Nonoperative or Surgical Treatment of Acute Achilles' Tendon Rupture.

BACKGROUND: Whether surgical repair of an acute Achilles' tendon rupture by an open-repair or minimally invasive approach is associated with better outcomes than nonsurgical treatment is not clear. METHODS: We performed a multicenter, randomized, controlled trial that compared nonoperative treatment, open repair, and minimally invasive surgery in adults with acute Achilles' tendon rupture who presented to four trial centers. The primary outcome was the change from baseline in the Achilles' tendon Total Rupture Score (scores range from 0 to 100, with higher scores indicating better health status) at 12 months. Secondary outcomes included the incidence of tendon rerupture. RESULTS: A total of 554 patients underwent randomization, and 526 patients were included in the final analysis. The mean changes in the Achilles' tendon Total Rupture Score were -17.0 points in the nonoperative group, -16.0 points in the open-repair group, and -14.7 points in the minimally invasive surgery group (P = 0.57). Pairwise comparisons provided no evidence of differences between the groups. The changes from baseline in physical performance and patient-reported physical function were similar in the three groups. The number of tendon reruptures was higher in the nonoperative group (6.2%) than in the open-repair or minimally invasive surgery group (0.6% in each). There were 9 nerve injuries in the minimally invasive surgery group (in 5.2% of the patients) as compared with 5 in the open-repair group (in 2.8%) and 1 in the nonoperative group (in 0.6%). CONCLUSIONS: In patients with Achilles' tendon rupture, surgery (open repair or minimally invasive surgery) was not associated with better outcomes than nonoperative treatment at 12 months. (Funded by the South-Eastern Norway Regional Health Authority and Akershus University Hospital; ClinicalTrials.gov number, NCT01785264.).

B7H6 is a functional ligand for NKp30 in rat and cattle and determines NKp30 reactivity toward human cancer cell lines.

NK cells kill cancer cells and infected cells upon activation by cell surface receptors. Human NKp30 is an activating receptor expressed by all mature NK cells. The B7 family member B7H6 has been identified as one ligand for NKp30. Several alternative ligands have also been reported, and the field remains unsettled. To this end, we have identified full-length functional B7H6 orthologs in rat and cattle, demonstrated by phylogenetic analysis and transfection experiments. In cell-cell contact-dependent assays, chimeric NKp30 reporter cells responded strongly to B7H6 in rat and cattle. Likewise, rat NKp30 expressing target cells induced strong activation of B7H6 reporter cells. Together, these observations demonstrate that B7H6 is conserved as a functional ligand for NKp30 in mammalian species separated by more than 100 million years of evolution. B7H6 and NKp30 are pseudogenes in laboratory mice. The rat thus represents an attractive experimental animal model to study the NKp30-B7H6 interaction in vivo. B7H6 was widely expressed among human cancer cell lines, and the expression level correlated strongly with the activation of human NKp30 reporter cells. Furthermore, siRNA knockdown of B7H6 abolished NKp30 reporter responses, suggesting that B7H6 is the major functionally relevant expressed ligand for NKp30 on these cancer cell lines.

The Activating Human NK Cell Receptor KIR2DS2 Recognizes a ß2-Microglobulin-Independent Ligand on Cancer Cells.

The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of ß2-microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a ß2-microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after ß2-microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same ß2-microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies.

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.

NK cell receptors in rodents and cattle.

Natural killer (NK) cells discriminate between normal syngeneic cells and infected, neoplastic or MHC-disparate allogeneic cells. The reactivity of NK cells appears to be regulated by a balance between activating receptors that recognize non-self or altered self, and inhibitory receptors recognizing normal, self-encoded MHC class I molecules. Subfamilies of NK receptors undergo rapid evolution, and appear to co-evolve with the MHC. We here review present views on the evolution and function of NK cell receptors, with an emphasis on knowledge gained in cattle and rodents.

