Wiskott-Aldrich syndrome: a study of 577 patients defines the genotype as a biomarker for disease severity and survival.

ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

Characterization of the central nervous system penetrant and selective purine P2X7 receptor antagonist JNJ-54175446 in patients with major depressive disorder.

JNJ-54175446 is a selective purine P2X7 receptor (P2X7R) antagonist that attenuates microglial IL-1ß/IL-18 release. In healthy volunteers, JNJ-54175446 suppressed peripheral interleukin (IL)-1ß release, and attenuated dexamphetamine-induced improvements of mood and (visuo)motor performance in a human dexamphetamine-challenge paradigm. In depression, P2X7R inhibition may dampen immune-related dysregulation of mood. These results suggest that the impact of P2X7R inhibition is most prominent in situations where mood regulation is disrupted. Total sleep deprivation (TSD) results in an acute emotional perturbation, which yields a transient antidepressant effect. In the current study, TSD was applied as a behavioral challenge to investigate whether such effects could be modulated by JNJ-54175446. This was a double-blind, placebo-controlled, randomized study to assess the safety and pharmacokinetics of JNJ-54175446 and explore its effects in patients with single episode and recurrent major depressive disorder (MDD) (N = 69) and baseline total Inventory of Depressive Symptomatology Clinician Rated (IDS-C) > 30. Patients were randomized to receive JNJ-54175446 throughout the 10-day treatment period, placebo for days 1-3 followed by JNJ-54175446 or placebo throughout. All patients underwent 36 h of TSD starting on day three until the evening of day four. The early start group was hypothesized to experience a reduced effect from TSD whilst the late starting group was hypothesized to experience prolonged effects from the TSD. JNJ-54175446 was well-tolerated and adverse events were mild to moderate. JNJ-54175446 reduced IL-1ß release by LPS-stimulated peripheral white blood cells in the presence of the P2X receptor agonist benzyl adenosine triphosphate (BzATP). JNJ-54175446 did not have a significant effect on mood as assessed using the Hamilton Depression Rating Scale, 17 items (HDRS17) and the Self-rated Quick Inventory of Depressive Symptoms (QIDS-SR). However, JNJ-54175446 blunted an acute reduction of anhedonia that occurred as a result of TSD, assessed by the Snaith-Hamilton Pleasure Scale (SHAPS) and the Probabilistic Instrumental Learning Task (PILT).

In vitro Generation of Cytotoxic T Cells With Potential for Adoptive Tumor Immunotherapy of Multiple Myeloma.

Multiple myeloma is a life-threatening hematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using tumor antigen-specific, cytotoxic T-lymphocytes, may represent an alternative or additional treatment for multiple myeloma. In this study, we used hybrid cell lines, generated by fusion of an EBV B-lymphoblastoid cell line (B-LCL) and myeloma cells, to stimulate in vitro peripheral blood lymphocytes (PBLs) from patients with multiple myeloma. We investigated induction of antigen-specific, cytotoxic T-lymphocytes to the well-defined tumor associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1, which have been shown to be expressed in a high proportion of cases of multiple myeloma. HLA-A2-peptide pentamer staining, interferon-γ and perforin ELISpot assays, as well as cytotoxicity assays were used. Following several rounds of in vitro stimulation, the hybrid cell lines induced antigen-specific, cytotoxic T-lymphocytes to four candidate TAAs in PBLs from HLA-A2+ multiple myeloma patients, using known HLA-A2 restricted peptide epitopes of the TAAs. In contrast, the HLA-A2+ myeloma cell line U266 failed to induce antigen-specific, cytotoxic T-lymphocytes in vitro. Our data indicate that B-LCL/myeloma hybrid cell lines induce antigen-specific, cytotoxic T-lymphocytes in PBLs isolated from multiple myeloma patients in vitro and may represent a novel strategy for use in adoptive immunotherapy of multiple myeloma.

Case presentation: persistent adenovirus B3 infections associated with bronchiolitis obliterans treated with cidofovir in a child with mosaic tetrasomy 9p.

