What effective technique to differentiate radiation brain necrosis from a tumor progression in patients treated with radiation: A monocentric retrospective study combining the MRI TRAMs technique and the (18F)-dopa PET/CT.

PURPOSE: Brain necrosis after radiotherapy is a challenging diagnosis, since it has similar radiological appearance on standard MRI to tumor progression. Consequences on treatment decisions can be important. We compare recent imaging techniques in order to adopt a reliable diagnostic protocol in doubtful situations. PATIENTS AND METHOD: This is a retrospective study comparing the performance of three imaging techniques after radiotherapy of brain metastasis: Perfusion-MRI, TRAMs technique and F-dopa PET-CT. The evolution of the treated metastasis volume was also analyzed by contouring all patients MRIs. All included patients were suspected of relapse and had the three exams once the volume of treated metastasis increased. RESULTS: The majority of our patients were treated by stereotactic radiotherapy. Suspicion of relapse was on average around 17months after treatment. Four cases of radionecrosis were diagnosed and six cases of real tumor progression. Neurological symptoms were less present in radionecrosis cases. All of our radionecrosis cases had relative cerebral blood volume below 1. F-dopa PET-CT succeeded to set the good diagnosis in eight cases, although we found one false positive and one false negative exam. The TRAMs technique failed in one case of false negative exam. CONCLUSIONS: Perfusion-MRI showed high performance in the diagnosis of radionecrosis, especially when calculating relative cerebral blood volume rate. The TRAMs technique showed interesting results and deserves application in daily routine combined with the perfusion-MRI. F-dopa CT might induce false results because of different metabolic uptake according to tumor type, medication and brain blood barrier leak.

Future prophylactic treatments in migraine: Beyond anti-CGRP monoclonal antibodies and gepants.

Migraine is ranked as a leading cause of years lived with disability among all neurological disorders. Therapies targeting the calcitonin gene-related peptide (CGRP) signaling pathway, including monoclonal antibodies against the receptor or ligand and small molecule CGRP receptor antagonists (gepants), are today approved for migraine prophylaxis with additional compounds expected to be introduced to the market soon. In this review, we consider other putative prophylactic migraine drugs in development, including compounds targeting G-protein coupled receptors, glutamate, ion channels, and neuromodulatory devices. Emergence of these new interventions could complement our current treatment armamentarium for migraine management.

VAV1 regulates experimental autoimmune arthritis and is associated with anti-CCP negative rheumatoid arthritis.

Rheumatoid arthritis (RA) patients can be stratified into two subgroups defined by the presence or absence of antibodies against citrullinated circular peptides (anti-CCP) with most of the genetic association found in anti-CCP positive RA. Here we addressed the role of VAV1, previously associated to multiple sclerosis (MS), in the pathogenesis of RA in experimental models and in a genetic association study. Experimental arthritis triggered by pristane or collagen type II was induced in DA rats and in the DA.BN-R25 congenic line that carries a polymorphism in Vav1. Difference in arthritis severity was observed only after immunization with pristane. In a case-control study, 34 SNPs from VAV1 locus were analyzed by Immunochip genotyping in 11475 RA patients (7573 anti-CCP positive and 3902 negative) and 15,870 controls in six cohorts of European Caucasians. A combination of the previous MS-associated haplotype and two additional SNPs was associated with anti-CCP negative RA (alleles G-G-A-A of rs682626-rs2546133-rs2617822-rs12979659, OR=1.13, P=1.27 × 10-5). The same markers also contributed to activity of RA at baseline with the strongest association in the anti-CCP negative group for the rs682626-rs12979659 G-A haplotype (ß=-0.283, P=0.0048). Our study suggests a role for VAV1 and T-cell signaling in the pathology of anti-CCP-negative RA.

The impacts of dental filling materials on RapidArc treatment planning and dose delivery: challenges and solution.