KLRE/I1 and KLRE/I2: a novel pair of heterodimeric receptors that inversely regulate NK cell cytotoxicity.

NK cells identify infected, neoplastic, or MHC-disparate target cells via several different receptors. The NK cell receptor KLRE1 lacks known signaling motifs but has nevertheless been shown to regulate NK cell-mediated cytotoxicity. Here we demonstrate that KLRE1 forms functional heterodimers with either KLRI1 or KLRI2. Cotransfection with KLRE1 was necessary for surface expression of the NK cell receptor chains KLRI1 and KLRI2 in 293T cells. Moreover, KLRE1 can be coimmunoprecipitated with KLRI1 or KLRI2 from transfected NK cell lines. By flow cytometry, KLRE1 and KLRI1 showed colinear expression on NK cells, suggesting surface expression as heterodimers. Unlike other killer cell lectin-like receptors, KLRE1/KLRI1 and KLRE1/KLRI2 heterodimers predominantly migrated as single chains in SDS-PAGE, indicating noncovalent association. KLRI1 was coimmunoprecipitated with the tyrosine phosphatase Src homology region 2 domain-containing phosphatase 1. In accordance with an inhibitory function, anti-HA Ab induced reduced killing of FcR-bearing targets by KLRI1-HA-transfected NK cell lines in a redirected cytotoxicity assay. Reciprocally, KLRI2-HA transfectants displayed increased killing in this assay. Finally, Ab to KLRE1 induced inhibition in KLRI1-transfected cells but increased cytotoxicity in KLRI2 transfectants, demonstrating that KLRE/I1 is a functional inhibitory heterodimer in NK cells, whereas KLRE/I2 is an activating heterodimeric receptor.

The rat orthologue to the inhibitory receptor gp49B is expressed by neutrophils and monocytes, but not by NK cells or mast cells.

Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.

Molecular cloning of KLRI1 and KLRI2, a novel pair of lectin-like natural killer-cell receptors with opposing signalling motifs.

We here report the molecular cloning of a novel family of killer-cell lectin-like (KLR) receptors in the rat and the mouse, termed KLRI. In both species, there are two members, KLRI1 and KLRI2. While the extracellular lectin-like domains of KLRI1 and KLRI2 are similar [74% (rat) and 83% (mouse) amino acid identity], they differ intracellularly. KLRI1 has two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic domain, suggesting an inhibitory function. KLRI2 has no ITIM, but a positively charged lysine residue in the transmembrane region, suggesting association with activating adapter molecules. Klri1 and Klri2 are localized within the natural killer (NK) cell gene complex on rat chromosome 4 and mouse chromosome 6. By RT-PCR and Northern blot analysis KLRI1 and KLRI2 were selectively expressed by NK cells in both rat and mouse. Epitope-tagged expression constructs of rat KLRI1 and rat KLRI2 induced surface expression of a nondisulphide-linked protein of M(r) 36,000/39,000 and M(r) 34,000, respectively.

Cutting edge: molecular cloning of a killer cell Ig-like receptor in the mouse and rat.

We report the molecular cloning of a KIR3DL1 receptor in the mouse and the rat, between 37.4 and 45.4% identical with primate killer cell Ig-like receptors (KIRs/CD158). Both mouse and rat molecules contain a pair of immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic regions, suggesting an inhibitory function. Southern blot analysis indicated a single KIR gene in the rat, whereas the mouse genome contains more than one KIR-related element. The rat Kir3dl1 locus was mapped to the leukocyte receptor gene complex on chromosome 1, whereas mouse Kir3dl1 was localized to the X chromosome. RT-PCR demonstrated that KIR3DL1 was selectively expressed by NK cells in both rat and mouse. An epitope-tagged expression construct of mouse KIR3DL1 transfected into 293T cells induced expression of a approximately 55-kDa protein. Our data indicate that KIR receptors may contribute to the NK cell receptor repertoire in rodents, alongside the Ly-49 family.