Search

Gating of positron emission tomography images has been shown to reduce the motion effects, especially when imaging small targets, such as coronary plaques. However, the selection of optimal number of gates for gating remains a challenge. Selecting too high number of gates results in a loss of signal-to-noise ratio, while too low number of gates does remove only part of the motion. Here, we introduce a respiratory-cardiac motion model to determine the optimal number of respiratory and cardiac gates. We evaluate the model using a realistic heart phantom and data from 12 cardiac patients (47–77 years, 64.5 on average). To demonstrate the benefits of our model, we compared it with an existing respiratory model. Based on our study, the optimal number of gates was determined to be five respiratory and four cardiac gates in the phantom and patient studies. In the phantom study, the diameter of the most active hot spot was reduced by 24% in the dual gated images compared to non-gated images. In the patient study, the thickness of myocardium wall was reduced on average by 21%. In conclusion, the motion model can be used for estimating the optimal number of respiratory and cardiac gates for dual gating.

Continuous power supply for wearable electronics can be facilitated using wireless power transfer (WPT). We use a 3D knitted helical coil as the receiver coil in the wrist or the waist of a garment. This 3D knitted helical coil is a novel approach to integrate coils in garments that maintains full flexibility of the garment. Measurements and simulations of coil-coil coupling give compelling evidence of the feasibility of this approach for wearable WTP. The coupling factor between a closely wound and knitted coil is found to be ~0.25 and ~0.55 for adjacent coils for a knit in the border of a cuff and waist, respectively. Using a simple circuit, we demonstrate a 30% efficiency between a closely wound transmitter coil worn on the wrist and a 3D knitted helical receiver coil integrated in the cuff of a garment at 6 mm distance.

Positron emission tomography (PET) respiratory motion correction has been a subject of great interest for the last twenty years, prompted mainly by the development of multimodality imaging devices such as PET/computed tomography (CT) and PET/magnetic resonance imaging (MRI). PET respiratory motion correction involves a number of steps including acquisition synchronization, motion estimation and finally motion correction. The synchronization steps include the use of different external device systems or data driven approaches which have been gaining ground over the last few years. Patient specific or generic motion models using the respiratory synchronized datasets can be subsequently derived and used for correction either in the image space or within the image reconstruction process. Similar overall approaches can be considered and have been proposed for both PET/CT and PET/MRI devices. Certain variations in the case of PET/MRI include the use of MRI specific sequences for the registration of respiratory motion information. The proposed review includes a comprehensive coverage of all these areas of development in field of PET respiratory motion for different multimodality imaging devices and approaches in terms of synchronization, estimation and subsequent motion correction. Finally, a section on perspectives including the potential clinical usage of these approaches is included.

Objective: The efficacy of therapeutic vaccines against HIV-1 infection has been modest, New inerts to redirect responses to vulnerable sites are urgently needed to improve these results. Design: We performed the first-in-human clinical trial with naked mRNA (IFIIVARNA) combining a dendritic cell activation strategy (TriMix:CD40L+CD70+caTLR4 RNA) with a novel HIV immunogen sequences (HTI immunogen). Methods: A dose escalation, phase I clinical trial was performed in 21 chronic HIV-1-infected patients under ART who received three intranodal doses of mRNA (weeks 0, 2 and 4) as follow: TriMix-100g, TriMix-300g, TriMix-300g with HTI 300g, TriMix-300 g with HTI-600g, TriMix-300g with HTI-900g. Primary end-point was safety and secondary exploratory end-points were immunogenicity, changes in viral reservoir and transcriptome. Results: Overall, the vaccine was secure and well tolerated. There were 31 grade 1/2 and 1 grade 3 adverse events, mostly unrelated to the vaccination. Patients who received the highest dose showed a moderate increase in T-cell responses spanning HTI sequence at week 8, In addition, the proportion of responders receiving any dose of HTI increased from 31% at w0 to 80% postvaccination. The intervention had no impact on caHIV-DNA levels, however, cal-IIV-RNA expression and usVL were transiently increased at weeks 5 and 6 in the highest dose of iHIVARNA, and these changes were positively correlated with HIV-1-specific-induced immune responses. Conclusion: This phase I dose-escalating trial showed that iHIVARNA administration was safe and well tolerated, induced moderate HIV-specific T-cell responses and transiently increased different viral replication readouts. These data support further exploration of iHIVARNA in a phase II study. Copyright (C) 2018 The Authon(s). Published by Wolters Kluwer Health, Inc.

Dendritic cell therapy has been optimized a lot aiming to induce a strong and broad immune response in terms of the recognized epitopes by both CD8+ and CD4+ T cells and the use of the patients' complete unique set of HLA molecules. We here give an overview of our approach for antigen loading and maturation of dendritic cells and describe the consequences to evaluate the immune response after treatment as well as the Brussels experience in clinical trials.

