Regulation of TGF-β1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-β-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-β are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-β-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-β stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-β-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells.
Fibrosis; Extracellular Matrix; Fibroblast; Epithelial; mesenchymal; ERK MAP kinase
CT pulmonary angiography (CTPA) has become widely adopted to detect pulmonary embolism (PE) after total joint arthroplasty (TJA). CTPA is a sensitive tool, which has the ability to detect emboli that may be clinically insignificant. This may lead to iatrogenic harm from overtreatment.
The purpose of this study was to assess changing prevalence, mortality, treatment complications, and resource consumption associated with PE after TJA before and after the introduction of CTPA.
The Nationwide Inpatient Sample was used to identify 2,335,248 patients undergoing TJA from 1993 to 1998 before the introduction of CTPA and 6,321,671 patients from 1999 to 2008 after the introduction of CTPA. Bivariate and multivariate regression analysis was performed to compare changing prevalence of PE, mortality, potential treatment complications of anticoagulation, length of stay, and total charges before and after the introduction of CTPA in patients with PE.
In-hospital diagnosis of PE after TJA increased (p < 0.001) from an average of 0.27% to 0.37% after the introduction of CTPA. All-cause mortality in patients with a diagnosis of PE decreased (p < 0.001) from 11.5% to 4.6% (odds ratio, 2.3; 95% confidence interval, 2.1–2.6) after the introduction of CTPA. Overall, PE was associated with increased (p < 0.001) risks for hematoma/seroma, postoperative infection, gastrointestinal bleed, and drug-related thrombocytopenia, although the prevalence of these complications has decreased after 1998 (p < 0.001). Length of stay doubled for patients with PE (both before and after CTPA) and total charges increased over 69% in both study periods for these patients.
Adoption of CTPA appears to be associated with an increase in the diagnosis of PE after TJA and an associated decrease in case-fatality. Although CTPA may improve our ability to diagnose PE and possibly reduce mortality, the observed decrease in case-fatality could also be explained by the overdiagnosis of clinically unimportant emboli. The diagnosis of PE was strongly associated with potential iatrogenic harm from anticoagulation and increased length of stay and hospital charges in this study, emphasizing the importance of further investigation to define the role of CTPA in the diagnosis and treatment of PE after TJA.
Level of Evidence
Level III, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
Age-related muscle weakness due to atrophy and fatty infiltration in orofacial muscles may be related to swallowing deficits in older adults. An important component of safe swallowing is the geniohyoid (GH) muscle, which helps elevate and stabilize the hyoid bone, thus protecting the airway. This study aimed to explore whether aging and aspiration in older adults were related to GH muscle atrophy and fatty infiltration.
Eighty computed tomography scans of the head and neck from 40 healthy older (average age 78 years) and 40 younger adults (average age 32 years) were analyzed. Twenty aspirators and 20 nonaspirators from the 40 older adults had been identified previously. Two-dimensional views in the sagittal and coronal planes were used to measure the GH cross-sectional area and fatty infiltration.
GH cross-sectional area was larger in men than in women (p < .05). Decreased cross-sectional area was associated with aging (p < .05), and cross-sectional area was significantly smaller in aspirators compared with nonaspirators, but only among the older men (p < .01). Increasing fatty infiltration was associated with aging in the middle (p < .05) and posterior (p < .01) portions of the GH muscle. There was no significant difference in fatty infiltration of the GH muscle among aspirators and nonaspirators.
GH muscle atrophy was associated with aging and aspiration. Fatty infiltration in the GH muscle was increased with aging but not related to aspiration status. These findings suggest that GH muscle atrophy may be a component of decreased swallowing safety and aspiration in older adults and warrants further investigation.
Atrophy; Geniohyoid muscle; Older adults; Fatty infiltration; Aspiration; Swallow; CT scans.