BACKGROUND: Adenoviruses (AdV) are non-enveloped, double-stranded DNA viruses with multiple serotypes, which cause a variety of end-organ disease in both immunocompetent and immunocompromised individuals. Some adenoviruses can become latent in the mucosa-associated lymphoid tissue (e.g. adenoids and tonsils), with the potential to reactivate sporadically, leading to upper or lower respiratory tract infection and disease. Bronchiolitis Obliterans (BO) is a rare chronic lung disorder which usually follows a severe insult to the respiratory tract. In children, it is a complication of severe infections (as post-infectious BO), typically manifesting after a severe respiratory infection, in previously healthy pre-school children. Symptoms and signs of air trapping (hyperinflated chest, expiratory wheeze) with persistent oxygen requirement are characteristic. The presence of the unusual mosaic tetrasomy 9p genotype in this case, despite standard cidofovir therapy for persistent or chronic adenovirus infection, may have impacted on the child's long-term clinical outcomes. CASE PRESENTATION: We present a case of persistent AdV B3 infection in a 14-month old boy with mosaic tetrasomy 9p, which persisted for 10 weeks, resulting in radiologically-confirmed BO, requiring cidofovir to control the persistent AdV B3 infection and standard therapy with pulsed steroids. We argue that in the presence of the mosaic tetrasomy 9p, earlier antiviral therapy may have decreased the severity of BO, as this mutation is known to be associated with some degree of immune dysregulation. CONCLUSIONS: Adenovirus infections are common in children and may persist as latent infections, with subsequent reactivations during loss of immune control, related to systemic illness arising from other causes. In chronic, reactivated AdV infection with pneumonia, BO is a recognised complication. However, in this case, with the presence of the mosaic tetrasomy 9p mutation, earlier antiviral therapy may have reduced such longer term complications, due to the immune dysregulatory nature of this mutation.

Dissociable temporal effects of bupropion on behavioural measures of emotional and reward processing in depression.

Antidepressants remediate negative biases in emotional processing early in treatment, prior to mood improvement. However, the effects on reward processing potentially relevant to the treatment of anhedonia are less clear. Here we investigate the early and sustained effects of the dopamine and noradrenaline reuptake inhibitor bupropion on behavioural measures of emotional and reward processing in currently depressed individuals. Forty-six currently depressed patients and 42 healthy controls participated in a repeated measures study, during which open-label bupropion was administered to only the patient group over a six week period without a placebo group. All participants completed the Emotional Test Battery and a probabilistic instrumental learning task at week 0, week 2 and week 6. Currently depressed patients displayed negative biases in emotional processing and blunted response bias for high-probability wins compared to the healthy controls at baseline. Bupropion was found to reduce the negative biases in emotional processing early in treatment, including a significant decrease in the percentage misclassification of other face emotions as sad and the number of negative self-referent words falsely recalled between baseline and week 2. Conversely, bupropion was found to initially further reduce the response bias for high-probability wins between baseline and week 2. This effect reversed with six weeks' bupropion treatment and reward processing was normalized compared to the healthy controls. Early in treatment, bupropion acts to reduce negative biases in emotional processing but exacerbates impaired reward processing. The beneficial actions of bupropion on reward processing then occur later in treatment. Such dissociation in the temporal effects of bupropion on emotional and reward processing has implications for the treatment of the different symptom domains of negative affect and anhedonia in depression.This article is part of a discussion meeting issue 'Of mice and mental health: facilitating dialogue between basic and clinical neuroscientists'.

Thymus transplantation for complete DiGeorge syndrome: European experience.

BACKGROUND: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS). METHODS: Twelve patients with cDGS underwent transplantation with allogeneic cultured thymus. OBJECTIVE: We sought to confirm and extend the results previously obtained in a single center. RESULTS: Two patients died of pre-existing viral infections without having thymopoiesis, and 1 late death occurred from autoimmune thrombocytopenia. One infant had septic shock shortly after transplantation, resulting in graft loss and the need for a second transplant. Evidence of thymopoiesis developed from 5 to 6 months after transplantation in 10 patients. Median circulating naive CD4 counts were 44 × 106/L (range, 11-440 × 106/L) and 200 × 106/L (range, 5-310 × 106/L) at 12 and 24 months after transplantation and T-cell receptor excision circles were 2,238/106 T cells (range, 320-8,807/106 T cells) and 4,184/106 T cells (range, 1,582-24,596/106 T cells). Counts did not usually reach normal levels for age, but patients were able to clear pre-existing infections and those acquired later. At a median of 49 months (range, 22-80 months), 8 have ceased prophylactic antimicrobials, and 5 have ceased immunoglobulin replacement. Histologic confirmation of thymopoiesis was seen in 7 of 11 patients undergoing biopsy of transplanted tissue, including 5 showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator expression was also demonstrated. Autoimmune complications were seen in 7 of 12 patients. In 2 patients early transient autoimmune hemolysis settled after treatment and did not recur. The other 5 experienced ongoing autoimmune problems, including thyroiditis (3), hemolysis (1), thrombocytopenia (4), and neutropenia (1). CONCLUSIONS: This study confirms the previous reports that thymus transplantation can reconstitute T cells in patients with cDGS but with frequent autoimmune complications in survivors.