PURPOSE: The presence of high-density material in the oral cavity creates dose perturbation in both downstream and upstream directions at the surfaces of dental filling materials (DFM). In this study, the authors have investigated the effect of DFM on head and neck RapidArc treatment plans and delivery. Solutions are proposed to address (1) the issue of downstream dose perturbation, which might cause target under dosage, and (2) to reduce the upstream dose from DFM which may be the primary source of mucositis. In addition, an investigation of the clinical role of a custom-made plastic dental mold∕gutter (PDM) in sparing the oral mucosa and tongue reaction is outlined. METHODS: The influence of the dental filling artifacts on dose distribution was investigated using a geometrically well-defined head and neck intensity modulated radiation therapy (IMRT) verification phantom (PTW, Freiberg, Germany) with DFM inserts called amalgam, which contained 50% mercury, 25% silver, 14% tin, 8% copper, and 3% other trace metals. Three RapidArc plans were generated in the Varian Eclipse System to treat the oral cavity using the same computer tomography (CT) dataset, including (1) a raw CT image, (2) a streaking artifacts region, which was replaced with a mask of 10 HU, and (3) a 2 cm-thick 6000 HU virtual filter [a volume created in treatment planning system to compensate for beam attenuation, where the thickness of this virtual filter is based on the measured percent depth dose (PDD) data and Eclipse calculation]. The dose delivery for the three plans was verified using Gafchromic-EBT2 film measurements. The custom-made PDM technique to reduce backscatter dose was clinically tested on four head and neck cancer patients (T3, N1, M0) with DFM, two patients with PDM and the other two patients without PDM. The thickness calculation of the PDM toward the mucosa and tongue was purely based on the measured upstream dose. Patients' with oral mucosal reaction was clinically examined initially and weekly during the course of radiotherapy. RESULTS: For a RapidArc treatment technique, the backscatter dose from the DFM insert was measured to be 9.25±2.17 in the IMRT-verification-phantom. The measured backscatter upstream dose from DFM for a single-field was 22% higher than without the DFM, whereas the downstream dose was lower by 14%. The values of homogeneity index for the plans with and without the application of mask were 0.09 and 0.14, respectively. The calculated mean treatment planning volume (PTV) dose differed from the delivered dose by 13% and was reduced to 2% when using the mask and virtual filter together. A grade 3 mucosa reaction was observed in the control group after 22-24 fractions (44-48 Gy). In contrast, no grade 3 mucositis was observed in the patients wearing the PDM after 25-26 fractions (50-52 Gy). CONCLUSIONS: The backscatter from the DFM for a single, parallel-opposed fields, and RapidArc treatment technique was found significant. The application of mask in replacing streaking artifacts can be useful in improving dose homogeneity in the PTV. The use of a virtual filter around the teeth during the planning phase reduces the target underdosage issue in the phantom. Furthermore, a reduction in mucositis is observed in the head and neck patients with the use of PDM.

Genetic control of HgCl2-induced IgE and autoimmunity by a 117-kb interval on rat chromosome 9 through CD4 CD45RChigh T cells.

Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl(2)-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl(2)-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl(2) administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl(2)-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

SU-E-I-56: Threshold Effect of ASIR Before Which Image Improve and After Which Image Degrades.

PURPOSE: This study showed to what extent ASIR improves CT-image and to what extent it degrades it. METHODS: In our study we used GE HD750 CT-scanner, Siemens Sensation CT-scanner, Catphan, PTW-pin-ion- chamber, CTDI-phantom. We measured the CT-dose using the PTW-pinion-chamber and CTDI-phantom. Image-quality and noise were evaluated using catphan and GE water phantom. RESULTS: Image noise reduce as higher levels of ASIR are applied. A phantom scan showed that 50%ASIR with 50% lower-dose (10.8mGy) achieved the same image noise of standard FBP image with full dose 21.7mGy (noise∼5). To confirm that the two same-noise images retain same image-quality, two scans were compared; one with full dose 260mAs(21.7mGy) and the other one with 50% lower dose 130mAs(10.8mGy). The results showed that ASIR failed to retain the same quality. For high contrast resolution, 50%ASIR reduced the resolution of patterns = 71p/cm, however it improved the detectability of patterns = 61p/cm. ASIR has degraded the CNR of the low-contrast-objects of = 5HU (CNR of 1.4 at 260mAs STND to CNR of 1.08 at 130mAs ASIR), however it improved the CNR of the low-contrast-objects of = 10HU (CNR of 2.35 at 260mAs STND to CNR of 2.63 at 130mAs ASIR). ASIR degraded the edges and killed some of the small objects. This shows that ASIR has a critical point of improve/degrade. Also, ASIR can improve images for the same dose, but with high levels of ASIR (e.g. 100%ASIR), cause disapear of small low contrast objects (e.g. 2mm). CONCLUSIONS: People think that ASIR only improves image and reduces patient dose. Our study showed that ASIR has some drawbacks. There is a threshold before wich ASIR is positive and after which ASIR is negative. Recently only GE provide ASIR in the market but our study showed that other CTs such as Siemens can do similar performance like ASIR.

Role of IFNgamma in allograft tolerance mediated by CD4+CD25+ regulatory T cells by induction of IDO in endothelial cells.