A critical part of generating robust chromatin immunoprecipitation (ChIP) data is the optimization of chromatin purification and size selection. This is particularly important when ChIP is combined with next-generation sequencing (ChIP-seq) to identify targets of DNA-binding proteins, genome-wide. Current protocols refined by the ENCODE consortium generally use a two-step cell lysis procedure that is applicable to a wide variety of cell types. However, the isolation and size selection of chromatin from primary human epithelial cells may often be particularly challenging. These cells tend to form sheets of formaldehyde cross-linked material in which cells are resistant to membrane lysis, nuclei are not released and subsequent sonication produces extensive high molecular weight contamination. Here we describe an optimized protocol to prepare high quality ChIP-grade chromatin from primary human bronchial epithelial cells. The ENCODE protocol was used as a starting point to which we added the following key steps to separate the sheets of formaldehyde-fixed cells prior to lysis. (1) Incubation of the formaldehyde-fixed adherent cells in Trypsin-EDTA (0.25% room temperature) for no longer than 5 min. (2) Equilibration of the fixed cells in detergent-free lysis buffers prior to each lysis step. (3) The addition of 0.5% Triton X-100 to the complete cell membrane lysis buffer. (4) Passing the cell suspension (in complete cell membrane lysis buffer) through a 25-gauge needle followed by continuous agitation on ice for 35 min. Each step of the modified protocol was documented by light microscopy using the Methyl Green-Pyronin dual dye, which stains cytoplasm red (Pyronin) and the nuclei grey-blue (Methyl green). This modified method is reproducibly effective at producing high quality sheared chromatin for ChIP and is equally applicable to other epithelial cell types.
T cells bearing receptors made up of α and β chains (TCRs) usually react with peptides bound to major histocompatibility complex proteins (MHC). This bias could be imposed by positive selection, the phenomenon that selects thymocytes to mature into T cells only if the TCRs they bear react with low but appreciable affinity with MHC + peptide combinations in the thymus cortex. However, it is also possible that the polypeptides of TCRs themselves do not have random specificities but rather are biased toward reaction with MHC. Evolution would therefore have selected for a collection of TCR variable elements that are prone to react with MHC. If this were to be so, positive selection would act on thymocytes bearing a pre biased collection of TCRs to pick out those that react to some extent, but not too well, with self MHC + self-peptides. A problem with studies of this evolutionary idea is the fact that there are many TCR variable elements and that these differ considerably in the amino acids with which they contact MHC. However, recent experiments by our group and others suggest that one group of TCR variable elements, those related to the mouse Vβ8 family, has amino acids in their CDR2 regions that consistently bind a particular site on an MHC α-helix. Other groups of variable elements may use different patterns of amino acids to achieve the same goal. Mutation of these amino acids reduces the ability of T cells and thymocytes to react with MHC. These amino acids are present in the variable regions of distantly related species such as sharks and human. Overall the data indicate that TCR elements have indeed been selected by evolution to react with MHC proteins. Many mysteries about TCRs remain to be solved, including the nature of auto-recognition, the basis of MHC allele specificity, and the very nature and complexity of TCRs on mature T cells.
comparative immunology; T cells; T-cell receptors; thymus
Ikaros is important in the development and maintenance of the lymphoid system, functioning in part by associating with chromatin-remodeling complexes. We have studied the functions of Ikaros in the transition from pre-T cell to the CD4+CD8+ thymocyte using an Ikaros null CD4−CD8− mouse thymoma cell line (JE131). We demonstrate that this cell line carries a single functional TCR β gene rearrangement and expresses a surface pre-TCR. JE131 cells also carry non-functional rearrangements on both alleles of their TCR α loci. Retroviral re-introduction of Ikaros dramatically increased the rate of transcription in the α locus and TCR Vα/Jα recombination resulting in the appearance of many new αβTCR+ cells. The process is RAG dependent, requires SWI/SNF chromatin-remodeling complexes and is coincident with the binding of Ikaros to the TCR α enhancer. Furthermore, knockdown of Mi2/NuRD complexes increased the frequency of TCR α rearrangement. Our data suggest that Ikaros controls Vα/Jα recombination in T cells by controlling access of the transcription and recombination machinery to the TCR α loci. The JE131 cell line should prove to be a very useful tool for studying the molecular details of this and other processes involved in the pre-T cell to αβTCR+ CD4+CD8+ thymocyte transition.
