Streptococcal superantigen-induced expansion of human tonsil T cells leads to altered T follicular helper cell phenotype, B cell death and reduced immunoglobulin release.

Streptococcal pyrogenic exotoxin (Spe) A expression is epidemiologically linked to streptococcal tonsillo-pharyngitis and outbreaks of scarlet fever, although the mechanisms by which superantigens confer advantage to Streptococcus pyogenes are unclear. S. pyogenes is an exclusively human pathogen. As the leucocyte profile of tonsil is unique, the impact of SpeA production on human tonsil cell function was investigated. Human tonsil cells from routine tonsillectomy were co-incubated with purified streptococcal superantigens or culture supernatants from isogenic streptococcal isolates, differing only in superantigen production. Tonsil cell proliferation was quantified by tritiated thymidine incorporation, and cell surface characteristics assessed by flow cytometry. Soluble mediators including immunoglobulin were measured using enzyme-linked immunosorbent assay. Tonsil T cells proliferated in response to SpeA and demonstrated typical release of proinflammatory cytokines. When cultured in the absence of superantigen, tonsil preparations released large quantities of immunoglobulin over 7 days. In contrast, marked B cell apoptosis and abrogation of total immunoglobulin (Ig)A, IgM, and IgG production occurred in the presence of SpeA and other superantigens. In SpeA-stimulated cultures, T follicular helper (Tfh) cells showed a reduction in C-X-C chemokine receptor (CXCR)5 (CD185) expression, but up-regulation of OX40 (CD134) and inducible T cell co-stimulator (ICOS) (CD278) expression. The phenotypical change in the Tfh population was associated with impaired chemotactic response to CXCL13. SpeA and other superantigens cause dysregulated tonsil immune function, driving T cells from Tfh to a proliferating phenotype, with resultant loss of B cells and immunoglobulin production, providing superantigen-producing bacteria with a probable survival advantage.

Impact of contusion injury on intramuscular emm1 group a streptococcus infection and lymphatic spread.

Invasive group A Streptococcus (iGAS) is frequently associated with emm1 isolates, with an attendant mortality of around 20%. Cases occasionally arise in previously healthy individuals with a history of upper respiratory tract infection, soft tissue contusion, and no obvious portal of entry. Using a new murine model of contusion, we determined the impact of contusion on iGAS bacterial burden and phenotype. Calibrated mild blunt contusion did not provide a focus for initiation or seeding of GAS that was detectable following systemic GAS bacteremia, but instead enhanced GAS migration to the local draining lymph node following GAS inoculation at the same time and site of contusion. Increased migration to lymph node was associated with emergence of mucoid bacteria, although was not specific to mucoid bacteria. In one study, mucoid colonies demonstrated a significant increase in capsular hyaluronan that was not linked to a covRS or rocA mutation, but to a deletion in the promoter of the capsule synthesis locus, hasABC, resulting in a strain with increased fitness for lymph node migration. In summary, in the mild contusion model used, we could not detect seeding of muscle by GAS. Contusion promoted bacterial transit to the local lymph node. The consequences of contusion-associated bacterial lymphatic migration may vary depending on the pathogen and virulence traits selected.

De novo variants in CDK13 associated with syndromic ID/DD: Molecular and clinical delineation of 15 individuals and a further review.

De novo variants in the gene encoding cyclin-dependent kinase 13 (CDK13) have been associated with congenital heart defects and intellectual disability (ID). Here, we present the clinical assessment of 15 individuals and report novel de novo missense variants within the kinase domain of CDK13. Furthermore, we describe 2 nonsense variants and a recurrent frame-shift variant. We demonstrate the synthesis of 2 aberrant CDK13 transcripts in lymphoblastoid cells from an individual with a splice-site variant. Clinical characteristics of the individuals include mild to severe ID, developmental delay, behavioral problems, (neonatal) hypotonia and a variety of facial dysmorphism. Congenital heart defects were present in 2 individuals of the current cohort, but in at least 42% of all known individuals. An overview of all published cases is provided and does not demonstrate an obvious genotype-phenotype correlation, although 2 individuals harboring a stop codons at the end of the kinase domain might have a milder phenotype. Overall, there seems not to be a clinically recognizable facial appearance. The variability in the phenotypes impedes an à vue diagnosis of this syndrome and therefore genome-wide or gene-panel driven genetic testing is needed. Based on this overview, we provide suggestions for clinical work-up and management of this recently described ID syndrome.