In this technical note we propose a rapid and scalable software solution for the processing of PET list-mode data, which allows the efficient integration of list mode data processing into the workflow of image reconstruction and analysis. All processing is performed on the graphics processing unit (GPU), making use of streamed and concurrent kernel execution together with data transfers between disk and CPU memory as well as CPU and GPU memory. This approach leads to fast generation of multiple bootstrap realisations, and when combined with fast image reconstruction and analysis, it enables assessment of uncertainties of any image statistic and of any component of the image generation process (e.g. random correction, image processing) within reasonable time frames (e.g. within five minutes per realisation). This is of particular value when handling complex chains of image generation and processing. The software outputs the following: (1) estimate of expected random event data for noise reduction; (2) dynamic prompt and random sinograms of span-1 and span-11 and (3) variance estimates based on multiple bootstrap realisations of (1) and (2) assuming reasonable count levels for acceptable accuracy. In addition, the software produces statistics and visualisations for immediate quality control and crude motion detection, such as: (1) count rate curves; (2) centre of mass plots of the radiodistribution for motion detection; (3) video of dynamic projection views for fast visual list-mode skimming and inspection; (4) full normalisation factor sinograms. To demonstrate the software, we present an example of the above processing for fast uncertainty estimation of regional SUVR (standard uptake value ratio) calculation for a single PET scan of (18)F-florbetapir using the Siemens Biograph mMR scanner.

The output power of a thin-film thermoelectric generator consisting of a Cu-20- μm nanowire (NW) array (NWA)-Si bulk-Cu sandwich with Si or Si0.8Ge0.2 NWs is measured and compared to Cu-Si bulk-Cu for small temperature differences around room temperature. The array of NWs is made by metal-assisted chemical etching that retains the Ge concentration in the wires. The conversion of one surface of Si bulk into an array of short-relative to the remaining bulk-Si NWs improves the maximum output power by a factor of two. Using an array of Si NWs in combination with Si0.8Ge0.2 increases the maximum output power by a factor of 20. The increased output power, under the same heating power, is due to the lower thermal conductivity and contact resistance of the NWA and SiGe compared to Si bulk. A matrix model is developed to solve the coupled thermal transport equations for an arbitrary number of layers. Fits to the measurement allow the extraction of the electrical contact resistance and the effective internal temperature drop across the semiconductor composite.

HIV infection is characterized by a number of abnormalities in several components of the immune system. For example, during HIV infection, a massive decrease of CD4(+) T cells is observed, as well as a progressive depletion of naïve CD8(+) T cells. Furthermore, elevated numbers of apoptotic B and T cells are present in HIV-infected patients, and a systemic immune activation results in T-cell exhaustion. Finally, HIV infection is characterized by the presence of functionally impaired dendritic cells, with decreased expression of maturation markers, decreased secretion of cytokines and defects in antigen processing and presentation. All these characteristics result in the occurrence of non-functional cytotoxic T lymphocytes, that fail to control HIV-replication in most individuals during progressive disease. Costimulatory and co-inhibitory molecules are involved in the activation, differentiation and survival of several cell-types of the immune system. Each costimulatory receptor (generally expressed on effector cells) can conjugate with one or more specific ligands (expressed on antigen-presenting cells), which leads to an activation of intracellular signaling pathways inside the cells on which they are expressed. HIV infection is characterized by an aberrant expression of these molecules on cells of the immune system. Many of the immune deficiencies mentioned in the previous paragraph can be explained by abnormal expression of costimulatory molecules, and could consequently be overcome by interfering with their interactions. In this review, we give an overview of the functions and expression patterns of the receptor/ligand pairs of the tumor necrosis factor and the B7 super-families of costimulatory and co-inhibitory molecules in HIV-infected patients. We will also discuss possibilities for manipulating their signaling as a therapeutic anti-HIV tool.

Characterization of the Ge concentration in a Si1−xGex heterojunction for x varying from 5% to 40% in steps of 5% is done by bevelling and wet thermal oxidation of the exposed layers. The resulting difference in oxide thickness as a function of Ge concentration is visualized due to light interference. Different Ge concentrations are seen as different colours through an optical microscope. The technique—CABOOM—stands for characterization of alloy concentration by bevelling, oxidation and optical microscopy. The oxidation temperature and time to visualize 5% differences in Ge concentration has been optimized for CABOOM. We established that the bevelling method and smoothness do not have a significant influence on the results. A mathematical model was developed in Mathematica™ to study the limits and strengths of the technique. The results of the calculations are in excellent agreement with the measurements. Mathematical calculations are used to study the limit in layer thickness and Ge concentration resolution.

Interaction of the CD40L (CD154) molecule on activated T cells with its receptor, CD40, on macrophages and dendritic cells (DC) provides a strong signal for interleukin (IL)-12 production. As IL-12 is the most important factor in driving Th precursor (Thp) cells into T(h)elper 1 cells, CD40-CD40L interactions strongly promote Th1 differentiation. Th2 cytokines (IL-4, IL-13, IL-10) on the other hand, are known to inhibit Th1 differentiation, and to promote either directly or indirectly, Th2 differentiation. Inhibition of lipopolysaccharide (LPS)-induced IL-12 production by IL-4, IL-13 and IL-10 is supposed to be one such mechanism. However, we here report that IL-4 and IL-13 enhance p70 IL-12 production and p40 mRNA transcription by human monocytes when the latter are stimulated trough triggering of CD40. This effect on IL-12 induction is most clear in the presence of interferon (IFN)-gamma, which upregulates CD40 expression. IL-10 potently inhibits IL-12 production. The increased IL-12 production in the presence of IL-4 and IL-13 is however, not the indirect result of a reduction in IL-10 production, but is most likely owing to a direct effect of IL-4 and IL-13. We conclude that IL-4 and IL-13 enhance rather than decrease the IL-12 production by human monocytes during interaction with T cells. This effect can potentially contribute in vivo to switching of an ongoing Th2 response towards a Th1 response and the findings also support the dominant effect of CD40/CD40L interaction on Th1 development, even in the presence of Th2 cytokines.

