Exploiting Exosomes for Differential Diagnosis of Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by a clonal plasma cell proliferation. Usually, all MM are preceded by an asymptomatic premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS). Differential diagnosis requires the evidence of end-organ damage, but recently new biomarkers are emerging to help clinicians to distinguish MM from the premalignant phase. Circulating exosomes in serum seem to be a powerful tool to be analyzed for liquid biopsy, and in this chapter, we show that MM and MGUS exosomes are different in concentration, biological activity, and biochemical markers. These differences seem to be related to the free light chains (FLCs) associated with exosomes and their propathogenic properties. The cellular processing FLC-decorated exosomes and their ability to activate proinflammatory mechanisms are different in MM and MGUS patients. These elements can be evaluated to create an innovative multiparameter panel to monitor MGUS to MM switching

Effects of glossy privet's invasion on the water dynamics of the Argentinean Yungas forest

Las Yungas argentinas (bosques de montaña) tienen alta biodiversidad y un rol clave en la regulación hídrica. El ligustro (Ligustrum lucidum) es una de las especies exóticas que dominan sus bosques secundarios. Debido a sus altas tasas de crecimiento y follaje verde todo el año, se planteó la hipótesis de que la disponibilidad de agua en el suelo se reduciría en bosques de ligustro en comparación con bosques nativos, principalmente durante la época seca (invierno-primavera). Se trabajó en seis pares de parcelas de bosques nativos y bosques secundarios monodominantes de ligustro en la Sierra San Javier, Tucumán, Argentina. Se registró la humedad edáfica mediante mediciones de contenido volumétrico y potencial hídrico y se estimó la productividad de los distintos tipos de bosque mediante el índice de vegetación de diferencia normalizada (NDVI). Durante la época seca de 2011 y 2012, los suelos en ligustrales presentaron un contenido volumétrico de agua 32,5 % y 24 % y un potencial hídrico en el suelo ocho y cuatro veces menor. Se observaron mayores valores de NDVI (8,5 %) en bosques de ligustro que en bosques nativos, principalmente en las selvas localizadas a menor altura y con mayor déficit hídrico inverno-primaveral. Los resultados sugieren que el bosque de ligustro estaría consumiendo mayor cantidad de agua, sobre todo en la época seca, debido a una actividad fenológica más prolongada respecto al bosque nativo y que su invasión en las Yungas podría alterar el funcionamiento hídrico de este ecosistema.Argentinean Yungas (subtropical montane forests) have high biodiversity and play a key role in regional watershed regulation. Ligustrum lucidum (glossy privet), native of China, is one of the dominant exotic tree species in secondary forests of this ecoregion. Due to its high growth rates and green foliage throughout the year -in contrast to native forests which are partially deciduous- the hypothesis is that water availability is reduced in glossy privet forests, especially during dry season (winter-spring). Six pairs of plots both in native and secondary glossy privet dominated forests in Sierra San Javier, Tucumán, Argentina were sampled. Soil moisture, by measuring both volumetric water content and water potential, and estimated productivity of different types of forests, through normalized difference vegetation index (NDVI derived from satellite imagery), were monitored. During 2011 - 2012 dry seasons, soil volumetric water content in glossy privet forests was 32.5 % and 24 % lower than in native forests; while soil water potential was respectively eight and four times lower. In glossy privet forests NDVI values were 8.5 % higher than in native forests; particularly in lower altitude forests and those with a higher winter-spring water deficit. These results suggest that the glossy privet forest would be consuming more water, especially during the dry season, due to more prolonged phenology activity compared to native forests. In consequence, glossy privet invasion in Argentinean Yungas could alter water functioning of this ecosystem.Fil: Zamora Nasca, Lucia Belén. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo. Instituto de Ecología Regional; ArgentinaFil: Montti, Lia Fernanda. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo. Instituto de Ecología Regional; Argentina. Centro de Investigaciones del Bosque Atlantico; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Grau, Hector Ricardo. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo. Instituto de Ecología Regional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Paolini, Leonardo. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales E Instituto Miguel Lillo. Instituto de Ecología Regional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

C-src Enriched Serum Microvesicles Are Generated in Malignant Plasma Cell Dyscrasia

Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM). MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC) are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicles and exosomes production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS) patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia

A Canine Gait Analysis Protocol for Back Movement Assessment in German Shepherd Dogs