Laparoscopic ventral rectopexy for rectal prolapse and rectal intussusception using a biological mesh.

AIM: Laparoscopic ventral rectopexy (LVR) is a nerve-sparing technique for the treatment of rectal prolapse. Concerns about the use of synthetic meshes in the pelvis and the associated risk of erosion have led to the recent use of biological meshes in some colorectal units. This retrospective study aims to assess the outcomes of patients undergoing LVR using a noncross-linked nondermal biological mesh. METHOD: The medical notes of all patients who underwent LVR between 1 December 2011 and 31 May 2014 were reviewed. The rate of obstructed defaecation before surgery was retrospectively determined from medical records using the Rome III criteria. The rates of obstructed defaecation and faecal incontinence following surgery were determined using a self-reported questionnaire. RESULTS: A total of 51 patients had LVR between 1 December 2011 and 31 May 2014. Their mean age was 57.3 ± 2.5 years and the mean follow-up was 23 ± 1 months. There were seven (13.7%) postoperative complications. In total, 45 (88%) patients completed the functional outcome questionnaires. Before surgery, 33 (73.3%) patients complained of symptoms of obstructed defaecation. At the end of follow-up, 22 (48.8%, P = 0.001) patients continued to have some symptoms of obstructed defaecation. Before surgery, 12 (26.7%) patients complained of faecal incontinence. At the end of follow-up, only three (6.7%, P = 0.004) patients reported faecal incontinence. At the end of follow-up, recurrence of symptoms had occurred in six (13.3%) patients. CONCLUSION: LVR using a biological mesh is safe and results in significant reduction in symptoms associated with external rectal prolapse and rectal intussusception.

Hic-5 remodeling of the stromal matrix promotes breast tumor progression.

The remodeling of the stromal extracellular matrix (ECM) has a crucial, but incompletely understood role during tumor progression and metastasis. Hic-5, a focal adhesion scaffold protein, has previously been implicated in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in breast tumor progression in vivo, Hic-5-/- mice were generated and crossed with the Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen mouse. Tumors from the Hic-5-/-;PyMT mice exhibited increased latency and reduced growth, with fewer lung metastases, as compared with Hic-5+/-;PyMT mice. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer-associated fibroblasts (CAFs). Further analysis revealed that the Hic-5-/-;PyMT tumor stroma contains fewer CAFs and exhibits reduced ECM deposition. The remodeling of the stromal matrix by CAFs has been shown to increase tumor rigidity to indirectly regulate FAK Y397 phosphorylation in tumor cells to promote their growth and invasion. Accordingly, the Hic-5-/-;PyMT tumor cells exhibited a reduction in FAK Y397 phosphorylation. Isolated Hic-5-/-;PyMT CAFs were defective in stress fiber organization and exhibited reduced contractility. These cells also failed to efficiently deposit and organize the ECM in two and three dimensions. This, in turn, impacted three-dimensional MDA-MB-231 tumor cell migration behavior. Thus, using a new knockout mouse model, we have identified Hic-5 expression in CAFs as a key requirement for deposition and remodeling of the stromal ECM to promote non-cell autonomous breast tumor progression.

Registrar wellness in Botswana: Measuring burnout and identifying ways to improve wellness

