Human leucocyte antigen class I-redirected anti-tumour CD4+ T cells require a higher T cell receptor binding affinity for optimal activity than CD8+ T cells.

CD4+ T helper cells are a valuable component of the immune response towards cancer. Unfortunately, natural tumour-specific CD4+ T cells occur in low frequency, express relatively low-affinity T cell receptors (TCRs) and show poor reactivity towards cognate antigen. In addition, the lack of human leucocyte antigen (HLA) class II expression on most cancers dictates that these cells are often unable to respond to tumour cells directly. These deficiencies can be overcome by transducing primary CD4+ T cells with tumour-specific HLA class I-restricted TCRs prior to adoptive transfer. The lack of help from the co-receptor CD8 glycoprotein in CD4+ cells might result in these cells requiring a different optimal TCR binding affinity. Here we compared primary CD4+ and CD8+ T cells expressing wild-type and a range of affinity-enhanced TCRs specific for the HLA A*0201-restricted NY-ESO-1- and gp100 tumour antigens. Our major findings are: (i) redirected primary CD4+ T cells expressing TCRs of sufficiently high affinity exhibit a wide range of effector functions, including cytotoxicity, in response to cognate peptide; and (ii) optimal TCR binding affinity is higher in CD4+ T cells than CD8+ T cells. These results indicate that the CD4+ T cell component of current adoptive therapies using TCRs optimized for CD8+ T cells is below par and that there is room for substantial improvement.

Global audit on bowel perforations related to transanal irrigation.

PURPOSE: Transanal irrigation is increasingly used against chronic constipation and fecal incontinence in selected patients. The aims were to estimate the incidence of irrigation-related bowel perforation in patients using the Peristeen Anal Irrigation(®) system, and to explore patient- and procedure-related factors associated with perforation. METHODS: External independent expert audit on the complete set of global vigilance data related to Peristeen Anal Irrigation from 2005 to 2013. RESULTS: In total, 49 reports of bowel perforation had been recorded. Based on sales figures, this corresponds to an average risk of bowel perforation of 6 per million procedures. The latest two-year data indicate a risk of 2 per million procedures. In 29 out of 43 evaluable cases (67 %), perforation happened within the first 8 weeks since start of treatment. After 8 weeks, long-term use has an estimated risk of less than 2 per million procedures. Among patients with non-neurogenic bowel dysfunction, 11 out of 15 (73 %) had a history of pelvic organ surgery compared to 5 out of 26 (19 %) in neurogenic bowel dysfunction. In 11 of 46 (24 %) evaluable cases, burst of the rectal balloon was reported. CONCLUSION: Enema-induced perforation is a rare complication to transanal irrigation with Peristeen Anal Irrigation, which increases the benefit risk ratio in support of the further use of transanal irrigation. Increased risk is present during treatment initiation and in patients with prior pelvic organ surgery. Careful patient selection, patient evaluation and proper training of patients are critical to safe practice of this technique.

T cell receptor binding affinity governs the functional profile of cancer-specific CD8+ T cells.

Antigen-specific T cell receptor (TCR) gene transfer via patient-derived T cells is an attractive approach to cancer therapy, with the potential to circumvent immune regulatory networks. However, high-affinity tumour-specific TCR clonotypes are typically deleted from the available repertoire during thymic selection because the vast majority of targeted epitopes are derived from autologous proteins. This process places intrinsic constraints on the efficacy of T cell-based cancer vaccines and therapeutic strategies that employ naturally generated tumour-specific TCRs. In this study, we used altered peptide ligands and lentivirus-mediated transduction of affinity-enhanced TCRs selected by phage display to study the functional properties of CD8(+) T cells specific for three different tumour-associated peptide antigens across a range of binding parameters. The key findings were: (i) TCR affinity controls T cell antigen sensitivity and polyfunctionality; (ii) supraphysiological affinity thresholds exist, above which T cell function cannot be improved; and (iii) T cells transduced with very high-affinity TCRs exhibit cross-reactivity with self-derived peptides presented by the restricting human leucocyte antigen. Optimal system-defined affinity windows above the range established for natural tumour-specific TCRs therefore allow the enhancement of T cell effector function without off-target effects. These findings have major implications for the rational design of novel TCR-based biologics underpinned by rigorous preclinical evaluation.