OBJECTIVES The purpose of this work was to study the clonal relationship between the cells that secrete monoclonal proteins in an IgA/ IgE double multiple myeloma patient. Double monoclonal gammopathy is a rare condition in which two types of monoclonal proteins can be found in the serum and/or urine of patients with multiple myeloma or gammopathy of undetermined significance. The study of the relationship between the cells expressing the different monoclonal proteins may provide insight in the pathogenesis of these disorders. METHODS The clonal relationship of the two tumoral plasma cell populations was examined by immunophenotyping and sequence analysis of the variable regions of the immunoglobulin heavy chain genes. Both immunoglobulin sequences were isolated from the bone marrow using a polymerase chain reaction (PCR)-based cloning strategy. Rare isotype-switch variants were detected by a myeloma-specific PCR in combination with different isotype-specific primers. An in vitro culture system, based on the activation of the CD40 molecule on the B cell, was used in order to isolate and expand myeloma-related B cells from peripheral blood that could possibly be regarded as myeloma precursor cells. RESULTS The variable parts of the immunoglobulin heavy chains linked to either Calpha or Cepsilon were exactly the same, including the same somatic mutations. From the in vitro CD40 cultures B cells could be isolated that either expressed IgA or IgE with exactly the same variable immunoglobulin part as the myeloma clone. No pre-switched IgM myeloma-related B cells could be found. CONCLUSION Both cell populations in this IgA/IgE myeloma patient shared a common clonal origin. No evidence for a pre-switched IgM precursor myeloma cell was found in this patient.

The 5T series of multiple myelomas (MM) and Waldenstrsom's macroglobulinaemia-like lymphomas (WM), which developed spontaneously in ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble their corresponding human diseases. In order to compare the patterns of somatic mutation in V(H) genes of mouse tumours with those of human counterparts, we have determined and analysed sequences of immunoglobulin V(H) genes in five cases of murine MM, two of WM and one of biclonal benign monoclonal gammopathy (BMG). Four of five MM and 2/2 WM cases used V(H) genes of the large J558 family; one MM used a gene of the VGAM3.8 family, and both clones of the BMG used genes of the 36-60 family. N-region insertions were observed in all cases, but D-segment genes were only identified in 6/9 cases, which were all from the D-SP family and translated in reading frame 3. Compared with human MM, in which the V(H) genes have been found to be consistently hypermutated (mean% ± SD = 8.8 ± 3.2), the degree of somatic mutation in the murine tumours was significantly lower (mean% ± SD=2.9 ± 2.3). There was no significant evidence of clustering of replacement mutations in complementarity determining regions (CDR), a feature considered to be characteristic of antigen-selected sequences. However, one clone of the biclonal BMG case showed intraclonal variation, a feature described in some cases of human BMG. These results indicate that murine V(H) genes in mature tumours differ from human counterparts in the level and distribution of somatic mutations, but support the concept that BMG may be distinct from MM.

Activation of natural killer (NK) cells in the presence of interleukin-12 (IL-12) augments the capacity of these effector cells to recognize B7-1– and B7-2–expressing target cells. These effector cells also efficiently lyse autologous B7-positive progenitor or organ-derived dendritic cells, suggesting a physiologic regulatory pathway between IL-12, NK cells, and B7-expressing antigen-presenting cells. Although IL-12–activated NK cells secreted higher levels of interferon-γ, this cytokine did not play a role in synergistic effects of IL-12 and B7 on NK activation. The B7-counterreceptor was found to be selectively upregulated on IL-2/IL-12 as compared with IL-2–activated NK cells. CD28 is functionally involved in the recognition of B7 on target cells since IL-2/IL-12–activated NK cells derived from CD28 knockout mice were strongly reduced in their capacity to lyse syngeneic B7-positive tumor cells as well as antigen-presenting cells. However, recognition of B7 on allogeneic targets did not require the expression of CD28 on the IL-2/IL-12–activated NK cells. Hence, IL-12 triggers the expression of both CD28-dependent and CD28-independent mechanisms that allow NK cells to eliminate B7-positive target cells including autologous dendritic cells.

Background Autologous monocyte-derived dendritic cells (DCs) electroporated with synthetic messenger RNA (mRNA) encoding a CD40 ligand, a constitutively active Toll-like receptor 4 and CD70, together with mRNA encoding fusion proteins of a human leukocyte antigen (HLA)-class II targeting signal (DC-LAMP) and a melanoma-associated antigen (MAA); either MAGE-A3, MAGE-C2, tyrosinase or gp100) (TriMixDC-MEL) are superiorly immunogenic. Patients and methods In this phase IB clinical trial, 24 million viable DCs were administered by four biweekly combined intradermal (id) and intravenous (iv) administrations, and a fifth administration on week 16. The number of iv-administered DCs was escalated in four sequentially treated cohorts. Immune responses were assessed by analysis of antigen specificity of blood-derived T-cells and skin infiltrating lymphocytes (SKILs). Results Fifteen patients with pretreated advanced melanoma tolerated administration of TriMixDC-MEL well. Two patients achieved a complete response and two patients a partial response. All objective responders are progression-free after a follow-up of, respectively, 24+, 28+, 33+, and 34+ months. Post-therapy antigen-specific SKILs were documented in 6 of 12 patients, and antigen-specific CD8+ T-cells were detected in the blood of 4 of 5 patients. Conclusions Cellular immunotherapy with TriMixDC-MEL is safe and immunogenic. Antitumor activity with durable disease control is observed across the investigated iv-dose levels. ClinicalTrials.gov Identifier NCT01066390.