Background. Burnout during registrar training is high, especially in resource-limited settings where stressors are intensified. Burnout leads to decreased quality of life for doctors, poor job and patient satisfaction, and difficulty retaining doctors.Objectives. Primary: to measure burnout among registrars working at Princess Marina Hospital in Gaborone, Botswana. Secondary: to determine factors contributing to burnout and identify potential wellness interventions.Methods. The validated Maslach Burnout Inventory was used to measure the degree of emotional exhaustion, depersonalisation and personal accomplishment. Work-related difficulties and potential wellness interventions were explored through multiple-choice and open-ended questions.Results. Of 40 eligible registrars, 20 (50%) completed the survey. High levels of burnout were reported for emotional exhaustion in 65% (13/20), depersonalisation in 45% (9/20), and personal accomplishment in 35% (7/20) of registrars. A high degree of burnout was reported by 75% (15/20) of registrars in one or more domains. In the previous 7 days, registrars worked an average of 77 hours, took 1.5 overnight calls, slept 5.7 hours per night, and 53% (10/19) had ≥1 of their patients die. Five (25%) registrars considered leaving Botswana to work in another country, which correlated with those with the highest degree of burnout. The most common frustrations included insufficient salary and limited medical resources. Suggested interventions included improved mentorship and wellness lectures.Conclusions. There is a high degree of burnout, especially emotional exhaustion, among registrars. Encouragingly, most registrars have a desire to work in Botswana after training. Future research on improving registrar wellness in low-resource settings is urgently needed

The effect of two levels of hemospermia on stallion fertility.

Hemospermia can occur consistently or intermittently in stallion ejaculates and may cause a reduction in the fertility of the affected ejaculate. It is unknown what amount of blood in an ejaculate leads to subfertility. This study investigated the effect of higher and lower levels of hemospermia (50% and 5%, respectively) on fertility using 24 reproductively normal mares inseminated over three consecutive estrous cycles with fresh extended semen. Mares inseminated with a 5% blood-contaminated ejaculate became pregnant at the same rate (75% per cycle; 18 of 24) as the mares inseminated with blood-free (control) semen (75% per cycle; 18 of 24). The ejaculates containing 50% blood were sterile (0% per cycle, 0 of 24). We concluded that it is the amount of blood, not the mere presence of blood, in an ejaculate that impacts fertility.

Vaginal delivery compared with elective caesarean section: the views of pregnant women and clinicians.

OBJECTIVE: To quantify the risk of morbidity from vaginal delivery (VD) that pregnant women would be prepared to accept before requesting an elective caesarean section and to compare these views with those of clinicians. DESIGN: Cross-sectional survey. SETTING: Major teaching hospital (nulliparas and midwives) and national samples of medical specialists. SAMPLE: Nulliparas (n = 122), midwives (n = 84), obstetricians (n = 166), urogynaecologists (n = 12) and colorectal surgeons (n = 79). METHODS: Face-to-face interviews (nulliparas) and mailed questionnaire (clinicians). MAIN OUTCOME MEASURES: Maximum level of risk participants would be prepared to accept before opting for an elective caesarean section for each of 17 potential complications of VD. Utility scores for each complication were calculated with higher scores (closer to 1) indicating a greater acceptance of risk. RESULTS: Pregnant women were willing to accept higher risks than clinicians for all 17 potential complications. They were least accepting of the risks of severe anal incontinence (mean utility score 0.32), emergency caesarean section (0.51), moderate anal incontinence (0.56), severe urinary incontinence (0.56), fourth-degree tears (0.59) and third-degree tears (0.72). The views of midwives were closest to those of pregnant women. Urogynaecologists and colorectal surgeons were the most risk averse, with 42 and 41%, respectively, stating that they would request an elective caesarean for themselves or their partners. CONCLUSIONS: Pregnant women were willing to accept significantly higher risks of potential complications of VD than clinicians involved in their care. Pregnant women's views were more closely aligned to midwives than to medical specialists.

Cell motility: ARNOand ARF6 at the cutting edge.

The transition of epithelial cells from a stationary to a motile state is important in embryogenesis, wound repair and metastasis. Recent studies have shown that ARNO and ARF6 play significant roles in coordinating this transition, providing new insight into the interplay between the Rho and ARF families of GTPases.

Paxillin-ARF GAP signaling and the cytoskeleton.

Several new families of ARF GTPase activating proteins (ARF GAPs) have been described recently that associate with paxillin and other cytoskeletal and signaling proteins. Important insights have been gained regarding their subcellular distribution, enzymatic specificity and protein scaffold function. Evidence suggests an important role for ARF GAPs in mediating changes in the cell's actin cytoskeleton in response to adhesion and growth factor stimulation.