Communication: Slow supramolecular mode in amine and thiol derivatives of 2-ethyl-1-hexanol revealed by combined dielectric and shear-mechanical studies.

In this paper, we present results of dielectric and shear-mechanical studies for amine (2-ethyl-1-hexylamine) and thiol (2-ethyl-1-hexanethiol) derivatives of the monohydroxy alcohol, 2-ethyl-1-hexanol. The amine and thiol can form hydrogen bonds weaker in strength than those of the alcohol. The combination of dielectric and shear-mechanical data enables us to reveal the presence of a relaxation mode slower than the α-relaxation. This mode is analogous to the Debye mode seen in monohydroxy alcohols and demonstrates that supramolecular structures are present for systems with lower hydrogen bonding strength. We report some key features accompanying the decrease in the strength of the hydrogen bonding interactions on the relaxation dynamics close to the glass-transition. This includes changes (i) in the amplitude of the Debye and α-relaxations and (ii) the separation between primary and secondary modes.

Shear-modulus investigations of monohydroxy alcohols: evidence for a short-chain-polymer rheological response.

Liquids composed of small-molecule monohydroxy alcohols are demonstrated to display rheological behavior typical for oligomeric chains. This observation was made possible by rheological experiments in which more than seven decades in frequency and more than five decades on the mechanical modulus scale are covered. The singly hydrogen-bonded monohydroxy alcohols were chosen because they display significant, but surprisingly poorly understood effects of intermolecular association. Based on the present shear study, one can apply theoretical concepts of polymer science to understand the anomalous physical behavior of a wide range of hydrogen-bonded liquids.

Mechanical stability of the femoral fixation for single- and double-bundle ACL reconstruction in an in vitro experimental model.

Anterior cruciate ligament ACL reconstruction using the double-bundle (DB) technique is gaining popularity. A possible weak link in the DB technique could be that two tendon grafts of smaller diameters are used. The purpose of this study was to test different femoral fixation methods and graft diameters representing single-bundle (SB) and DB ACL reconstructions and compare their biomechanical properties. We hypothesized that SB 6-mm graft constructs had inferior biomechanical properties than SB 9-mm grafts or DB 2 × 6-mm grafts. Furthermore, we hypothesized that interference (IF) screw fixation would demonstrate less elongation and a higher stiffness than Endobutton (Smith & Nephew®, Inc., Andover, Massachusetts, USA) fixation (EBF). We performed an in vitro study using porcine knees and extensor tendons. The mechanical test consisted of a cyclic test followed by a load-to-failure test. We found that 6-mm graft constructs had an ultimate failure load that was up to 40% less than both the 9-mm and 2 × 6-mm graft constructs, despite the fixation method (P-values ≥ 0.004). Comparing fixation methods, EBF was superior to IF concerning maximum load to failure (P < 0.001); IF resulted in a higher stiffness of the femur/graft complex than the EBF (P < 0.001) but no significant difference in elongation between fixation methods. Since the two graft strands are subjected to different loads in different knee flexion angles, the reduced strength of the individual graft strands in DB ACL reconstruction could be a concern.

The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity.

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.

The combination of radiostereometric analysis and the telos stress device results in poor precision for knee laxity measurements after anterior cruciate ligament reconstruction.

PURPOSE: Several devices for measuring knee laxity following anterior cruciate ligament ACL reconstruction exist, but the precision of the methods has never been optimal. Therefore, a new standardized protocol (NSP) was made, aiming at ensuring a reliable positioning of the Telos Stress Device (TSD) which theoretically could result in precise knee laxity measurements when using radiostereometric analysis (RSA) in combination with TSD. METHOD: The TSD was applied to the knee of 30 healthy persons, using both the NSP and the official company instructions. The position of the stress arms of the TSD was marked following each measurement. The reliability of each protocol was calculated as the difference in length between the first and second markings. The NSP for the TSD was then used in a clinical study. Thirty-five patients underwent ACL reconstruction. Double measurements of knee laxity by RSA were performed at a 3-month follow-up. RESULTS: Using the NSP for TSD positioning, the prediction interval at the marking sites ranged from ±0.4 to ±1.1 mm. Following the company instructions, the prediction interval ranged from ±0.8 to ±3.9 mm depending on marking site. Thus, the precision of positioning the stress arms of the TSD was improved at all marking sites using the NSP compared with the original company protocol. The double measurements of the knee laxity in the clinical study resulted in a mean difference of 0.0 mm and a prediction interval of ±5.2 mm. CONCLUSION: Even though the NSP improved the positioning of the TSD on patients' extremities, the combination of NSP-TSD and RSA was not able to provide acceptable knee laxity measurements in a clinical setting compared with published precision data for other devices on the market. Therefore, the Telos Stress Device is not recommendable for use in knee laxity measurements following ACL reconstruction.