Regulatory T cells have been described to specifically accumulate at the site of regulation together with effector T cells and antigen-presenting cells, establishing a state of local immune privilege. However the mechanisms of this interplay remain to be defined. We previously demonstrated, in a fully MHC mismatched rat cardiac allograft combination, that a short-term treatment with a deoxyspergualine analogue, LF15-0195, induces long-term allograft tolerance with a specific expansion of regulatory CD4+CD25+T cells that accumulate within the graft. In this study, we show that following transfer of regulatory CD4+T cells to a secondary irradiated recipient, regulatory CD25+Foxp3+ and CD25+Foxp3(-) CD4+T cells accumulate at the graft site and induce graft endothelial cell expression of Indoleamine 2, 3-dioxygenase (IDO) by an IFNgamma-dependent mechanism. Moreover, in vivo transfer of tolerance can be abrogated by blocking IFNgamma or IDO, and anti-IFNgamma reduces the survival/expansion of alloantigen-induced regulatory Foxp3+CD4+T cells. Together, our results demonstrate interrelated mechanisms between regulatory CD4+CD25+T cells and the graft endothelial cells in this local immune privilege, and a key role for IFNgamma and IDO in this process.

In the rat, citrullinated autologous fibrinogen is immunogenic but the induced autoimmune response is not arthritogenic.

Conversion of arginyl to citrullyl residues (citrullination) is essential for the formation of the epitopes recognized by rheumatoid arthritis (RA)-associated autoantibodies to citrullinated proteins (ACPA). ACPA are secreted by plasma cells of the rheumatoid synovial tissue where their major target, citrullinated fibrin, is abundant. Although numerous arguments suggest that ACPA play an important role in RA, their pathological relevance remains to be established. In the present study, we assessed the immunogenicity and arthritogenicity of complete Freund's adjuvant-emulsified autologous citrullinated (C-rFBG) or non-citrullinated (NC-rFBG) fibrinogen in Lewis (LEW) and Brown-Norway rats, which exhibit drastic differences in their susceptibility to induced autoimmune diseases. NC-rFBG induced no antibody response. In contrast, a single injection of C-rFBG induced an IgG response directed mainly to citrullinated determinants of rFBG. However, all rat strains remained devoid of clinical and histological signs of arthritis up to 3 months after C-rFBG inoculation. Next, in LEW rats, we tested whether autoimmunity to C-rFBG could aggravate acute ankle arthritis triggered by intra-articular injection of incomplete Freund's adjuvant (IFA). However, such arthritis evolved identically in the presence or absence of anti-C-rFBG autoantibodies. However, IFA-injected joints were devoid of citrullinated fibrin deposits. Therefore, citrullination allows breakdown of immunological tolerance but the autoimmune response developed is not spontaneously arthritogenic. Whether or not it can aggravate arthritis with citrullinated fibrin deposits remains to be evaluated.

Chronomodulation of topotecan or X-radiation treatment increases treatment efficacy without enhancing acute toxicity.

PURPOSE: Topotecan (TPT), a camptothecin analog, is currently used to treat human ovarian and small-cell lung cancer and is in clinical trials for other tumor sites. However, it is unknown whether chronomodulation of TPT treatment is beneficial. We examined the effects of administering TPT or X-radiation (XR) alone at different times of the day or night. METHODS: We treated mice bearing human colorectal tumor xenografts at four different times representing the early rest period (9 am or 3 HALO [hours after light onset]), late rest period (3 pm or 9 HALO), early active period (9 pm or 15 HALO), and late active period (3 am or 21 HALO) of the mice. We gave either TPT (12 mg/kg, injected i.p.) or XR (4 Gy, directed to the tumor) twice weekly on Days 0, 4, 7, 10 within 2 weeks. RESULTS: Treatment with either TPT or XR at 3 am demonstrated the greatest efficacy (measured by a tumor regrowth assay) without significantly increasing acute toxicity (assessed by a decrease in leukocyte counts or body weight). Conversely, treatment at 3 pm, in particular, showed increased toxicity without any enhanced efficacy. CONCLUSIONS: Our study provided the first evidence that chronomodulation of TPT treatments, consistent with the findings of other camptothecin analogs, is potentially clinically beneficial. Additionally, our findings suggest that chronomodulation of fractionated XR treatments is also potentially clinically beneficial.

Analysis of CD4+CD25+ cell population in the thymus from myasthenia gravis patients.

The present study is aimed at exploring the regulatory CD4(+)CD25(+) T cells in the thymus from myasthenia gravis (MG) patients. In early-onset MG, the thymus is hyperplastic and contains autoreactive activated T cells. Preliminary studies indicate that these CD4(+)CD25(+) cells include activated autoreactive T cells. Studies to characterize the phenotype and suppressive capacity of these cells will be discussed.

Gold is a T cell polyclonal activator in BN and LEW rats but favors IL-4 expression only in autoimmune prone BN rats.