Tumour cell conditioned medium reveals greater M2 skewing of macrophages in the absence of properdin.

INTRODUCTION: The tumour microenvironment is shaped by the interaction of immune, non immune, and tumour cells present in close proximity. Tumour cells direct the development of a locally immune suppressed state, affecting the activity of anti tumour T cells and preparing the escape phase of tumour development. Macrophages in the tumour typically develop into so-called tumour associated macrophages with a distinct profile of activities which lead to a reduction in inflammation and antigen presentation. The direct impact of tumour cell conditioned medium on the activity profile of macrophages in dependence of their complement component expression has not yet been investigated. METHODS: In our in vitro study, macrophages differentiated from bone marrows of properdin deficient and wildtype mice were stimulated with conditioned medium of a syngeneic tumour cell line, B16F10, a mouse melanoma subline. RESULTS: In comparison with macrophages from wildtype mice, those from congenic properdin deficient mice showed skewing towards M2 profile, encompassing mRNA expression for genes involved in arginine metabolism, production of type 2 cytokines, and relatively lower surface expression of molecules needed for antigen presentation. CONCLUSIONS: These data suggest that properdin insufficiency promotes a tumour environment that helps the tumour evade the immune response.

The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation.

Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft-versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201(+) hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1235-243, hTERT615-624 or PRAME100-108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting.

Cowden's syndrome with immunodeficiency.

BACKGROUND: Cowden's syndrome is a rare, autosomal dominant disease caused by mutations in the phosphoinositide 3-kinase and phosphatase and tensin homolog (PTEN) gene. It is associated with hamartomatous polyposis of the gastrointestinal tract, mucocutaneous lesions and increased risk of developing certain types of cancer. In addition to increased risk of tumour development, mutations in PTEN have also been associated with autoimmunity in both mice and humans. Until now, however, an association between Cowden's syndrome and immune deficiency has been reported in a single patient only. METHODS AND RESULTS: Two patients with Cowden's syndrome and an increased frequency of infections were investigated for possible underlying immunodeficiency. In one patient, hypogammaglobulinaemia with a functional antibody deficiency was identified, while the other patient had a persisting CD4+ T cell lymphopenia (with normal antibody production). CONCLUSIONS: Our data indicate that Cowden's syndrome may be associated with both T cell and B cell immune dysfunction. We recommend that patients with Cowden's syndrome and an increased frequency of infections are investigated for associated immunodeficiency.

Pneumococcal polysaccharide vaccine responses are impaired in a subgroup of children with cystic fibrosis.

BACKGROUND: Pneumococcal immunization is recommended in children with cystic fibrosis (CF). To date, however, there are no published studies on the efficacy of pneumococcal vaccination in this group of patients. METHODS: We carried out a retrospective study of serotype-specific pneumococcal antibody responses to immunization with Prevenar 7 and Pneumovax II in a cohort of children with CF. RESULTS: Nine children had been immunized with Prevenar 7, and all had serotype-specific pneumococcal antibody levels in the protective range (>0.35mg/L) to all 7 immunizing serotypes. In contrast, only 7 of 33 patients (21%) immunized with Pneumovax II made protective antibody responses to all 7 serotypes, and 3 failed to make protective antibodies to any of the serotypes. Controlling for age as a confounder in the analysis, children with impaired antibody responses to pneumococcal polysaccharide (Pneumovax II) immunization had lower Shwachman-Kulczycki scores than children with normal polysaccharide antibody responses. All isolates of Pseudomonas aeruginosa occurred in patients with impaired anti-pneumococcal antibody responses, and a broader range of respiratory pathogens was isolated from these children. CONCLUSIONS: Impaired antibody responses to immunization with Pneumovax II are common in children with CF and this may be associated with increased disease severity.