In this paper we study propagation of electromagnetic waves in media with an imaginary Kerr-type nonlinearity. We show numerically that the slowly varying envelope approximation can still be used. It is proved that theoretical problems described by this type of differential equations are Hamiltonian systems. This characteristic is used to predict the variation of the electromagnetic field in the nonlinear material. Furthermore, we find a closed expression for the electromagnetic waves in a nonlinear medium with a linear absorption and an imaginary Kerr-type nonlinearity, for both a localized and a diffusive nonlinearity. Calculating the transmission probability of light through a Fabry-P\'erot structure shows that the introduction of an imaginary Kerr-type nonlinearity describes an intensity-dependent optical gain or absorption in a nonlinear medium. This system also shows bistable behavior comparable to real Kerr-type nonlinear materials. \textcopyright{} 1996 The American Physical Society.

Movement degrades image quality in PET/CT. Two types of motion can be described: respiratory and global body motion, also called patient motion. In this study, an analysis of both types of movement is carried out in the context of dynamic PET acquisition for oncology research, using [18F]FL T and [18F]FDG data from patients with various types of tumours. To investigate the influence of the two types of motion during the acquisition, the PET data were partitioned in time frames and phases of the respiratory cycle ("gating"). Assuming the tumour does not deform during acquisition, we have used locally rigid registration to estimate its motion. Registrations were validated visually, using profiles and by checking alignment of the centre-of-mass of the segmented tumour. Dynamic patient motion of up to 1.5cm and respiratory motion of up to 1cm were observed, indicating that both types of motion in PET images cannot be neglected for certain applications. In addition, we show that patient movement changes the relation between tumour position and RPM position. This needs to be taken into account when using displacement gating for respiratory motion correction.

Over the years, there has been an exponential increase in the number of gene therapy approaches that are under investigation for the treatment of cancer. This can be attributed to our growing understanding of the molecular mechanisms that contribute to the onset and maintenance of cancer as well as to the development of gene delivery vectors. In this review, we will focus on the use of lentiviral vectors (LVs) in immuno gene therapy of cancer, as these efficacious gene delivery vehicles have come to the fore front because of their many attractive features. LVs have been successfully applied to generate potent dendritic cell based anti-cancer vaccines and to deliver cancer-specific receptors to T-cells. Moreover, LVs are under investigation for the modulation of cancer cells. We will describe various strategies of this 'genuine' cancer gene therapy, amongst which transfer of suicide genes, modulation of pro- and anti-apoptotic molecules, strategies to optimize chemo- and radiotherapy, expression of molecules that affect angiogenesis or affect the immunogenicity of tumor cells. These will be discussed in view of our current knowledge of tumor immunology. Finally we will discuss some important issues and future directions to push the field forward.

Antigen-specific immunity is crucially important for containing viral replication in human immunodeficiency virus (HIV)-1-infected hosts. Several epitopes have been predicted for the early expressed HIV-1 proteins Tat and Rev, but few have been studied in detail. We characterized the human leukocyte antigen (HLA)-B44-restricted Rev epitope EELLKTVRL (EL9) in an HIV-1-infected subject treated with antiretroviral therapy. Interestingly, a high sequence similarity was found between the EL9 epitope and the human nucleolar protein 6 (NOL6). However, this similarity does not seem to impede immunogenicity as CD8(+) T-cells, previously stimulated with EL9-pulsed dendritic cells, were able to specifically recognize the HIV-1 Rev epitope without cross-recognizing the human self-antigen NOL6. After the subject interrupted antiretroviral therapy and virus rebounded, mutations within the EL9 epitope were identified. Although the emerging mutations resulted in decreased or abolished T-cell recognition, they did not impair Rev protein function. Mutations leading to escape from T-cell recognition persisted for up to 124 weeks following treatment interruption. This study shows that the HLA-B44-restricted Rev CD8(+) T-cell epitope EL9 is immunogenic notwithstanding its close resemblance to a human peptide. The epitope mutates as a consequence of dynamic interaction between T-cells and HIV-1. Clinical status, CD4(+) T-cell count and viral load remained stable despite escape from T-cell recognition.

Lentiviral vectors (LVs) provide unique opportunities for the development of immunotherapeutic strategies, as they transduce a variety of cells in situ, including antigen-presenting cells (APCs). Engineering LVs to specifically transduce APCs is required to promote their translation towards the clinic. We report on the Nanobody (Nb) display technology to target LVs to dendritic cells (DCs) and macrophages. This innovative approach exploits the budding mechanism of LVs to incorporate an APC-specific Nb and a binding-defective, fusion-competent form of VSV. G in the viral envelope. In addition to production of high titer LVs, we demonstrated selective, Nb-dependent transduction of mouse DCs and macrophages both in vitro and in situ. Moreover, this strategy was translated to a human model in which selective transduction of in vitro generated or lymph node (LN)-derived DCs and macrophages, was demonstrated. In conclusion, the Nb display technology is an attractive approach to generate LVs targeted to specific cell types.

