produced contractile force when stimulated with a Ca2+ ionophore.

Smitin, a novel smooth muscle titin–like protein, interacts with myosin filaments in vivo and in vitro

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.

Cytotoxicity of free versus multilayered polyelectrolytes.

The cytotoxicity of polyelectrolytes commonly employed for layer-by-layer deposition of polyelectrolyte multilayers (PEMUs) was assessed using rat smooth muscle A7r5 and human osteosarcoma U-2 OS cells. Cell growth, viability, and metabolic assays were used to compare the responses of both cell lines to poly(acrylic acid), PAA, and poly(allylamine hydrochloride), PAH, in solution at concentrations up to 10 mM and to varying thicknesses of (PAA/PAH) PEMUs. Cytotoxicity correlated with increasing concentration of solution polyelectrolytes for both cell types and was greater for the positively charged PAH than for the negatively charged PAA. While metabolism and proliferation of both cell types was slower on PEMUs than on tissue culture plastic, little evidence for direct toxicity on cells was observed. In fact, evidence for more extensive adhesion and cytoskeletal organization was observed with PAH-terminated PEMUs. Differences in cell activity and viability on different thickness PEMU surfaces resulted primarily from differences in attachment for these adhesion-dependent cell lines.

Cell Durotaxis on Polyelectrolyte Multilayers with Photogenerated Gradients of Modulus

Behaviors of rat aortic smooth muscle (A7r5) and human osteosarcoma (U2OS) cells on photo-cross-linked polyelectrolyte multilayers (PEMUs) with uniform, or gradients of, moduli were investigated. The PEMUs were built layer-by-layer with the polycation poly(allylamine hydrochloride) (PAH) and a polyanion poly(acrylic acid) (PAA) that was modified with photoreactive 4-(2-hydroxyethoxy) benzophenone (PAABp). PEMUs with different uniform and gradients of modulus were generated by varying the time of uniform ultraviolet light exposure and by exposure through optical density gradient filters. Analysis of adhesion, morphology, cytoskeletal organization, and motility of the cells on the PEMUs revealed that A7r5 cells established a polarized orientation toward increasing modulus on shallow modulus gradients (approximately 4.7 MPa mm(-1)) and durotaxed toward stiffer regions on steeper gradients (approximately 55 MPa mm(-1)). In contrast, U2OS cells exhibited little orientation or durotaxis on modulus gradients. These results demonstrate the utility of photo-cross-linked PEMUs to direct vascular and osteoblast cell behavior, a potential application for PEMU coatings on biomedical implants.

CLONING AND EXPRESSION OF CHICKEN CLIP-170 AND RESTIN ISOFORMS

We have cloned cDNA for the chicken homologues of human CLIP-170 and Restin and characterized expression of chicken CLIP-170 and Restin messages in a variety of chicken tissues. Chicken CLIP-170 and Restin, like the human homologues, differ only in a stretch of 35 amino acids present in Restin but missing from CLIP-170. This Restin-specific insert is perfectly conserved between the chicken and human sequences at both the protein and nucleotide level and contributes an additional five heptads to one of the heptad repeat regions in the central alpha-helical coiled-coil rod domain. Other highly conserved chicken and human CLIP-170/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-binding motifs in the N-terminal globular head domain and two CCHC metal-binding motifs in the C-terminal globular tail domain. We have used Southern DNA blot analysis and PCR amplification of exon-intron junctions of chicken genomic DNA to confirm that CLIP-170 and Restin are isoforms encoded by the same gene. Semiquantitative RT-PCR analysis of CLIP-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissues tested, except brain, the message for CLIP-170 was more abundant than that for Restin. Comparison of the levels of CLIP-170 and Restin messages in RNA from chicken and human intestinal epithelial cells revealed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and that the two isoforms have distinct functions in a wide variety of cells.

Role of titin in nonmuscle and smooth muscle cells.

Extensive investigation of vertebrate striated muscle titin has yielded significant insight into its structure and function in striated muscle. We have begun to investigate other members of the titin protein family found in vertebrate smooth muscle and nonmuscle cells. Smooth and nonmuscle titins resemble striated muscle titin in molecular size and morphology but differ in their interactions with myosin II filaments and in the structural contexts in which they exist in vivo. Divergence of these titins from the muscle titin paradigm demonstrates the versatility of this remarkable family of giant proteins.

Identification and expression of two novel CLIP-170/Restin isoforms expressed predominantly in muscle

CLIP-170 and Restin, microtubule-binding proteins originally cloned from human cells, are identical except for a stretch of 35 amino acids present in Restin, but missing from CLIP-170. Here we present the discovery of two novel isoforms of the CLIP-170/Restin gene in both chickens and humans. One of the new isoforms, named CLIP-170(11), contains an 11 amino acid insert instead of the 35 amino acid insert found in Restin. Eight of these 11 amino acids, including a helix-breaking proline residue, are perfectly conserved between chickens and humans. The second new isoform, named CLIP-170(11+35), contains both the 11 and 35 amino acid inserts in tandem. PCR analysis of chicken genomic DNA revealed that all four isoforms result from differential splicing of two exons in a region of the CLIP-170 gene that contains approximately 8.6 kb of intervening sequence. We found that the CLIP-170(11) and CLIP-170(11+35) are expressed preferentially in muscle tissues. Chicken and human skeletal muscle express predominantly CLIP-170(11) and to a lesser extent CLIP-170 and CLIP-170(11+35). Adult chicken cardiac and smooth muscles also express CLIP-170(11) and CLIP-170(11+35), but CLIP-170 is the predominant isoform in these muscles as it is in all other tissues except brain. The ratios of CLIP-170 isoform expression found in embryonic and adult chicken cardiac muscles reveal that isoform expression is regulated differentially in different developmental stages as well as in different tissues.

Smooth Muscle Titin Zq Domain Interaction with the Smooth Muscle α-Actinin Central Rod

Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein α-actinin. In striated muscle Z-disks, α-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in α-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the α-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to α-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in α-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for α-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the α-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the α-actinin central rod.