Antigen presenting cell/ tumor cell fusion vaccines for cancer immunotherapy.

Fusions of antigen presenting cells and tumor cells have been investigated in animal models and phase I/II clinical trials as candidate cancer vaccines. In animal studies there have been numerous reports of induction of protective immunity against a wide range of tumor types. Results of clinical trials have been less dramatic, but tumor-specific immune responses have been reported in many patients, with clinical responses to the vaccination in a subset. In this commentary article, I review the current status of antigen presenting cell/tumor cell fusion vaccines for cancer immunotherapy.

Toll-like receptor 9 activation induces expression of membrane-bound B-cell activating factor (BAFF) on human B cells and leads to increased proliferation in response to both soluble and membrane-bound BAFF.

OBJECTIVES: Activation of TLR7 and TLR9 and high serum levels of BAFF have been implicated in the pathogenesis of SLE. However, little is known about the effects of TLR9 activation on BAFF expression by human B cells. We investigated the effect of the TLR9 agonist, CpG-ODN 2006, on the expression of BAFF and its receptors BAFF-R, TACI and BCMA, in isolated B cells from healthy donors. METHODS: We used RT-PCR, flow cytometry and ELISA to investigate the expression of BAFF, and flow cytometry for BAFF-R, TACI and BCMA. Functional assays assessed the responses of resting and CpG-ODN-activated B cells to exogenous soluble and membrane-bound BAFF. RESULTS: CpG-ODN did not induce BAFF secretion, but increased expression of membrane-bound BAFF on B cells. CpG-ODN also induced the expression of TACI and BCMA, but did not up-regulate BAFF-R expression. In functional studies, CpG-ODN sensitized human B cells to proliferate in response to exogenous BAFF. This effect was inhibited by a blocking antibody against BAFF-R, but was not inhibited by anti-TACI or anti-BCMA antibodies. Membrane-bound BAFF, induced by CpG-ODN, co-stimulated the proliferation of B cells stimulated with anti-IgM in a manner that was dependent on the expression of surface BAFF on the CpG-ODN-treated B cells. CONCLUSION: TLR9 activation induces expression of membrane-bound BAFF on human B cells and leads to increased proliferation in response to both soluble and membrane-bound BAFF. These data extend our understanding of the role of TLR9 activation on human B cells and provide insights into the mechanisms by which TLR9 may participate in the pathogenesis of SLE.

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies.

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

Radiation induces acute alterations in neuronal function.

Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage. In the past it was accepted that radiation-induced normal tissue injury resulted from a progressive reduction in the survival of clonogenic cells. Moreover, because radiation-induced brain dysfunction is believed to evolve over months to years, most studies have focused on late changes in brain parenchyma. However, clinically, acute changes in cognition are also observed. Because neurons are fully differentiated post-mitotic cells, little information exists on the acute effects of radiation on synaptic function. The purpose of our study was to assess the potential acute effects of radiation on neuronal function utilizing ex vivo hippocampal brain slices. The cellular localization and functional status of excitatory and inhibitory neurotransmitter receptors was identified by immunoblotting. Electrophysiological recordings were obtained both for populations of neuronal cells and individual neurons. In the dentate gyrus region of isolated ex vivo slices, radiation led to early decreases in tyrosine phosphorylation and removal of excitatory N-methyl-D-aspartate receptors (NMDARs) from the cell surface while simultaneously increasing the surface expression of inhibitory gamma-aminobutyric acid receptors (GABA(A)Rs). These alterations in cellular localization corresponded with altered synaptic responses and inhibition of long-term potentiation. The non-competitive NMDAR antagonist memantine blocked these radiation-induced alterations in cellular distribution. These findings demonstrate acute effects of radiation on neuronal cells within isolated brain slices and open new avenues for study.

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro.

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

Optimized protocol to make phospho-specific antibodies that work.

Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state. These phospho-specific antibodies can thus be used to track the activity of a protein, not simply its level of expression. In this review, we will discuss both the design of the phosphopeptide immunogen and immunization. The affinity purification of the phospho-specific antibody as well as the methods most suitable for characterizing the phosphospecificity of the antibody will be described here. Taken together, these methods will cover the key procedures and protocols required to produce a phospho-specific antibody that works.