Background: Autologous monocyte derived dendritic cells (DC) electroporated with synthetic messenger RNA (smRNA) encoding CD40 ligand, a constitutively active TLR4, and CD70 (TriMixDC), together with smRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen are immunogenic when administered ID (Wilgenhof et al. ASCO AM 2009). The TriMixIDIV clinical trial investigates the safety, immunogenicity, and activity of combined ID and IV administration in patients with pretreated melanoma. Methods: Following leukapheresis, immature DC (derived from adherent PBMC cultured for 6 days in IL-4/GM-CSF supplemented medium) are electroporated with smRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix smRNA. TriMix-DC are cryopreserved and 24.106 viable DC are administered by 4 ID/IV-injections q2w, and a 5th-injection on w16. The number of IV administered DC was escalated from 4.106, over 12.106, to 20.106 in cohort 1, 2 and 3 respectively. Tumor response assessments are performed by 18F-PET/CT at baseline and in w8, 16 and 24. Immunomonitoring is performed by analysis of DTH infiltrating lymphocytes (DIL) at an ID injection site. Results: 11 pts (9M/2F; med age 54, range 40-77) with stage IV pretreated melanoma, have initiated study treatment. TriMix-DC related AE's: gr2 local skin injection site reactions (all pts); fever & lethargy (gr1, 1 pt in cohort 1); chills (gr2, 2 pts in cohort 3). Vaccine-specific DIL were documented in 6/7 pts (5/7 pts had a CD137+CD8+ and 4/7 pts a CD4+ T-cell response). As of Jan 2011, 8 pts were evaluated for response (RECISTv1.1): 1 CR, 1 PR, 3 SD, and 3 PD; regression of metastases occurred in lung- (2 pts) and lymph node metastases (3 pts); 4/8 pts remain progression-free after respectively 3.1, 3.6, 7.7 and 8.2 mths of follow-up. Conclusions: TriMix-DC therapy by combined IV/ID administration is feasible, safe, and immunogenic. Durable anti-melanoma activity is observed across the investigated IV-dose levels and compares favorably with our prior observations with TriMix-DC administered ID-only.

Since decades, the main goal of tumor immunologists has been to increase the capacity of the immune system to mediate tumor regression. Considerable progress has been made in enhancing the efficacy of therapeutic anticancer vaccines. First, dendritic cells (DCs) have been identified as the key players in orchestrating primary immune responses. A better understanding of their biology and the development of procedures to generate vast amounts of DCs in vitro have accelerated the development of potent immunotherapeutic strategies for cancer. Second, tumor-associated antigens have been identified which are either selectively or preferentially expressed by tumor cells and can be recognized by the immune system. Finally, several studies have been performed on the genetic modification of DCs with tumor antigens. In this regard, loading the DCs with mRNA, which enables them to produce/process and present the tumor antigens themselves, has emerged as a promising strategy. Here, we will first overview the different aspects that must be taken into account when generating an mRNA-based DC vaccine and the published clinical studies exploiting mRNA-loaded DCs. Second, we will give a detailed description of a novel procedure to generate a vaccine consisting of tumor antigen-expressing dendritic cells with an in vitro superior capacity to induce anti-tumor immune responses. Here, immature DCs are electroporated with mRNAs encoding a tumor antigen, CD40 ligand (CD40L), CD70, and constitutively active (caTLR4) to generate mature antigen-presenting DCs.

9024 Background: Electroporation of dendritic cells (DC) with mRNA encoding fusion-proteins of a HLA-class II targeting signal and a melanoma associated antigen (MAA) together with mRNA encoding CD40 ligand, a constitutively active TLR4 and CD70 (TriMix) improves the immunostimulatory capacity of autologous DC. Methods: Following leukapheresis, immature DCs (derived from adherent PBMC cultured for 6 days in IL-4 / GM-CSF supplemented medium) are electroporated with mRNA encoding MAGE-A3, MAGE-C2, Tyrosinase and gp100 linked to DC-LAMP, and TriMix mRNA. TriMix-DC (12.5 106/antigen) are cryopreserved and administered by 4 ID-injections q2w, and q8w thereafter. After the 4th vaccination, interferon alfa-2b (IFN- a2b, 5 MIU TIW) is initiated. Immune monitoring is performed by skin biopsy of a vaccine injection site. Biopsies are investigated by IHC and by analyzing the activation (CD137+), cytolytic capacity (CD107a+), and cytokine release (IFN-γ and TNF-α) of DTH infiltrating T-cells in response to autologous EBV-B cells expressing MAA. Results: 29 pts (17M/12F; med age 49, range 28–75) with stage III/IV melanoma, nl LDH, and no CNS metastases were recruited. Vaccine related AE's (first 24 pts): gr2 local injection site reactions (all pts); fever & lethargy (gr2, 1 pt). Pts (20) who initiated IFN-a2b experienced constitutional side effects (gr3, 1 pt). Vaccine-specific DTH infiltrating T cells were documented post-vaccination in 13/17 pts (10/13 pts had a CD137+CD8+ and 2/13 pts a CD4+ response). Out of the 11 pts without evaluable disease, 2 had a local recurrence (salvaged by surgery). After a mFU of 7.8 mths (range 4.3–13.7) all pts remain disease-free. Out of the 13 pts with measurable disease, BOR (RECIST) was 8 SD and 5 PD; 1 pt with initial PD subsequently obtained a PR. Regression of metastases occurred in lung- (2 pts), orbita- (1 pt) and lymph node metastases (3 pts). After a mFU of 7 mths (range 1–14), the mPFS is 3,1 mths (95% CI 2,29–4,08); 4 pts remain progression-free after respectively 5, 8, 10 and 11 mths of follow-up. Conclusions: Therapeutic vaccination with TriMix-DC combined with sequential IFN-a2b is feasible, safe, immunogenic and associated with anti-tumor activity in patients with advanced melanoma. [Table: see text]

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8+ and CD4+ T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4+ T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA+ melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

This paper discusses how to estimate scatter when performing motion correction of PET data. It concentrates on head movement, but some results are valid for non-rigid motion as well. We show that rigid motion affects scattered events differently then unscattered events. This has consequences for the scatter estimation procedure. We show with simulations, phantom measurements and patient data that our proposed method obtains fully quantitative motion-corrected PET images.

It is a well-known fact that the maximum likelihood expectation maximization (MLEM) algorithm applied to either the original image or the image rescaled with the sensitivity, produces the same result. However, if an a posteriori function is maximized instead of the likelihood function, this rescaling procedure produces a different outcome. This abstract concentrates on the difference between these procedures applied in the maximum a posteriori (MAP) algorithm with the median root prior (MRP). It will be shown that depending on the way the prior is utilised, it is possible to achieve more noise reduction in the low sensitivity regions. The disadvantage being the fact that the weight prior, beta, must be tuned according to the maximum in the sensitivity.

Images reconstructed with iterative algorithms with conventional inter-iteration filtering as a method of regularisation have spatially varying resolution, which is also object dependent. Three different methods were previously proposed which overcome that problem. Here we compare these algorithms with inter-iteration filtering to penalised likelihood algorithms and in particular penalised likelihood with certainty based penalty (PLCB). We derive an analytic expression that relates the penalty term to the filter coefficients. The linearised local impulse response (LLIR) as well as noise properties present in these algorithms are nearly identical if this matching is performed. This is confirmed with simulations.

Ultimately, positron emission tomography (PET) image reconstruction boils down to solving an overdetermined system of linear equations with nonnegative variables. The latter requirement gets a natural treatment if the problem is reformulated as an optimization problem. The deviation /spl par/Ax-b/spl par/ can be measured by different vector norms leading to different optimization problems. In the paper we give some preliminary results of our investigations about the usefulness of advanced optimization techniques for the solution of different formulations of the problem.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.

VEGF is a key mediator of pathological cancer associated neoangiogenesis and immunosuppression. VEGFR targeted therapy holds promise to counteract cancer associated neoangiogenesis but also to alleviate cancer associated immune suppression. Melanoma patients are at high risk for developing brain metastases. These lesions are strongly vascularized and have a high risk for spontaneous bleeding. Melanoma is susceptible to immunotherapy and patients affected by brain metastases might benefit from combining immunotherapy with anti-VEGFR therapy. The aim of this study was to investigate the effect of axitinib, a small molecule tyrosine kinase inhibitor of VEGFRs, on MO4 melanoma cell growth in vitro and on the composition of tumor-infiltrating immune cells in a subcutaneous and intracranial melanoma syngeneic mouse model. We first treated MO4 melanoma cells in vitro with increasing concentrations of axitinib and found no induction of apoptosis. Furthermore, increasing amounts of axitinib had no effect on T-cell proliferation or DC maturation, thus excluding a potential suppressive effect of axitinib on antigen presenting and T-cell function. When subcutaneous tumor-bearing mice were treated with axitinib (oral gavage, 25 mg/kg, bid), tumor growth was strongly inhibited and survival was significantly improved as compared to the control group. Next, subcutaneous and intracranial tumor bearing mice were treated with axitinib. After 7 days of treatment a significant increase in the number of total immune cells (CD45 + cells) within the spleen, subcutaneous and intracranial tumor was observed. Whereas the increase in immune cells within the spleen was most marked for CD3 + CD8 + T cells, a significant increase in the number of CD11b + cells was noted within the tumors. Upon further characterizing of this CD11b+ population, we found that there was a significant increase in cells with a monocytic myeloid derived suppressor cell phenotype (moMDSCs,CD11b + Ly6ChighLy6G-). However, in vitro proliferation assays using highly purified splenic MDSCs showed that moMDSCs isolated from axitinib-treated mice to have a reduced suppressive capacity on CD4+ and CD8+ T cells than those isolated from vehicle-treated mice on a per cell basis. When highly purified tumor MDSCs were used in in vitro proliferation assays we found that moMDSCs isolated from axitinib-treated mice almost lost their suppressive capacity on CD4+ and CD8+ T cells. Based on these observations we conclude that axitinib delays tumor growth, increases survival and is associated with an increased infiltration of the tumor micro-environment with immune cells both in subcutaneous as well as intracranial localizations. Axitinib seems to specifically increase moMDSCs within the CD11b+ cell population but to reduce their suppressive capacity. Further investigation of combination therapies with axitinib and immunotherapy seems warranted.

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cmu region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.

Increasing evidence indicates that dendritic cells (DCs) are the antigen-presenting cells of the primary immune response. However, several reports suggest that B lymphocytes could be required for optimal T cell sensitization. We compared the immune responses of wild-type and B cell-deficient (muMT) mice, induced by antigen emulsified in adjuvant or pulsed on splenic dendritic cells. Our data show that lymph node cells from both control and muMT animals were primed, but each released distinct cytokine profiles. Lymph node T cells from control animals secreted interferon (IFN)-gamma, interleukin (IL)-2, and IL-4, whereas those from muMT mice produced IFN-gamma and IL-2 but no IL-4. To test whether B cells may influence the T helper cell type 1 (Th1)/Th2 balance by affecting the function of DCs, we immunized mice by transferring antigen-pulsed DCs from wild-type or mutant mice. Injection of control DCs induced the secretion of IL-4, IFN-gamma, and IL-2, whereas administration of DCs from muMT animals failed to sensitize cells to produce IL-4. Analysis of IL-12 production revealed that DCs from muMT mice produce higher levels of IL-12p70 than do DCs from wild-type animals. These data suggest that B lymphocytes regulate the capacity of DCs to promote IL-4 secretion, possibly by downregulating their secretion of IL-12, thereby favoring the induction of a nonpolarized immune response.

We previously reported on a murine T lymphoma cell line, BW-Sp3, with inherent immunogenicity. BW-Sp3 tumors can elicit an anti-tumor CD8(+) CTL response capable of mediating a regression of subcutaneous tumors. However, this immune response is inadequate to eliminate cancer cells completely in a significant percentage of the recipients, resulting in progressing tumors. In this tumor model, tumor progression correlated with a tolerization of tumor-reactive T cells and cellular immunotherapy of tumor bearing animals, with or without B7-mediated costimulation, even increased tumor progression (Raes et al, 1998). In the present study, we investigated whether the co-expression of IFN gamma, together with B7-1, could have beneficial effects on immunotherapy. Although immunotherapy with IFN gamma and B7-1 single transfectants tended to tolerize anti-tumor T-cells and consequently increased tumor growth, the B7-1/IFN gamma double transfectants resulted in a more beneficial outcome. This phenomenon correlated with an increased CTL-inducing potential of the double transfectants. Secondly, we wondered whether CTLA-4 signalling was involved in the down-regulation of the anti-tumor response. Indeed, when immunotherapy was provided along with anti-CTLA-4, the protection by B71/IFN gamma double transfectants was further improved and the tumor-promoting effect of BW-Sp3(B7-1) was compensated for. Our results indicate that B7-1, IFN gamma and the blockade of CTLA-4 cooperate to tilt the balance in favour of tumor elimination, while either factor alone fails to do so or even promotes tumor growth.

Substance P (SP) is an immunoregulatory tachykinin which augments antigen- and mitogen-induced lymphocyte proliferation via signaling through the neurokinin-1 receptor (NK1-R). Non-neuronal cells of the immune system such as monocytes, T lymphocytes and eosinophils can be a source of SP. We have investigated if antigen-presenting dendritic cells (DC) produce SP. DC were grown from bone marrow precursors using a cocktail of GM-CSF, IL-4 and Flt-3 ligand. Reverse transcriptase-PCR amplification using primers for the mouse preprotachykinin-A gene and direct DNA sequencing of amplified products from purified DC demonstrated the presence of the gamma-transcript of the gene, coding for SP and neurokinin A. At the protein level, mouse DC expressed SP as determined by an enzyme immunoassay and confirmed by immunostaining. The functional role of endogenous SP release was determined. During the interaction with syngeneic or allogeneic DC, the addition of a specific NK1-R antagonist partly reduced proliferation in responding T lymphocytes. This was confirmed by using responders derived from NK1-R-deficient mice. In the absence of DC, proliferation of T cells induced by direct TCR ligation and soluble CD28 was partly dependent on signaling through NK1-R, revealing an autocrine effect of SP production by T cells. In conclusion, these results demonstrate that endogenously produced SP contributes to T cell proliferation induced by DC or TCR / CD28 stimulation.

Genes of the MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 families encode antigenic peptides that are presented by HLA class I molecules and that are recognized on human tumors by autologous cytolytic T lymphocytes. These genes are expressed in many solid tumor types but not in normal tissues, except male germline cells. Because the latter cells are devoid of HLA molecules, the derived antigens are strictly tumor-specific and should constitute safe immunogens for cancer immunotherapy. We detected a significant expression of these genes in a high proportion of bone marrow samples from patients with advanced multiple myeloma. This observation provides a basis for clinical trials aimed at inducing a cellular immune response directed at malignant plasma cells in advanced myeloma patients.

Clinical studies have demonstrated beneficial effects of interferon-beta (IFN-beta) therapy in multiple sclerosis (MS) patients. However, the mechanism of action of IFN-beta in MS remains unknown. IFN-beta has even been demonstrated to enhance isolated T cell secretion of IFN-gamma, a cytokine proven to be deleterious in MS. However, IFN-beta inhibits IFN-gamma secretion of T cells, when they are stimulated by antigen presenting cells (APC). We therefore decided to study the effects of IFN-beta on the in vitro differentiation of dendritic cells (DC), a major class of APC. First, we found that the addition of IFN-beta at the initiation of the differentiation did not modify DC morphology, but enhanced the expression of molecules involved in antigen presentation (HLA-DR, B7/1 and B7/2). However, DC, differentiated in the presence of IFN-beta, secreted less interleukin-12 (IL-12) both spontaneously and upon activation by CD40-ligand bearing cells. As a consequence, DC differentiated in the presence of IFN-beta induced less IFN-gamma secretion by alloreactive T cells. We conclude that the direct action of IFN-beta on DC results in inhibition of their ability to secrete IL-12 and to elicit Thelper-1 (Th-1) type responses. These results are of particular interest in MS, in which a critical role for IL-12 has recently been suggested by a number of clinical and experimental observations.

