Advancing Biomarker Development Through Convergent Engagement: Summary Report of the 2nd International Danube Symposium on Biomarker Development,Molecular Imaging and Applied Diagnostics; March 14–16, 2018; Vienna,Austria

Molecular Imaging and Biology - Here, we report on the outcome of the 2nd International Danube Symposium on advanced biomarker development that was held in Vienna, Austria, in early 2018. During...     

Imaging SERT Availability in a Rat Model of L-DOPA-Induced Dyskinesia

Purpose

The development of L-DOPA-induced dyskinesia (LID) is one of the most severe side effects of chronic L-DOPA treatment in Parkinson’s disease patients. [¹¹C]DASB positron emission tomography (PET) provides a prominent tool to visualize and quantify serotonin transporter (SERT) pathology in vivo in patients and in animal models. To evaluate the effect of chronic L-DOPA treatment on SERT availability in an animal model of LID, we performed a longitudinal PET study.

Procedures

Rats received a unilateral 6-hydroxydopamine (6-OHDA) lesion, and striatal and extrastriatal SERT expression levels were studied with [¹¹C]DASB, a marker of SERT availability, before and after daily treatment with L-DOPA. Dyskinesias were evaluated at different time points over a period of 21 days.

Results

[¹¹C]DASB binding was found to be decreased after 6-OHDA lesions in the striatum, cortex, and hippocampus 5 weeks after 6-OHDA injection in the lesioned hemisphere of the rat brain. Chronic L-DOPA priming resulted in a relative preservation of SERT availability in the lesioned and healthy hemisphere compared to baseline measurements.

Conclusions

Our longitudinal PET data support a preservation of SERT availability after the induction of L-DOPA-induced dyskinesia, which is in line with previous reports in dyskinetic PD patients.

     

Immunohistochemistry for c-myc and bcl-2 overexpression improves risk stratification in primary central nervous system lymphoma

Overexpression of bcl-2 and c-myc are defining features of double-expressor-lymphoma (DEL) but may also occur separately in patients with primary central nervous system lymphoma (PCNSL). Despite all progress in optimizing treatment regimen, there is lack of sufficient risk stratification models. Here, we first describe the relationship between DEL biology, the National Comprehensive Cancer Network International Prognostic Index (NCCN-IPI), treatment response, disease progression, and mortality in PCNSL. In this study, we determined c-myc and bcl-2 status immunohistochemically in samples of 48 patients with newly diagnosed PCNSL and followed these patients for a median interval of 6.2 years. Twelve, 18, and 17 patients harbored none, one, or both DEL features. Corresponding overall response rates after first-line therapy were strongly associated with DEL biology (100%, 42%, and 44% in patients with 0, 1, or 2 DEL features). Patients with one or both DEL features had a 5-fold and 13-fold higher 5-year risk of progression and/or death than patients without DEL features. These associations prevailed after adjusting for the NCCN-IPI. DEL improved the discriminatory capability of the NCCN-IPI (P = .0001). Furthermore, we could show that addition of DEL biology to the NCCN-IPI significantly improved the score's discriminatory potential both toward progression-free survival (increase in Harell's c = 0.15, P = .005) and overall survival (increase in Harell's c = 0.11, P = .029). In conclusion, DEL biology is a strong and simple-to-use predictor of adverse outcome in PCNSL. Addition of DEL to the NCCN-IPI improves its prognostic potential. Disease progression from PCNSL harboring both DEL features is invariably fatal. This defines a novel PCNSL patient subset with a great unmet need for improved therapy.     

Characterization of drug-specific T cells in lamotrigine hypersensitivity

BACKGROUND: Lamotrigine is associated with hypersensitivity reactions, which are most commonly characterized by skin rash. An immune etiology has been postulated, though the nature of this is unclear. OBJECTIVES: The aim of this study was to characterize the role of T cells in lamotrigine hypersensitivity. METHODS: A lymphocyte transformation test was performed on 4 hypersensitive patients. Lymphocytes from 3 of 4 lamotrigine-hypersensitive patients proliferated when stimulated with lamotrigine. T-cell clones were generated from one patient to further characterize the nature of the T-cell involvement. Cells were characterized in terms of their phenotype, functionality, and mechanisms of antigen presentation and cytotoxicity. RESULTS: Of the 44 drug-specific T-cell clones generated, most were CD4(+) with occasional CD8(+) cells. All clones expressed the alphabeta T-cell receptor; several Vbeta 5.1(+) or 9(+) T-cell clones were generated. All clones also expressed the skin-homing receptor cutaneous lymphocyte antigen. Lamotrigine-stimulated T cells were cytotoxic and secreted perforin, IFN-gamma, IL-5, and macrophage inflammatory protein 1alpha, macrophage inflammatory protein 1beta, RANTES, and I-309. Lamotrigine was present on HLA-DR and HLA-DQ by antigen-presenting cells in the absence of drug metabolism and processing. The T-cell receptor of certain clones could accommodate analogs of lamotrigine, but no cross-reactivity was seen with other anticonvulsants. CONCLUSIONS: Our data provide evidence that T cells are involved in the pathogenesis of some lamotrigine-hypersensitivity reactions. The identification of drug-specific cells that express cutaneous lymphocyte antigen and type 1 cytokines after T-cell receptor activation is consistent with the clinical symptoms. Furthermore, identification of large numbers of Vbeta 5.1(+) T cells suggests that polymorphisms within T-cell receptor genes might act as determinants of susceptibility.     

富血小板血浆对人骨髓间充质干细胞成骨分化的抑制效应

目的:一些理论质疑富血小板血浆对骨前体细胞成骨分化的作用,本实验拟验证富血小板血浆对体外培养的人骨髓间充质干细胞成骨分化的抑制效应。方法:实验于2005-05/11在南方医科大学组织工程试验室(省级)完成。①实验方法:抽取6名健康志愿者髂前上棘骨髓5mL进行体外细胞培养扩增,静脉血10mL以二次离心法制得富血小板血浆。诱导骨髓间充质干细胞时富血小板血浆与骨髓间充质干细胞均来自同一个体。②碱性磷酸酶染色:取第4代骨髓间充质干细胞,分为两组:富血小板血浆组加入富血小板血浆使终浓度为100g/L,单纯血清培养组仅加入等量胎牛血清。培养后第7天进行碱性磷酸酶染色,阳性细胞为胞质中呈现黑色颗粒或块状沉淀。③矿化结节染色:取第4代骨髓间充质干细胞,分组同上。培养后第19天以0.1%茜素红-TrisHcl(pH8.3)37℃下放置30min,矿盐沉积染色阳性为红色。④Cbfa1基因表达:取第4代骨髓间充质干细胞,分组同上。培养后第3,7,12,16天RT-PCR法检测骨髓间充质干细胞Cbfa1基因的表达。⑤形态学观察:实验过程中使用相差显微镜观察各组细胞生长情况及形态学变化。结果:①骨髓间充质干细胞碱性磷酸酶染色结果:培养后第7天,富血小板血浆组碱性磷酸酶阳性细胞数量较单纯血清培养组明显减少,且阳性细胞内灰黑色颗粒也明显减少,为弱阳性。②骨髓间充质干细胞矿化结节染色结果:培养后第19天,单纯血清培养组可见细胞表面有较多的矿盐沉积,但未形成明显的矿化结节。富血小板血浆组细胞表面只有稀少的矿盐沉积。③骨髓间充质干细胞cbfa1mRNA的表达:培养后第3,7,12,16天,随着培养时间的延长单纯血清培养组与富血小板血浆组cbfa1基因表达量均逐渐增高,同一时间点两组间cbfa1基因的表达基本相似。④骨髓间充质干细胞形态学变化:富血小板血浆组骨髓间充质干细胞增殖旺盛,细胞达到单层汇合的时间较单纯血清培养组明显缩短。单纯血清培养组细胞在完全汇合后开始出现聚合现象(14～16d),但趋向性不明显,未完全形成团簇;富血小板血浆组细胞在完全汇合后未出现聚合现象,细胞密集生长。培养初期两组细胞以梭形为主,多角形细胞较少,培养至14～16d单纯血清培养组多角形细胞较富血小板血浆组增多。结论:富血小板血浆可抑制人骨髓间充质干细胞碱性磷酸酶的分泌与矿盐沉积,对人骨髓间充质干细胞成骨分化的直接效应是抑制其分化。     

Molecular genetic rearrangements distinguish pre- and post-bone marrow transplantation lymphoproliferative processes

Chronic myelocytic leukemia (CML) may display a lymphoproliferative phase (lymphoid blast crisis) that is generally of B cell phenotype. Since lymphoproliferative disorders may occur following bone marrow transplantation (BMT), it may be difficult to distinguish posttransplant relapse of CML lymphoid blast crisis from de novo lymphoproliferation. Lymphoid blast crisis cells from a patient with CML displayed immunoglobulin heavy chain gene (C mu) rearrangement before BMT. Following BMT the patient developed a lymphoproliferative disorder involving multiple organs. Clonal rearrangement of C mu was demonstrated in several involved tissues. The rearranged C mu restriction fragment was distinct from that displayed before BMT. Additionally, rearrangement of the breakpoint cluster region (bcr) was demonstrated in the pretransplant blast crisis sample, but not in the posttransplant lymphoproliferation samples, thus confirming that these lymphoproliferative disorders were distinct. Molecular genetic techniques offer powerful diagnostic tools for monitoring the course of patients with CML undergoing BMT.     

The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.S358L mutation in TMEM43

To determine the phenotype and natural history of a founder genetic subtype of autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) caused by a p.S358L mutation in TMEM43. The age of onset of cardiac symptoms, clinical events and test abnormalities were studied in 412 subjects (258 affected and 154 unaffected), all of which occurred in affected males significantly earlier and more often than unaffected males. Affected males were hospitalized four times more often than affected females (p ≤ 0.0001) and died younger (p ≤ 0.001). The temporal sequence from symptoms onset to death was prolonged in affected females by 1–2 decades. The most prevalent electrocardiogram (ECG) manifestation was poor R wave progression (PRWP), with affected males twice as likely to develop PRWP as affected females (p ≤ 0.05). Left ventricular enlargement (LVE) occurred in 43% of affected subjects, with 11% fulfilling criteria for dilated cardiomyopathy. Ventricular ectopy on Holter monitor was common and occurred early: the most diagnostically useful clinical test. No symptom or test could rule out diagnosis. This ARVC subtype is a sex‐influenced lethal arrhythmogenic cardiomyopathy, with a unique ECG finding, LV dilatation, heart failure and early death, where molecular pre‐symptomatic diagnosis has the greatest clinical utility.     

Lung tumor-associated derepressed alloantigen coded for by the K region of the H-2 major histocompatibility complex

Transplacental induction of lung tumors in C3HfeB/HeN (C3Hf) strain mice can be readily achieved with the carcinogen 1-ethyl-1-nitrosourea. Several of these tumors express, as a tumor-associated transplantation antigen (TATA), a normal tissue alloantigen present in strain A and C3H/HeN (C3H) mice. In the present study it was shown that the tumor-associated alloantigen on the C3Hf-derived lung tumor 85 was present in all mice of H-2(a) and H-2(k) haplotypes tested and in CBA (532) strain mice (H-2(ka) haplotype). Studies using congenic-resistant and recombinant strains of mice indicated that the genetic locus controlling the expression of this antigen was either within or to the left of the H-2K region of the major histocompatibility complex (MHC). Thus the antigen was expressed in B10.A (4R) mice (kkbbbb MHC haplotype) but not in B10 (bbbbbb) or B10.AQR mice (qkkddd). The antigen was expressed in all tissues tested of C3H and A strain mice. It was not detected on any tissue tested including embryo tissue of C3Hf mice or mice of MHC haplotype other than H-2(k) or H-2(a). Because C3Hf strain mice were originally derived from C3H strain mice (H-2(k)), the MHC haplotype of C3Hf mice has been provisionally designated H-2(kb). The finding of a tumor-associated change in the expression of a H-2K region-coded antigen is consistent with the concept that MHC-coded antigens may act as targets for immunological surveillance of tumors.