T Cells; Transcription Factors; T Cell Receptor Genes; VDJ Recombination
Our objective was to compare outcomes (anesthesia time, total operative time, tourniquet time, duration of hospital stay, 90-day complication rate and transfusion rates) of patients with total knee arthroplasty (TKA) who underwent bariatric surgery before or after TKA. One-hundred-twenty-five patients were included: TKA before bariatric surgery (group 1; n=39); TKA within two years of bariatric surgery (group 2; n=25); and TKA more than 2 years after bariatric surgery (group 3; n=61). Patients with TKA more than 2 years after bariatric surgery had shorter anesthesia, total operative and tourniquet times than other groups; differences were significant between groups. Ninety-day complication and transfusion rates approached but did not meet statistical significance. Ninety-day complication rates and duration of hospital stay did not differ significantly between the three groups.
Total knee arthroplasty; morbidly obese; bariatric surgery; outcomes
The degree of T cell self-reactivity considered dangerous by the immune system, thereby requiring thymic selection processes to prevent autoimmunity, is unknown. Here, we analyzed a panel of T cell receptors (TCRs) with a broad range of reactivity to ovalbumin (OVA323-339) in the rat insulin promoter (RIP)-mOVA self-antigen model for their ability to trigger thymic self-tolerance mechanisms. Thymic regulatory T (Treg) cell generation in vivo was directly correlated with in vitro TCR reactivity to OVA-peptide in a broad ~1,000-fold range. Interestingly, higher TCR affinity was associated with a larger Treg cell developmental “niche” size, even though the amount of antigen should remain constant. The TCR-reactivity threshold to elicit thymic negative selection and peripheral T cell responses was ~100 fold higher than that of Treg cell differentiation. Thus, these data suggest that the broad range of self-reactivity that elicits thymic Treg cell generation is tuned to secure peripheral tolerance to self.
Posterior soft tissue repair after posterior THA reportedly decreases the risk of dislocation. Previously described techniques often require drill holes through the greater trochanter, do not include both the short external rotators and the capsule, or require a complex series of multiple sutures. We therefore describe a technique to address these issues.
Description of Technique
The posterior soft tissues were repaired with a single nonabsorbable suture passed through the external rotators and posterior capsule and then through the capsule and posterior border of minimus in a figure-of-eight pattern. This repair remains pliable and obliterates the dead space.
We retrospectively reviewed 165 patients who underwent 178 primary THAs through a mini-posterior THA and also underwent soft tissue repair using our technique. We determined the rate of dislocation and complications associated with this technique. The minimum clinical followup was 1 year (mean, 23 months; range, 12–37 months).
This repair was associated with a low risk of dislocation at 1 year (one of 178 hips, 0.56%) and no apparent complications related to the technique.
This soft tissue to soft tissue repair technique after posterior-approach THA is technically straightforward and reliable with a low associated dislocation rate.
Level of Evidence
Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.
Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.
αβ T cell receptors (TCRs) bind specifically to foreign antigens presented by major histocompatibility complex proteins (MHC) or MHC-like molecules. Accumulating evidence indicates that the germline-encoded TCR segments have features that promote binding to MHC and MHC-like molecules, suggesting co-evolution between TCR and MHC molecules. Here, we assess directly the evolutionary conservation of αβ TCR specificity for MHC. Sequence comparisons showed that some Vβs from distantly related jawed vertebrates share amino acids in their complementarity determining region 2 (CDR2). Chimeric TCRs containing amphibian, bony fish or cartilaginous fish Vβs can recognize antigens presented by mouse MHC class II and CD1d (an MHC-like protein), and this recognition is dependent upon the shared CDR2 amino acids. These results indicate that features of the TCR that control specificity for MHC and MHC-like molecules were selected early in evolution and maintained between species that last shared a common ancestor over 400 million years ago.
This article summarizes reconstruction options available for acetabular revision following total hip arthroplasty. A thoughtful methodology to the evaluation and treatment of patients with implant failure after joint replacement is essential to guarantee accurate diagnoses, appropriate triage to reconstruction options, and optimal clinical outcomes. In the majority of patients who undergo acetabular revision, factors such as bone loss and pelvic discontinuity provide a challenge in the selection and implementation of the proper reconstruction option. With advanced evaluation algorithms, imaging techniques, and implant designs, techniques have evolved to rebuild the compromised acetabulum at the time of revision surgery. However, clinical outcomes data for these techniques continue to lag behind the exponential increase in revision hip arthroplasty cases predicted to occur over the next several years. We encourage those involved in the treatment of patients undergoing hip replacement surgery to participate in well-designed clinical studies to enhance evidence-based knowledge regarding revision acetabular reconstruction options.
Hip; Arthoplasty; Revision; Acetabulum; Tantalum
The theoretical advantages of metal-on-metal (MOM) bearing couples in total hip arthroplasty (THA) have been recently balanced by concerns regarding adverse local and systemic effects. Higher than anticipated early revision rates have been reported by several joint registries. Failed MOM hips present with a spectrum of symptoms and findings and traditional methods of failure must be considered in addition to the failure modes that appear to be unique to the MOM bearing couple. Metal hypersensitivity and soft tissue immune reactions remain incompletely understood and require careful ongoing study. The tools available to evaluate MOM THAs and the indications for revision surgery remain to be defined. Outcomes following revision of MOM hips appear to depend on appropriate evaluation, early identification, and appropriate surgical management.
Hip arthroplasty; Metal-on-metal; Revision joint arthroplasty; Pseudotumor; Aseptic lymphocytic-vasculitis-associated lesions; Metal hypersensitivity
The antigen receptor for natural killer T cells (NKT TCR) bind CD1d-restricted microbial and self lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags), and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.
Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented the first comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα β-strand, switching Vα’s position relative to Vβ. In several other structures either Vα or Vβ undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands.
A number of recent reports have described novel failure mechanisms of metal-on-metal bearings in total and resurfacing hip arthroplasty. Hip arthroplasties with metal-on-metal articulations are also subject to the traditional methods of failure seen with different bearing couples. There is currently little information in the literature to help guide timely clinical evaluation and management of these patients.
We therefore describe the (1) clinical presentations; (2) reasons for failure; (3) operative findings; and (4) histologic findings in patients with failed metal-on-metal hip arthroplasties.
We retrospectively identified all 37 patients (37 hips) with metal on metal total hip or resurfacing arthroplasties who underwent revision over the past 3 years at our institution. Relevant clinical, radiographic, laboratory, intraoperative, and histopathologic findings were analyzed for all patients.
Of the 37 patients, 10 were revised for presumed hypersensitivity specific to the metal-on-metal articulation. This group included eight patients with tissue histology confirming chronic inflammation with lymphocytic infiltration, eight with aseptic loosening of a monoblock screwless uncemented acetabular component, two with iliopsoas impingement associated with a large-diameter femoral head, and three with femoral neck fracture after resurfacing arthroplasty; the remainder of the patients were revised for infection, instability, component malposition, and periprosthetic fracture.
Increased awareness of the modes of failure will bring to light the potential complications particular to metal-on-metal articulations while placing these complications into the context of failures associated with all hip arthroplasties. This novel clinical information should be valuable for the practicing surgeon faced with this patient population.
Level of Evidence
Level IV, therapeutic study. See Guidelines for Authors for a complete description of levels of evidence.