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

Integrin-linked kinase (ILK) binding to paxillin LD1 motif regulates ILK localization to focal adhesions.

Paxillin is a focal adhesion adapter protein involved in integrin signaling. Paxillin LD motifs bind several focal adhesion proteins including the focal adhesion kinase, vinculin, the Arf-GTPase-activating protein paxillin-kinase linker, and the newly identified actin-binding protein actopaxin. Microsequencing of peptides derived from a 50-kDa paxillin LD1 motif-binding protein revealed 100% identity with integrin-linked kinase (ILK)-1, a serine/threonine kinase that has been implicated in integrin, growth factor, and Wnt signaling pathways. Cloning of ILK from rat smooth muscle cells generated a cDNA that exhibited 99.6% identity at the amino acid level with human ILK-1. A monoclonal antibody raised against a region of the carboxyl terminus of ILK, which is identical in rat and human ILK-1 protein, recognized a 50-kDa protein in all cultured cells and tissues examined. Binding experiments showed that ILK binds directly to the paxillin LD1 motif in vitro. Co-immunoprecipitation from fibroblasts confirmed that the association between paxillin and ILK occurs in vivo in both adherent cells and cells in suspension. Immunofluorescence microscopy of fibroblasts demonstrated that endogenous ILK as well as transfected green fluorescent protein-ILK co-localizes with paxillin in focal adhesions. Analysis of the deduced amino acid sequence of ILK identified a paxillin-binding subdomain in the carboxyl terminus of ILK. In contrast to wild-type ILK, paxillin-binding subdomain mutants of ILK were unable to bind to the paxillin LD1 motif in vitro and failed to localize to focal adhesions. Thus, paxillin binding is necessary for efficient focal adhesion targeting of ILK and may therefore impact the role of ILK in integrin-mediated signal transduction events.

Paxillin interactions.

UNLABELLED: Cell Science at a Glance on the Web: electronic copies of the full-size poster insert are available in various formats Paxillin is a multi-domain protein that localizes in cultured cells primarily to sites of cell adhesion to the extracellular matrix (ECM) called focal adhesions. Focal adhesions form a structural link between the ECM and the actin cytoskeleton and are also important sites of signal transduction; their components propagate signals arising from the activation of integrins following their engagement with ECM proteins, such as fibronectin, collagen and laminin. Importantly, focal adhesion proteins including paxillin also serve as a point of convergence for signals resulting from stimulation of various classes of growth factor receptor. Paxillin localizes to focal adhesions through its LIM domains, possibly through a direct association with &bgr;-integrin tails or an intermediate protein 'X'. In lymphoid cells it can bind directly to *(4)-integrin (not shown). Its primary function is as a molecular adapter or scaffold protein that provides multiple docking sites at the plasma membrane for an array of signaling and structural proteins. For example, it provides a platform for protein tyrosine kinases such as FAK and SRC, which are activated as a result of adhesion or growth factor stimulation. Phosphorylation of residues in the N-terminus of paxillin by these kinases permits the regulated recruitment of downstream effector molecules such as CRK, which (via association with CAS) is important for transduction of external signals into changes in cell motility and for modulation of gene expression by the various MAP kinase cascades. LIM-domain-associated kinases regulate recruitment of paxillin to focal adhesions. In addition, negative regulators of these pathways, including CSK (an inhibitor of SRC activity) and PTP-PEST (a phosphatase that dephosphorylates p130Cas), bind directly to paxillin, thereby bringing them into close proximity with their targets. Paxillin binds to many proteins that are involved in effecting changes in the organization of the actin cytoskeleton, which are necessary for cell motility events associated with embryonic development, wound repair and tumor metastasis. These range from structural proteins such as vinculin and actopaxin that bind actin directly to regulators of actin cytoskeletal dynamics such as the ARF GAP, PKL, the exchange factor PIX and the p21-activated kinase, PAK. These proteins serve as modulators/effectors of the ARF and RHO GTPase families. Several of the paxillin-binding proteins have oncogenic equivalents, such as v-Src, v-Crk and BCR-ABL (not shown). These proteins probably use paxillin both as a substrate and as a docking site to perturb, and even bypass, the normal adhesion and growth factor signaling cascades necessary for controlled cell proliferation. Others, such as the E6 protein from Papillomavirus, facilitate transformation by disrupting the normal links between paxillin and the actin cytoskeleton by displacing paxillin-LD-motif-binding proteins. ABBREVIATIONS: CAS, CRK-associated substrate; CH, calponin-homology domain; CSK, C-terminal SRC kinase; E6, Papillomavirus E6 protein; FAK, focal adhesion kinase; GIT, GRK interacter; GPCR, heterotrimeric-G-protein-coupled receptor; GRK, G-protein-coupled-receptor kinase; MAPK, mitogen-activated protein kinase (ERK, p38, JNK); PAK, p21-activated kinase; PBS, paxillin-binding subdomain; PIX, PAK-interacting exchange factor; PKL, paxillin kinase linker; POR1, partner of Rac; PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine; RTK, growth factor receptor tyrosine kinase; SH, SRC-homology domain.