Gold salts are beneficial in the treatment of rheumatoid arthritis but may induce immune-mediated disorders in predisposed patients. Gold salts induce Th2-dependent autoimmunity in Brown-Norway (BN) rats but not in Lewis (LEW) rats. The aim of this study was to define molecular targets of gold salts and to approach why LEW rats are resistant. Gold salts act on early steps of transduction in T cells from BN and LEW rats since they trigger tyrosine phosphorylation of numerous proteins including p56(lck) and a calcium signal which results in IL-4 and IFN-gamma expression by BN and LEW T cells. However, the IL-4 response was favored in BN spleen cells in vitro and in vivo. IFN-gamma, produced in part by CD8(+) cells, contributes to the resistance of LEW rats since gold salt-injected LEW rats receiving anti-CD8 or anti-IFN-gamma mAb displayed the parameters characteristics of gold salt-induced Th2 autoimmunity although to a lesser extent than in BN rats. Gold salts transduce a signal in BN and LEW spleen cells resulting in IL-4 and IFN-gamma gene transcription with a preferential IL-4 response in BN rats, a Th2-prone strain, while IFN-gamma contributes to the resistance of LEW rats.

Induction of autoimmunity through bystander effects. Lessons from immunological disorders induced by heavy metals.

Autoreactive T cells exist in healthy individuals and represent a potential reservoir of pathogenic effectors which, when stimulated by microbial adjuvants, could trigger an autoimmune disease. Experimental studies have indicated that xenobiotics, well defined from a chemical point of view, could promote the differentiation of autoreactive T cells towards a pathogenic pathway. It is therefore theoretically possible that compounds present in vaccines such as thiomersal or aluminium hydroxyde can trigger autoimmune reactions through bystander effects. Mercury and gold in rodents can induce immunological disorders with autoimmune reactions. In vitro, both activate signal transduction pathways that result in the expression of cytokines, particularly of IL-4 and IFNgamma. In a suitable microenvironment heavy metals could therefore favour the activation of autoreactive T cells. In that respect, genetic background is of major importance. Genome-wide searches in the rat have shown that overlapping chromosomal regions control the immunological disorders induced by gold salt treatment, the development of experimental autoimmune encephalomyelitis and the CD45RC(high)/CD45RC(low)CD4(+)T cells balance. The identification and functional characterization of genes controlling these phenotypes may shed light on key regulatory mechanisms of immune responses. This should help to improve efficacy and safety of vaccines.

Essential role of TGF-beta in the natural resistance to experimental allergic encephalomyelitis in rats.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease induced in susceptible rat strains by a single immunization with myelin basic protein (MBP). The Lewis (LEW) strain is susceptible to disease induction while the Brown Norway (BN) strain is resistant. This resistance involves non-MHC genes since congenic BN-1L rats, with LEW MHC on a BN-derived background, are also resistant. In the present study we show that, upon immunization with MBP, the non-MHC-encoded resistance to develop clinical EAE in BN-1L rats is associated with a decreased production of IFN-gamma. This may be due to a difference between LEW and BN-1L rats in their ability to produce regulatory cytokines such as IL-4, IL-10 and TGF-beta. In comparison to LEW rats, immune lymph node cells from BN-1L rats express an increased amount of IL-4 mRNA but produce less IL-10. Furthermore, the sera from BN-1L rats contain higher amounts of active TGF-beta1. Therefore, we have investigated the involvement of IL-4 and TGF-beta in the resistance of BN-1L rats to develop EAE using neutralizing mAb. Neutralization of TGF-beta, but not IL-4, renders BN-1L rats susceptible to clinical EAE without affecting the proliferation or the cytokine repertoire of immune lymph node cells. With respect to the origin of the endogenous TGF-beta production, we excluded the involvement of CD8 T cells and discuss a possible role of platelets and of CD4 T cells exhibiting the CD45RC(low) phenotype.

The balance between CD45RChigh and CD45RClow CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RC(high)/CD45RC(low) CD4 T cell ratios. The CD45RC(high) subpopulation predominates in LEW rats, and the CD45RC(low) subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RC(low) CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC(high)/CD45RC(low) CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW x BN) F(2) intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (approximately 17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (approximately 28 cM; LOD score 3.1) and 20 (approximately 40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RC(high)/CD45RC(low) Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

Mercuric chloride-induced autoimmunity.

This unit describes methods for inducing autoimmune disease in Brown Norway rats through HgCl(2) injections as well for assessing parameters that characterize the disease by serum IgE concentration assays, anti-laminin antibody measurement, and renal immunofluorescence studies to detect autoantibodies. Also covered are disease induction using autoreactive CD4(+) T(H)2 anti-self MHC class II molecules and preparation of T cell lines. IL-4 is produced very early after the first HgCl(2) injection (beginning at day 3, peaking at day 14, and continuing up to day 30). Thus, IL-4 mRNA expression may be detected in spleen and lymph nodes from HgCl(2)-injected BN rats. The fact that HgCl(2) induces in vitro mRNA IL-4 gene expression in normal BN T cells but not in LEW T cells is probably crucial to susceptibility to the development of autoimmunity in the sense that it may condition the development of autoreactive T cells into pathogenic T(H)2 cells; a test for this condition is therefore also included.