The implications of the detection of residual disease after treatment of acute lymphoblastic leukemia (ALL) are unclear. We conducted a prospective study at 11 centers to determine the predictive value of the presence or absence of detectable residual disease at several points in time during the first six months after complete remission of childhood ALL had been induced. Junctional sequences of T-cell-receptor or immunoglobulin gene rearrangements were used as clonal markers of leukemic cells. Residual disease was quantitated with a competitive polymerase-chain-reaction (PCR) assay. Of 246 patients enrolled at diagnosis and treated with a uniform chemotherapy protocol, 178 were monitored for residual disease with one clone-specific probe (in 74 percent) or more than one probe (in 26 percent). The median follow-up period was 38 months.The presence or absence and level of residual leukemia were significantly correlated with the risk of early relapse at each of the times studied (P0.001). PCR measurements identified patients at high risk for relapse after the completion of induction therapy (those withor =10(-2) residual blasts) or at later time points (those withor =10(-3) residual blasts). Multivariate analysis showed that as compared with immunophenotype, age, risk group (standard or very high risk), and white-cell count at diagnosis, the presence or absence and level of residual disease were the most powerful independent prognostic factors.Residual leukemia after induction of a remission is a powerful prognostic factor in childhood ALL. Detection of residual disease by PCR should be used to identify patients at risk for relapse and should be taken into account in considering alternative treatment.

As demonstrated in several preclinical models, bispecific Abs are attractive immunotherapeutic agents for tumor treatment. We have previously reported that a bacterially produced anti-CD3 x antitumor bispecific single chain variable fragment of Ab fragment (BsscFv), which is capable of retargeting CTLs toward BCL1 tumor cells, exhibits antitumor activity in vitro. To further facilitate BsscFv production, the coding sequence was subcloned in a eukaryotic expression vector and introduced into Chinese hamster ovary cells for large-scale production. In this report, we have determined the serum stability and the clearance rate from the circulation of BsscFv. Most important, we prove here the therapeutic value of BsscFv in the treatment of BCL1 lymphoma, a murine model for human non-Hodgkin's lymphoma. Tumor-bearing mice that were treated with rscFv in combination with staphylococcal enterotoxin B superantigen, human rIL-2, or murine rIL-12 showed long-term survival, whereas untreated mice all died. This is the first report of the successful in vivo use of BsscFv as an immunotherapeutic agent. Furthermore, long-term survival was the result of complete tumor removal and was not due to the induction of dormancy.

The development of an immune response towards factor VIII (fVIII) remains a major complication for hemophilia A patients receiving fVIII infusions. The design of a specific therapy to restore unresponsiveness to fVIII has been hampered by the diversity of the anti-fVIII antibody. Molecular analysis of the specific immune response is therefore required. To this end, we have characterized an fVIII-specific human IgG4kappa monoclonal antibody (BO2C11) produced by a cell line derived from the memory B-cell repertoire of a hemophilia A patient with inhibitor. BO2C11 recognizes the C2 domain of fVIII and inhibits its binding to both von Willebrand factor (vWF) and phospholipids. It completely inhibits the procoagulant activity of native and activated fVIII, with a specific activity of approximately 7,000 Bethesda units/mg. vWF reduces the rate of fVIII inactivation by BO2C11. The antibody-fVIII association rate constant (kass approximately 7.4 x 10(5) M-1 s-1) is eightfold lower than that for vWF-fVIII association, whereas its dissociation rate constant (kdissor = 1 x 10(-5) s-1) is 100-fold lower than that for the vWF-fVIII complex, which suggests that BO2C11 almost irreversibly neutralizes fVIII after its dissociation from vWF. BO2C11 is the first human monoclonal anti-fVIII IgG antibody that has been isolated and allows the study of fVIII inactivation at the molecular level.

The 5T series of multiple myelomas (MM) and Waldenstrsöm's macroglobulinaemia-like lymphomas (WM), which developed spontaneously in ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble their corresponding human diseases. In order to compare the patterns of somatic mutation in VH genes of mouse tumours with those of human counterparts, we have determined and analysed sequences of immunoglobulin VH genes in five cases of murine MM, two of WM and one of biclonal benign monoclonal gammopathy (BMG). Four of five MM and 2/2 WM cases used VH genes of the large J558 family; one MM used a gene of the VGAM3.8 family, and both clones of the BMG used genes of the 36-60 family. N-region insertions were observed in all cases, but D-segment genes were only identified in 6/9 cases, which were all from the D-SP family and translated in reading frame 3. Compared with human MM, in which the VH genes have been found to be consistently hypermutated (mean% +/- SD = 8.8 +/- 3.2), the degree of somatic mutation in the murine tumours was significantly lower (mean% +/- SD = 2.9 +/- 2.3). There was no significant evidence of clustering of replacement mutations in complementarity determining regions (CDR), a feature considered to be characteristic of antigen-selected sequences. However, one clone of the biclonal BMG case showed intraclonal variation, a feature described in some cases of human BMG. These results indicate that murine VH genes in mature tumours differ from human counterparts in the level and distribution of somatic mutations, but support the concept that BMG may be distinct from MM.

The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease. Images Figure 2 Figure 3 Figure 5 Figure 7