Can neuroimaging help us to understand and classify somatoform disorders? A systematic and critical review.

OBJECTIVE: Debate about the nature of somatoform disorders and their current diagnostic classification has been stimulated by the anticipation of new editions of Diagnostic and Statistical Manual of Mental Disorders and International Statistical Classification of Diseases and Related Health Problems diagnostic classifications. In the current paper, we review systematically the literature on the neuroimaging of somatoform disorders and related conditions with the aim of addressing two specific questions: Is there evidence of altered neural function or structure that is specifically associated with somatoform disorders? What conclusions can we draw from these findings about the etiology of somatoform disorders? METHODS: Studies reporting neuroimaging findings in patients with a somatoform disorder or a functional somatic syndrome (such as fibromyalgia) were found using Pubmed, PsycINFO, and EMBASE database searches. Reported structural and functional neuroimaging findings were then extracted to form a narrative review. RESULTS: A relatively mature literature on symptoms of pain and less developed literatures on conversion and fatigue symptoms were identified. The available evidence indicates that, when compared with nonclinical groups, somatoform diagnoses are associated with increased activity of limbic regions in response to painful stimuli and a generalized decrease in gray matter density; however, methodological considerations restrict the interpretation of these findings. CONCLUSIONS: Whereas the neuroimaging literature has provided evidence about the possible mechanisms underlying somatoform disorders, this is not yet sufficient to provide a basis for classification. By adopting a wider variety of experimental designs and a more dynamic approach to diagnosis, there is every reason to be hopeful that neuroimaging data will play a significant role in future taxonomies.

An androgenic steroid delivery vector that imparts activity to a non-conventional platinum(II) metallo-drug.

A range of androgen conjugates with non-conventional platinum(II) complexes have been synthesised with the aim of targeting tumour cells since many display elevated levels of the androgen receptor. The androgenic platinum conjugates are delivered into selected cells with improved efficiency (when compared to their non-steroidal analogues). The act of conjugating an androgen to a platinum(II) complex resulted in synergistic effects between the metallic centre and the steroidal ligand, creating highly potent platinum(II) complexes from the inactive components.

A threshold level of TLR9 mRNA predicts cellular responsiveness to CpG-ODN in haematological and non-haematological tumour cell lines.

The human toll like receptor 9 (TLR9) detects differences between microbial and host DNA, based on unmethylated deoxycytidyl deoxyguanosine dinucleotide (CpG) motifs, leading to activation of both innate and adaptive immune mechanisms. The synthetic TLR9 agonist, CpG-ODN, can substitute for microbial DNA in these responses, and is in clinical trials as an immunomodulatory agent in diseases as diverse as infections, cancer and allergic disorders. Human TLR9 is expressed on cells of haematopoietic origin (principally plasmacytoid dendritic cells and B cells), but has also been described as being expressed on a number of other cell types. In order to clarify the expression and function of TLR9 in a range of cells of both haematopoietic and non-haematopoietic origin, we investigated the level of expression of TLR9 mRNA, and the ability of the cells to respond to CpG-ODN by upregulation of cell surface markers, cytokine production, cellular proliferation and activation of NFkappaB. Our data show that the cellular response to CpG-ODN depended on a threshold level of expression of TLR9. TLR9 was widely expressed amongst B cell tumours (with the exception of myeloma cell lines), but we did not find either threshold levels of expression of TLR9 or responses to CpG-ODN in several myeloma or myeloid tumour cell lines or any non-haematological tumour cell lines tested in our study. TLR9-positive cells varied significantly in their responses to CpG-ODN, and the level of TLR9 expression beyond the threshold did not correlate with the magnitude of the response to CpG-ODN. Finally, CpG-ODN induced NFkappaB activation and increased cellular proliferation in Hek293 cells that had been stably transfected with hTLR9, but did not affect the expression of surface markers or synthesis of IL-6, IL-10 or TNF-alpha. Thus both haematological and non-haematological cells expressing appropriate levels of TLR9 respond to CpG-ODN, but the nature of the TLR9-mediated response is dependent on cell type.

Preclinical development of hybrid cell vaccines for multiple myeloma.

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines.