Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56dimCD16+ and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56brightCD16− phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin− NK cell compartment in lymph nodes and related tissues probably outnumbers perforin+ NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.

Here we use time-lapse microscopy to analyse cell-matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP-paxillin to analyse focal contacts and GFP-tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometers per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain alpha5beta1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometers per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell-matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

We find that CD11c ؉ cells with many markers of dendritic cells (DCs) are a major cell type in the skin lesions of psoriasis. These CD11c ؉ cells, which are evident in both epidermis and dermis, are the sites for the expression of two mediators of inflammation, inducible nitric oxide synthase (iNOS) and TNF-␣ in diseased skin. These cells express HLA-DR, CD40, and CD86, lack the Langerin and CD14 markers of Langerhans cells and monocytes, respectively, and to a significant extent express the DC maturation markers DC-LAMP and CD83. Treatment of psoriasis with efalizumab (antiCD11a, Raptiva) strongly reduces infiltration by these DCs in patients responding to this agent. Disease activity after therapy was more related to DC infiltrates and iNOS mRNA levels than T cell infiltrates, and CD11c ؉ cells responded more quickly to therapy than epidermal keratinocytes. Our results suggest that a type of DC, which resembles murine ''Tip-DCs'' that can accumulate during infection, has proinflammatory effects in psoriasis through nitric oxide and TNF-␣ production, and can be an important target for suppressive therapies.autoimmune disease ͉ CD11c ͉ Tip-DC

No abstract

Objective. To investigate the effect of etanercept therapy on radiographic progression in patients with ankylosing spondylitis (AS).Methods Conclusion. Unlike other inflammatory rheumatic diseases such as rheumatoid arthritis and psoriatic arthritis, structural progression in AS seems to be independent of TNF, despite the fact that TNF is responsible for the signs and symptoms due to inflammation in this disease.Ankylosing spondylitis (AS) belongs to a family of rheumatic diseases known as spondylarthritides that characteristically cause spinal joint inflammation and bony fusion of the spine. AS is the prototype of the spondylarthritides and is typified by ankylosis of the axial skeleton. Radiographic damage known to result from AS primarily includes fusion of entheses of the sacroiliac joints and of the posterior articulations and ligaments of the spine. These fusions can lead to impaired spinal mobility and in turn decreased ability to perform daily activities and severely reduced healthrelated quality of life (1).Tumor necrosis factor ␣ (TNF␣) has been shown to play an important role in the inflammatory response observed in AS. It has been found at increased levels in the serum and synovium of patients with AS (2,3), and treatment with TNF␣-blocking agents (etanercept, adalimumab, and infliximab) has been shown to safely and effectively reduce the signs and symptoms of AS (4-6) and significantly improve health-related quality of life (1). In addition, these agents have been shown to ClinicalTrials.gov identifier: NCT00356356.

Conclusion. These data suggest that the acceptable safety profile of etanercept therapy is maintained for up to 8 years in this population of JRA patients. Improvements in the signs and symptoms of JRA were also maintained for up to 8 years.Juvenile rheumatoid arthritis (JRA) is the most common rheumatic disease in children (1,2). JRA is typified by joint inflammation that can cause joint damage, retard normal growth, and lead to long-term disability and decreased quality of life (3,4). Traditional treatment of children with JRA includes the use of nonsteroidal antiinflammatory drugs (NSAIDs), corticoClinicalTrials.gov identifier: NCT00357903.

Fibronectin matrix assembly is a multistep, integrin-dependent process. To investigate the role of integrin dynamics in fibronectin fibrillogenesis, we developed an antibody-chasing technique for simultaneous tracking of two integrin populations by different antibodies. We established that whereas the vitronectin receptor αvβ3 remains within focal contacts, the fibronectin receptor α5β1 translocates from focal contacts into and along extracellular matrix (ECM) contacts. This escalator-like translocation occurs relative to the focal contacts at 6.5 ± 0.7 μm/h and is independent of cell migration. It is induced by ligation of α5β1 integrins and depends on interactions with a functional actin cytoskeleton and vitronectin receptor ligation. During cell spreading, translocation of ligand-occupied α5β1 integrins away from focal contacts and along bundles of actin filaments generates ECM contacts. Tensin is a primary cytoskeletal component of these ECM contacts, and a novel dominant-negative inhibitor of tensin blocked ECM contact formation, integrin translocation, and fibronectin fibrillogenesis without affecting focal contacts. We propose that translocating α5β1 integrins induce initial fibronectin fibrillogenesis by transmitting cytoskeleton-generated tension to extracellular fibronectin molecules. Blocking this integrin translocation by a variety of treatments prevents the formation of ECM contacts and fibronectin fibrillogenesis. These studies identify a localized, directional, integrin translocation mechanism for matrix assembly.

INTRODUCTION DNA replication has been studied since the 1950s. It is well established that double helical DNA needs to be separated for replication by a helicase. Each strand is then copied by a DNA polymerase, continuously on the leading and discontinuously (via Okazaki fragments) on the lagging strand, where each DNA synthesis initiates from an RNA primer provided by primase. After six decades, how DNA polymerases, helicase, primase, and their accessory factors form a replisome and perform concerted leading and lagging strand synthesis at a replication fork had never been visualized in atomic detail. RATIONALE Bacteriophage T7 presents the simplest known DNA replication system, consisting of only three proteins. Helicase and primase reside in one polypeptide chain that forms a hexamer in the presence of DNA and ATP or dTTP. T7 DNA polymerase, aided by E. coli thioredoxin as its processivity factor, carries out both leading and lagging strand DNA synthesis. Based on published biochemical data, we designed a minimal DNA fork to trap these essential proteins in replication competent states. RESULTS We determined cryogenic-electron microscopy (cryo-EM) structures of the T7 replisome and showed how its essential enzymatic functions are coordinated in three dimensions. The hexameric helicase adopts a spiral “lock washer” form that encircles the coil-like lagging DNA strand, with two nucleotides (nt) bound to each protein subunit and adjacent helicase subunits linked by domain swapping. ATP hydrolysis propels each helicase domain to translocate sequentially and coaxially along DNA in a hand-over-hand fashion, advancing 2 nt per step in the 5′ to 3′ direction (Fig. A). Instead of all enzymes moving in the same direction parallel to the downstream parental DNA, a β-hairpin from the leading-strand polymerase separates the two parental DNA strands into a T-shaped fork that enables the closely coupled helicase to unspool the downstream DNA tangentially (Fig. B). By protein-protein and DNA-mediated interactions, the leading-strand DNA polymerase and helicase cooperate to determine the rate of replication. For every ATP hydrolyzed and 2 nt advanced on DNA by the helicase, the DNA polymerase incorporates two deoxyribonucelotides. T7 primase, separated from the leading-strand polymerase by the helicase domain, synthesizes the RNA primers needed to initiate lagging-strand DNA synthesis. Transfer of a short RNA primer from the primase to DNA polymerase is facilitated by a zinc-binding-domain at the N-terminus of the T7 primase-helicase protein. Two lagging strand polymerases can be attached to the hexameric primases with one actively synthesizing DNA and the other waiting for a primer (Fig. B). Such a relay system may allow the discontinuous lagging-strand synthesis to keep pace with the leading-strand synthesis. CONCLUSION We note the similarity between hexameric DNA helicases and AAA+ protein chaperones and unfoldases, which form spiral-shaped hexamers around protein substrates, bind two amino-acid residues with each su...