Serial dilation reduces graft slippage compared to extraction drilling in anterior cruciate ligament reconstruction: a randomized controlled trial using radiostereometric analysis.

PURPOSE: This study tested the hypothesis that serial dilation of the tibial tunnel could provide a stronger anchorage of the graft-fixation-device complex compared to traditional extraction drilling. METHODS: Forty patients (22 men and 18 women) undergoing ACL reconstruction were randomized to either extraction drilling (group ED) or compaction by serial dilation (group SD) of the tibial tunnel. Tantalum beads were placed in the tibia, femur, and in the hamstring graft. Radiostereometric analysis (RSA) was performed postoperatively and again after 6, 12, and 24 weeks. Migration of graft in the bone tunnels as well as knee laxity was assessed using RSA and a TELOS stress device. RESULTS: Six patients (three men and three women) were excluded during follow-up, which resulted in 17 patients in group ED [median age 30 years (range 20-50)] and 17 patients in group SD [median age 32 years (range 20-49)]. The mean migration of the graft in the tibial bone canal after 3 months was 1.3 (SD 0.6) mm in group ED and 0.8 (SD 0.5) mm in group SD (P = 0.02). The overall knee laxity after 3 months was 13.0 (SD 4.0) mm in group ED and 10.9 (SD 3.1) mm in group SD. CONCLUSION: This study found less slippage of the hamstring graft in the tibial bone canal in the serial dilated group compared to the extraction drilling group. The clinical relevance of the difference is unknown. No difference in stress radiographic knee laxity was found between the two groups.

Antagonist HIV-1 Gag peptides induce structural changes in HLA B8.

In the cellular immune response, recognition by CTL-TCRs of viral antigens presented as peptides by HLA class I molecules, triggers destruction of the virally infected cell (Townsend, A.R.M., J. Rothbard, F.M. Gotch, G. Bahadur, D. Wraith, and A.J. McMichael. 1986. Cell. 44:959-968). Altered peptide ligands (APLs) which antagonise CTL recognition of infected cells have been reported (Jameson, S.C., F.R. Carbone, and M.J. Bevan. 1993. J. Exp. Med. 177:1541-1550). In one example, lysis of antigen presenting cells by CTLs in response to recognition of an HLA B8-restricted HIV-1 P17 (aa 24-31) epitope can be inhibited by naturally occurring variants of this peptide, which act as TCR antagonists (Klenerman, P., S. Rowland Jones, S. McAdam, J. Edwards, S. Daenke, D. Lalloo, B. Koppe, W. Rosenberg, D. Boyd, A. Edwards, P. Giangrande, R.E. Phillips, and A. McMichael. 1994. Nature (Lond.). 369:403-407). We have characterised two CTL clones and a CTL line whose interactions with these variants of P17 (aa 24-31) exhibit a variety of responses. We have examined the high resolution crystal structures of four of these APLs in complex with HLA B8 to determine alterations in the shape, chemistry, and local flexibility of the TCR binding surface. The variant peptides cause changes in the recognition surface by three mechanisms: changes contributed directly by the peptide, effects transmitted to the exposed peptide surface, and induced effects on the exposed framework of the peptide binding groove. While the first two mechanisms frequently lead to antagonism, the third has more profound effects on TCR recognition.

Serial dilation versus extraction drilling in anterior cruciate ligament reconstruction: a biomechanical study.

The hamstring tendon graft has become increasingly popular in anterior cruciate ligament reconstruction because of low donor-site morbidity. However, the tibial fixation is considered difficult, mainly because of low tibial mineral bone density. Therefore, we tested whether preparation of the tibial tunnel with compaction by serial dilation provided a stronger anchorage of the graft-fixation-device complex than does traditional extraction drilling of the tibial tunnel. In 20 bovine tibiae, the bone tunnels were created with either extraction drilling (group 1) or compaction by serial dilation (group 2). Twenty bovine digital extensor tendons were fixated in the bone tunnel with an Intrafix tibial fastener. The graft-fixation-device complexes were mounted in a hydraulic test machine. The fixation strength was evaluated after cyclic loading. The difference between the serial dilation group and the extraction drilling group ranged from a mean slippage of 0 mm at 70-220 N, to a mean slippage of 0.1 mm at 70-520 N. We found no significant difference in slippage of the graft-fixation-device complex after 1,600 cycles. This study failed to show a significant difference between compaction by serial dilation and extraction drilling of the tibia bone tunnel in anterior cruciate ligament reconstruction.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity: a role for microglia.

