Peri-operative administration of tranexamic acid in lower limb arthroplasty: a multicentre, prospective cohort study.

In the UK, tranexamic acid is recommended for all surgical procedures where expected blood loss exceeds 500 ml. However, the optimal dose, route and timing of administration are not known. This study aimed to evaluate current practice of peri-operative tranexamic acid administration. Patients undergoing primary total hip arthroplasty, total knee arthroplasty or unicompartmental knee arthroplasty during a 2-week period were eligible for inclusion in this prospective study. The primary outcome was the proportion of patients receiving tranexamic acid in the peri-operative period. Secondary outcomes included: dose, route and timing of tranexamic acid administration; prevalence of pre- and postoperative anaemia; estimated blood loss; and red blood cell transfusion rates. In total, we recruited 1701 patients from 56 NHS hospitals. Out of these, 1523 (89.5%) patients received tranexamic acid and of those, 1052 (69.1%) received a single dose of 1000 mg intravenously either pre- or intra-operatively. Out of the 1701 patients, 571 (33.6%) and 1386 (81.5%) patients were anaemic (haemoglobin < 130 g.l-1 ) in the pre- and postoperative period, respectively. Mean (SD) estimated blood loss for all included patients was 792 (453) ml and 54 patients (3.1%) received a red blood cell transfusion postoperatively. The transfusion rate for patients with pre-operative anaemia was 6.5%, compared with 1.5% in patients without anaemia. Current standard of care in the UK is to administer 1000 mg of tranexamic intravenously either pre- or intra-operatively. Approximately one-third of patients present for surgery with anaemia, although the overall red blood cell transfusion rate is low. These data provide useful comparators when assessing the efficacy of tranexamic acid and other patient blood management interventions in future studies.

CD2F-10: a new member of the CD2 subset of the immunoglobulin superfamily.

The CD2 subset of the immunoglobulin superfamily consists of a rapidly expanding family of leukocyte cell surface receptors, at least five of which (CD2, CD48, CD58, CD150, and CD244) are involved in lymphocyte activation as either receptors or ligands. Completion of the draft sequence of the human genome offers the possibility of systematically identifying the full set of proteins and interactions of this important family. Here we describe the identification and characterization of the first new member of the subset, CD2F-10, found exclusively by genome searching.

Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1.

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Signaling lymphocytic activation molecule (CDw150) is homophilic but self-associates with very low affinity.

Signaling lymphocytic activating molecule ((SLAM) CDw150) is a glycoprotein that belongs to the CD2 subset of the immunoglobulin superfamily and is expressed on the surface of activated T- and B-cells. It has been proposed that SLAM is homophilic and required for bidirectional signaling during T- and B-cell activation. Previous work has suggested that the affinity of SLAM self-association might be unusually high, undermining the concept that protein interactions mediating transient cell-cell contacts, such as those involving leukocytes, have to be weak in order that such contacts are readily reversible. Using surface plasmon resonance-based methods and analytical ultracentrifugation (AUC), we confirm that SLAM is homophilic. However, we also establish a new theoretical treatment of surface plasmon resonance-derived homophilic binding data, which indicates that SLAM-SLAM interactions (solution K(d) approximately 200 micrometer) are in fact considerably weaker than most other well characterized protein-protein interactions at the cell surface (solution K(d) approximately 0.4-20 micrometer), a conclusion that is supported by the AUC analysis. Whereas further analysis of the AUC data imply that SLAM could form "head to head" dimers spanning adjacent cells, the very low affinity raises important questions regarding the physiological role and/or properties of such interactions.

LICOS, a primordial costimulatory ligand?

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.

Combined subcutaneous recombinant alpha-interferon and interleukin-2 in metastatic renal cell cancer: results of the Multicentre All Ireland Immunotherapy Study Group.

OBJECTIVE: To analyse the toxicity and efficacy of combined interferon-alpha and interleukin-2, administered subcutaneously in a general multicentre setting, as treatment for metastatic renal cell carcinoma. METHODS: Thirty-three patients with metastatic renal cell carcinoma were scheduled to receive 2 cyclical doses of subcutaneous interferon-alpha (week 1: 5 MU x 3 days) and interleukin-2 (week 2: 36 MU x 2 days, 9 MU x 3 days; weeks 3-5: 9 MU daily). Karnofsky scores ranged from 80 to 100 (median 90). Metastases occurred in multiple organs (lung 63%, retroperitoneal 39%, liver 24%). Patients were categorised according to the risk of disease progression. Treatment toxicity, therapeutic response and actuarial survival were analysed. RESULTS: All patients received recommended doses of treatment, but 6 received less than 2 cycles. Most were treated as outpatients, although hospitalisation was usual during the 1st week of a cycle. All complained of mild flu-like symptoms. Severe side effects developed in 13 patients (39%), and treatment was discontinued in 3 of these patients. No deaths occurred as a result of treatment. The overall median survival was 10 months. The overall actuarial survival rate at 3 years was 22%. On statistical analysis, actuarial survival rates were not influenced by either response to treatment or risk group category. CONCLUSION: Subcutaneously administered, combined interferon-alpha and interleukin-2 therapy achieves durable survival rates in a minority of patients with renal cell carcinoma. Toxicity is remedial, and not fatal, when subcutaneous therapy is administered by multiple medical disciplines at a variety of centres.

Structure and dimerization of a soluble form of B7-1.

B7-1 (CD80) and B7-2 (CD86) are glycoproteins expressed on antigen-presenting cells. The binding of these molecules to the T cell homodimers CD28 and CTLA-4 (CD152) generates costimulatory and inhibitory signals in T cells, respectively. The crystal structure of the extracellular region of B7-1 (sB7-1), solved to 3 A resolution, consists of a novel combination of two Ig-like domains, one characteristic of adhesion molecules and the other previously seen only in antigen receptors. In the crystal lattice, sB7-1 unexpectedly forms parallel, 2-fold rotationally symmetric homodimers. Analytical ultracentrifugation reveals that sB7-1 also dimerizes in solution. The structural data suggest a mechanism whereby the avidity-enhanced binding of B7-1 and CTLA-4 homodimers, along with the relatively high affinity of these interactions, favors the formation of very stable inhibitory signaling complexes.

Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation.

Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose-response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.

Effects of granulocyte-macrophage colony-stimulating factor and dose intensification of V-ICE chemotherapy in small-cell lung cancer: a prospective randomized study of 300 patients.

PURPOSE: To assess whether granulocyte-macrophage colony-stimulating factor (GM-CSF) reduces the toxicity of chemotherapy and alters delivered dose-intensity. To assess the feasibility of dose-intensification of chemotherapy in small-cell lung cancer (SCLC) and determine whether it has an impact on outcome. MATERIALS AND METHODS: Patients with good- or intermediate-prognosis SCLC entered a prospective multicenter study that involved a 2 x 2 factorial design with randomization to six cycles of chemotherapy with ifosfamide 5 g/m2, carboplatin 300 mg/m2, etoposide 120 mg/m2 intravenously (I.V.) on days 1 and 2 and 240 mg/m2 orally on day 3, and vincristine 0.5 mg/m2 I.V. on day 15 (V-ICE) every 3 weeks (intensified arm) or every 4 weeks (standard arm). A second double-blind randomization to subcutaneous GM-CSF (250 microg/m2/d) or placebo for 14 days between chemotherapy cycles was made. RESULTS: Three hundred patients were entered. Myelosuppression was the main toxicity, with no significant difference in the incidence or grade between treatment groups. The incidence of febrile neutropenia and bacteriologically confirmed sepsis was unaffected by chemotherapy schedule or use of GM-CSF. Twenty-six percent greater dose-intensity was delivered in the intensified arm, with a trend for greater dose-intensity for those who received GM-CSF. Eighty-three percent of patients achieved a response (51% complete response [CR] rate), with no significant difference in response rates between treatment groups. Survival was significantly increased in the intensified compared with the standard arm (P = .0014); median survival rates were 443 versus 351 days and 2-year survival rates were 33% versus 18%, respectively. CONCLUSION: GM-CSF does not reduce the incidence of complications from myelosuppression of aggressive chemotherapy. Dose intensification of V-ICE to a 3-week schedule in SCLC is not associated with increased toxicity, but appears to improve survival significantly. Future studies should aim to deliver chemotherapy in maximal-tolerated dose-intensities.

Ongoing Y-chromosome instability defines sub-clonal variants in radiation-induced leukaemias in the mouse.

Forty primary leukaemias that arose in vivo as a consequence of 3 Gy X-irradiation of inbred mouse strains were analysed for Y-chromosome aberrations by conventional cytogenetics and fluorescent in situ hybridization (FISH). Compared with control mice which were X-irradiated but which exhibited no overt signs of leukaemia, the loss and gain of Y-chromosomes in leukaemic spleen cells defined subclonal variants in the radiation-induced haemopoietic malignancies that arose in CBA/H, DBA/2 and (C57BL/6 x DBA/2)F1 mice. This Y-chromosome instability was significantly higher than that observed in spleen cells of age-matched (or older) irradiated control mice that had not developed overt leukaemia. The detection of Y-chromosome aberrations is considered in the context of the high numbers of potential gene regulatory sequences in the murine Y-chromosome and the potential for the insertional activation of cellular genes during multi-stage radiation leukaemogenesis.

Mini- and microsatellite mutations in radiation-induced acute myeloid leukaemia in the CBA/H mouse.

Radiation-induced acute myeloid leukaemia (AML) in the CBA/H mouse is a clonal disorder and therefore amenable to the analysis of genetic instability during radiation leukaemogenesis. The genotype of a single minisatellite and 20 microsatellite loci was compared in tail and leukaemic spleen DNA prepared from the same mouse. Somatic mutation at the Ms6-hm minisatellite locus was nearly seven times higher (27%, 4/15) than the spontaneous germline mutation rate (4%). Only 1/15 AMLs exhibited microsatellite mutations, but 5/20 loci were mutated in the same AML, indicating that it was deficient in mismatch repair. Thus, whereas somatic minisatellite mutations, which are associated with complex intra-allelic gene conversion events, occur at a very high rate in the radiation-induced AMLs, microsatellite instability, which has been associated with the acquisition of the replication error repair (RER+) phenotype, is infrequent but detectable.

Assay of E-cadherin by ELISA in human breast cancers.

E-cadherin is a membrane-bound adhesion glycoprotein. Loss of E-cadherin has been correlated with invasion and metastasis in model systems. Using a new ELISA, we found higher levels of E-cadherin in fibroadenomas than in primary breast cancers. Levels in primary cancers showed no significant relationship with either tumour size, nodal status or oestrogen receptor levels. Patients with breast cancers containing low levels of the adhesion protein had a significantly shorter disease-free interval than patients with high levels (P = 0.041). The prognostic value of E-cadherin, for disease-free interval, was also found in node-negative patients as well as in patients presenting with small tumors (< or = 2 cm). In conclusion, loss of E-cadherin expression in human breast cancers is associated with increased metastatic potential as has previously been found in model systems. Loss of E-cadherin is thus likely to contribute to breast cancer progression.

A polymorphic and hypervariable locus in the pseudoautosomal region of the CBA/H mouse sex chromosomes.

We have identified a genomic locus (DXYH1) that is polymorphic and hypervariable within the CBA/H colony. Using a panel of C57BL/6 x Mus spretus backcross offspring, it was mapped to the distal end of the X chromosome. Pseudoautosomal inheritance was demonstrated through three generations of CBA/H x CBA/H and CBA/H x C57BL/6 crosses and confirmed through linkage to the Sxr locus in X/Y Sxr x 3H1 crosses. Meiotic recombination frequencies place DXYH1 similar 28% into the pseudoautosomal region from the boundary. The de novo generation of CBA/H variant DXYH1 restriction fragment length polymorphisms during spermatogenesis is suggestive of the germline instability associated with hypermutable human minisatellites. The absence of DXYH1-related sequences in Mus spretus provides DNA sequence evidence to support the observed failure of X-Y pairing during meiosis and consequent hybrid infertility in C57BL/6 x Mus spretus male F1 offspring.

Co-amplification to tail-to-tail copies of MuRVY and IAPE retroviral genomes on the Mus musculus Y chromosome.

We have isolated a clone from a C57BL/6 genomic library that contains both part of the Y Chromosome-specific 8.7 kbp MuRVY genome (Hutchinson and Eicher, J. Virol. 63, 4043, 1989) and a full-length 8.3 kbp Intracisternal A Particle genome (IAPE-Y), in a tail-to-tail organization. Although IAPs are encoded by a disperse multigene family (approximately 1000 copies per haploid genome), we present evidence that a significant proportion of the IAP-related sequences are present on the Y Chromosome (Chr) and that a >25 kbp genomic sequence, which contains the two proviral genomes, has been amplified on the Y Chr. Two discrete amplified families of MuRVY retroviral genomes distinguishable by a polymorphic restriction site were detected, suggestive that amplification occurred in incremental stages. The presence of MuRVY-related DNA sequences, but absence of IAPE-Y-related DNA sequences in Mus spretus suggests that the IAPE-Y retrotransposition event occurred after the evolutionary divergence of the lineages leading to Mus musculus and Mus spretus, and that the amplification of MuRVY occurred independently in the two lineages.