A competitive enzyme-linked immunoassay using erythrocytes fixed to microtitre plates for anti-D quantitation in immunoglobulin products.

BACKGROUND AND OBJECTIVES: The batch control of anti-D immunoglobulin for prevention of haemolytic disease of the newborn necessitates assessment of its potency. Anti-D quantitation is usually performed using automated haemagglutination methodology although this can only be carried out in specialist centres. The aim of this study was to develop a simple and robust assay for anti-D quantitation. METHODS: We developed a competitive enzyme-linked immunoassay (EIA) in which unlabelled anti-D immunoglobulin and a biotinylated monoclonal anti-D compete for red cell binding. Binding of biotinylated anti-D is detected using an alkaline-phosphatase-labelled avidin preparation. The assay is conveniently carried out using erythrocytes fixed to microtitre plates. RESULTS: The competitive EIA was specific for anti-D activity, highly reproducible and showed good correlation with manufacturers' potency estimates using automated haemagglutination. The assay was quick and simple to perform using freshly prepared or stored plates, and the biotinylated monoclonal anti-D could be lyophilized in ampoules for distribution as a standardized reagent. CONCLUSIONS: The competitive EIA described can be used for the specific quantitation of anti-D and provides a robust alternative method to automated haemagglutination.

Paxillin localizes to the lymphocyte microtubule organizing center and associates with the microtubule cytoskeleton.

Paxillin is a focal adhesion-associated protein that functions as a multi-domain adapter protein, binding several structural and signaling molecules. alpha-Tubulin was identified as an interacting protein in a two-hybrid screen using the paxillin C-terminal LIM domain as a bait. In vitro binding assays with glutathione S-transferase-paxillin demonstrated an interaction of alpha-tubulin with the C terminus of paxillin. Another member of the tubulin family, gamma-tubulin, bound to both the N and the C terminus of paxillin. The interaction between paxillin and both alpha- and gamma-tubulin in vivo was confirmed by co-immunoprecipitation from human T lymphoblasts. Immunofluorescence studies revealed that, in adherent T cells, paxillin localized to sites of cell-matrix interaction as well as to a large perinuclear region. Confocal microscopy revealed that this region corresponds to the lymphocyte microtubule organizing center, where paxillin colocalizes with alpha- and gamma-tubulin. The localization of paxillin to this area was observed in cells in suspension as well as during adhesion to integrin ligands. These data constitute the first characterization of the interaction of paxillin with the microtubule cytoskeleton, and suggest that paxillin, in addition to its well established role at focal adhesions, could also be associated with the lymphocyte microtubule network.

Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

Paxillin and focal adhesion signalling.

To facilitate a rapid response to environmental change, cells use scaffolding - or adaptor - proteins to recruit key components of their signal-transduction machinery to specific subcellular locations. Paxillin is a multi-domain adaptor found at the interface between the plasma membrane and the actin cytoskeleton. Here it provides a platform for the integration and processing of adhesion- and growth factor-related signals.

Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M. C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851-863). In this study, we report the cloning and initial characterization of a new paxillin LD motif-binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell-cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.