Alpha/beta T cell receptors (TCRs) react with major histocompatibility complex proteins (MHC) plus peptides, a poorly understood phenomenon, probably because thymocytes bearing TCRs that manifest MHC-reactivity too well are lost by negative selection. Only TCRs with attenuated ability to react with MHC appear on mature T cells. Also, the interaction sites between TCRs and MHC may be inherently flexible and hence difficult to spot. Contacts between TCRs and MHC in the solved structures of their complexes were reevaluated with these points in mind. The results show that frequently used amino acids in TCR CDR1 and CDR2 regions are often used to bind MHC, in areas around small amino acids on the surfaces of MHC α helices that form a cup, allowing somewhat flexible binding of the TCRs. The TCR amino acids involved are specific to families of V regions and partially different rules govern recognition of MHC1 versus MHCII.
T cell receptor; MHC; evolution; conserved interactions; tolerance; selection; major histocompatibility complex
The ligands for αβ T cell receptors are usually major histocompatibility complex proteins (MHC) bound to peptides. Although there is evidence that T cell receptor variable regions have been selected evolutionarily to bind MHC, the rules governing this interaction have not previously been apparent. However, recent solved structures of T cell receptors with related variable regions bound to MHC plus peptides suggest that some amino acids in variable region CDR1 and CDR2s almost always react in a consistent way with MHC. These amino acids may therefore have been selected evolutionarily to predispose T cell receptors towards recognition of MHC ligands.
Mouse iNKT cell receptors (iNKT TCRs) use a single Vα14-Jα18 sequence and Vβs that are almost always Vβ8.2, Vβ7 or Vβ2, although the basis of this differential usage is unclear. We show that the Vβ bias occurs as a consequence of the CDR2β loops determining the affinity of the iNKT TCR for CD1d/glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent Vβ-CD1d affinities are further modulated by the hypervariable CDR3β loop, thereby defining a functional interplay between the two iNKT TCR CDRβ loops. These Vβ biases reveal a broadly hierarchical response in which Vβ8.2 > Vβ7 > Vβ2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR Vβ repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.
An αβ T cell response depends on the recognition of antigen plus major histocompatibility complex proteins (MHC)1 by its antigen receptor (TCR). The ability of peripheral αβ T cells to recognize MHC is at least partly determined by MHC dependent thymic selection, by which an immature T cell survives only if its TCR can recognize self MHC2–7. This process may allow MHC reactive TCRs to be selected from a repertoire with completely random and unbiased specificities. However, analysis of thymocytes prior to positive selection, suggested that TCR proteins might have a predetermined ability to bind MHC8–11. Here we show that specific germline-encoded amino acids in the TCR promote “generic” MHC recognition and control thymic selection. In mice expressing single, rearranged TCRβ chains, individual mutation of amino acids in CDR2β to Ala reduced development of the entire TCR repertoire. Altogether, these results show that thymic selection is controlled by germline-encoded MHC-contact points in the αβ TCR and suggest that the diversity of the peripheral T cell repertoire is enhanced by this “built-in” specificity.
Natural Killer T cells are a distinct lymphocyte lineage that regulates a broad range of immune responses. NKT cells recognize glycolipids presented by the non-classical MHC molecule CD1d. Structural insight into the TCR/glycolipid/CD1d tri-complex has revealed an unusual and unexpected mode of recognition. Recent studies have also identified some of the signaling events during NKT cell development that give NKT cells their innate phenotype. Pathogen-derived glycolipid antigens continue to be found, and new mechanisms of NKT cell activation have been described. Finally, NKT cells have been shown to be remarkably versatile in function during various immune responses. Whether these extensive functional capacities can be attributed to a single population sensitive to environmental cues or if functionally distinct NKT cell subpopulations exist remains unresolved.
Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how αβ T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR–CD1d–α-galactosylceramide (α-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of α-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR–CD1d–α-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jα18-encoded invariant CDR3α loop and Vβ11-encoded CDR2β loop were critical for recognizing CD1d. The residues within the Vα24-encoded CDR1α and CDR3α loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F′ pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR–CD1d–α-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.