Urokinase plasminogen activator as a predictor of aggressive disease in breast cancer.

Urokinase plasminogen activator (uPA) is a multifunctional protein involved in both extracellular proteolysis and signal transduction. uPA usually mediates its actions while attached to a membrane-bound receptor, termed uPAR. In this study, uPA and its receptor were measured at both protein and mRNA levels in breast cancer. At both levels, concentrations of uPA were significantly correlated with those for uPAR. uPA levels also correlated significantly with cathepsin B and cathepsin D but not with cathepsin L, MMP-8 or MMP-9 levels. Irrespective of the cut-off point used (e.g., median, tertile or quartile values), uPA was a significant prognostic marker for breast cancer.

Urokinase plasminogen activator and urokinase plasminogen activator receptor in breast cancer.

Urokinase plasminogen activator (uPA) is a serine protease involved in cancer invasion and metastasis. uPA mediates its action while attached to a membrane-bound receptor (uPAR). In this investigation we show that uPAR levels correlate with uPA levels in human breast cancers. uPAR levels, however, do not correlate with other components of the plasminogen activator system such as tissue-type plasminogen activator (t-PA), PAI-I or PAI-2. In addition, uPAR levels showed no correlation with tumor size, axillary-node status or estrogen-receptor status. On the basis of an optimum cut-off point, patients with breast cancers containing high levels of uPAR had a worse prognosis than patients with low levels of the receptor. However, as a prognostic marker in breast cancer, uPAR was not as strong as uPA. Our results are consistent with data from model systems suggesting that both uPA and uPAR are necessary for metastasis.

Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast cancer.

Results from model tumour systems suggest that either increased levels of certain metalloproteases (MMPs) or decreased levels of their inhibitors correlate with metastatic potential. In this study, levels of two MMPs, i.e. MMP-8 and -9, and their inhibitor tissue inhibitor of metalloprotease type 1 (TIMP-1) were measured by enzyme-linked immunosorbent assay in human breast tumours. Levels of MMP-8 and -9 correlated significantly with each other, but neither MMP correlated with urokinase plasminogen activator. Levels of both MMP-8 and -9 were also significantly related to levels of TIMP-1. In contrast, neither MMP correlated with plasminogen activator inhibitor. No relationship was found between MMP-8, MMP-9 or TIMP-1 and either tumour size or metastasis to axillary nodes. MMP-8 and -9 levels were inversely related to levels of oestrogen receptors. MMP-8 but not MMP-9 levels were also inversely correlated with progesterone receptor levels. It is concluded that the assay for MMP-8 and -9 described here will permit the evaluation of these proteases as prognostic markers in cancer.

Complex Y chromosome aberrations are a recurrent secondary event in radiation-induced murine acute myeloid leukaemia.

Arbitrarily primed-PCR analysis of DNA from male CBA/H radiation-induced leukaemic spleens revealed the loss of an approximately 350-bp sequence in several leukaemias. We have isolated a lambda EMBL3 C57BL/6 genomic subclone (pJB1) which hybridizes to the AP-PCR probe and is located on the Y chromosome. Southern blot analyses using the pJB1 probe indicate that the genomic sequence was deleted in five of 14 leukaemias. Cytogenetic analyses of 31 X-ray induced leukaemias in male CBA/H mice revealed, in addition to the characteristic partial deletion of chromosome 2 (28/31 leukaemias), a high incidence (16/31) of the loss of an intact Y chromosome. Comparison of the Southern blot and cytogenetic analyses of the leukaemias demonstrate a significant lack of correspondence between the loss of an intact Y chromosome and Y chromosome-specific DNA sequences, suggesting that Y chromosome aberrations are complex. Whereas partial deletion of chromosome 2 can be detected in 6% of bone marrow cells within 6-11 days of irradiation, no Y chromosome involvement was detected, indicating that Y chromosome aberrations are a late event in radiation-induced leukaemogenesis. These findings are comparable to the loss of sex chromosomes in human t(8;21) AML.