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants.

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkin's disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkin's lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy+/-fludarabine. The cumulative incidence of acute GVHD grade II-IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61-71%. Patients with MM, HD and MDS and a history of > or =5% blasts had a less favourable outcome with a PFS of 19-38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Constitutive binding of yeast heat shock factor to DNA in vivo.

We measured the binding of yeast heat shock factor (HSF) to DNA in vivo by using an interference assay in which HSF excludes GAL4 from a synthetic promoter element containing overlapping binding sites for each protein. The results show that HSF binds to DNA in unstressed cells and that binding is not sufficient for transcriptional activation.

Analysis of functional domain organization in DNA topoisomerase II from humans and Saccharomyces cerevisiae.

The functional domain structure of human DNA topoisomerase IIalpha and Saccharomyces cerevisiae DNA topoisomerase II was studied by investigating the abilities of insertion and deletion mutant enzymes to support mitotic growth and catalyze transitions in DNA topology in vitro. Alignment of the human topoisomerase IIalpha and S. cerevisiae topoisomerase II sequences defined 13 conserved regions separated by less conserved or differently spaced sequences. The spatial tolerance of the spacer regions was addressed by insertion of linkers. The importance of the conserved regions was assessed through deletion of individual domains. We found that the exact spacing between most of the conserved domains is noncritical, as insertions in the spacer regions were tolerated with no influence on complementation ability. All conserved domains, however, are essential for sustained mitotic growth of S. cerevisiae and for enzymatic activity in vitro. A series of topoisomerase II carboxy-terminal truncations were investigated with respect to the ability to support viability, cellular localization, and enzymatic properties. The analysis showed that the divergent carboxy-terminal region of human topoisomerase IIalpha is dispensable for catalytic activity but contains elements that specifically locate the protein to the nucleus.

Production of soluble alphabeta T-cell receptor heterodimers suitable for biophysical analysis of ligand binding.

A method to produce alphabeta T-cell receptors (TCRs) in a soluble form suitable for biophysical analysis was devised involving in vitro refolding of a TCR fusion protein. Polypeptides corresponding to the variable and constant domains of each chain of a human and a murine receptor, fused to a coiled coil heterodimerization motif from either c-Jun (alpha) or v-Fos (beta), were overexpressed separately in Escherichia coli. Following recovery from inclusion bodies, the two chains of each receptor were denatured, and then refolded together in the presence of denaturants. For the human receptor, which is specific for the immunodominant influenza A HLA-A2-restricted matrix epitope (M58-66), a heterodimeric protein was purified in milligram yields and found to be homogeneous, monomeric, antibody-reactive, and stable at concentrations lower than 1 microM. Using similar procedures, analogous results were obtained with a murine receptor specific for an influenza nucleoprotein epitope (366-374) restricted by H2-Db. Production of these receptors has facilitated a detailed analysis of viral peptide-Major Histocompatibility Complex (peptide-MHC) engagement by the TCR using both surface plasmon resonance (SPR) and, in the case of the human TCR, isothermal titration calorimetry (ITC) (Willcox et al., 1999). The recombinant methods described should enable a wide range of TCR-peptide-MHC interactions to be studied and may also have implications for the production of other heterodimeric receptor molecules.

Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2.

A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA. A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor. HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E. coli, refolded and purified. CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E.

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Production and crystallization of MHC class I B allele single peptide complexes.

Major histocompatibility complex class I B alleles, HLA B8, B53 and B3501 have been cloned, expressed, refolded and crystallized in specific complexes with a number of different 8-mer and 9-mer peptides. For some of these crystallization was initiated by cross-seeding between different B allele complexes. All crystallize in the space group P212121, with similar unit cell dimensions of approximately 52 A X 81 A X 112 A, contain one complex per asymmetric unit and diffract to approximately 2.0 A resolution.