Objective-To design and test a motion analysis protocol for the gait analysis of adult German Shepherd (GS) dogs with a focus in the analyses of their back movements. Animals-Eight clinically healthy adult large-sized GS dogs (age, 4 +/- 1.3 years; weight, 38.8 +/- 4.2 kg). Procedures-A six-camera stereo-photogrammetric system and two force platforms were used for data acquisition. Experimental acquisition sessions consisted of static and gait trials. During gait trials, each dog walked along a 6 m long walkway at self-selected speed and a total of six gait cycles were recorded. Results-Grand mean and standard deviation of ground reaction forces of fore and hind limbs are reported. Spatial-temporal parameters averaged over gait cycles and subjects, their mean, standard deviation and coefficient of variance are analyzed. Joint kinematics for the hip, stifle and tarsal joints and their average range of motion (ROM) values, and their 95% Confidence Interval (CI) values of kinematics curves are reported. Conclusions and Clinical Relevance-This study provides normative data of healthy GS dogs to form a preliminary basis in the analysis of the spatial-temporal parameters, kinematics and kinetics during quadrupedal stance posture and gait. Also, a new back movement protocol enabling a multi-segment back model is provided. Results show that the proposed gait analysis protocol may become a useful and objective tool for the evaluation of canine treatment with special focus on the back movement

The prognostic value of 18F-FDG PET-CT in the management of Hodgkin’s lymphoma: preliminary results of a prospective study

BACKGROUND: To date, Hodgkin’s lymphoma (HL) patients have achieved long-term survival of more than 80%. Unfortunately, longer follow-up has shown serious adverse effects of the treatments used. For this reason, therapeutic strategies are becoming more tailored to the individual patient´s prognosis. Pre-treatment risk factors for early-stage and advanced-stage HL are well known indicators of prognosis. Recently, early interim 18F-FDG PET has been shown as a strong and independent predictor of progression-free survival in HL. Our aim was to assess response to therapy by repeating 18F-FDG-PET/CT after four and six chemotherapy cycles. MATERIAL AND METHODS: We evaluated 21 consecutive patients affected by (HL) and presenting for assessment over a period of three years. All patients underwent initial staging with 18F-FDG-PET/CT along with standard staging procedures. We tailored an individual treatment plan dependent on pre-treatment risk factors and initial 18F-FDG-PET/CT. With the aim of the best definition of response to treatment, we repeated 18F-FDG-PET/CT after two (FDG-PET 2), four (FDG-PET 4) and six (FDG-PET 6) chemotherapy cycles. Chemotherapy was typically given for four cycles in early disease stages and was prolonged to six to eight cycles in advanced disease stages, depending on PET findings. RESULTS: Our results showed a strong negative predictive value in detecting responders in early stage HL and a positive predictive value in advanced-stage patients. Clinical stage, extra-nodal sites and the positivity of the 18F-FDG-PET/CT performed during chemotherapy were also noted as strong determinants of response to treatment. Moreover, in our series the 18F-FDG-PET/CT data obtained after only two chemotherapy cycles (FDG-PET 2) were the same of those obtained after FDGPET 4 and FDG-PET 6 controls. CONCLUSION: The preliminary data of the present study confirm those of previous published studies about the negative predictive value of 18F-FDG-PET/CT performed after four and six chemotherapy cycles, which contributed to the decision to stop treatment and to avoid radiotherapy in HL patients. Nonetheless, our preliminary data seems to suggest that only the 18F-FDG-PET/CT performed after two cycles of chemotherapy (FDG-PET 2) is able to provide the same prognostic information of the FDG-PET 4 and FDG-PET 6 earlier

Comparison of Hevylite™ IgA and IgG assay with conventional techniques for the diagnosis and follow-up of plasma cell dyscrasia

Background: Heavy/light chain assay allows the characterization and quantification of immunoglobulin light chains bound to heavy chains for each Ig’k and Ig’ immunoglobulin class, discriminating between the involved/uninvolved isotypes in plasma cell dyscrasia. The Ig’k/Ig’ ratio (heavy/light chain ratio) enables to monitor the trend of monoclonal component during therapy and disease evolution. Objective: In this study, we evaluate the impact of the heavy/light chain assay in monitoring multiple myeloma patients in comparison with conventional techniques. Methods: Serum samples of 28 patients with IgG or IgA monoclonal component were collected for a mean of 109 days and analyzed. The heavy/light chain assay was compared with classical immunoglobulin quantification (Ig’Tot), serum immunofixation electrophoresis, serum protein electrophoresis, and serum-free light chains quantification. Serum samples from 30 healthy patients were used as control (polyclonal). Results: Heavy/light chain ratio and serum immunofixation electrophoresis were comparable in 86% of the cases, and free light chain ratio and heavy/light chain ratio in 71.8%. Heavy/light chain assay and Ig’Tot measurements showed a concentration-dependent agreement in monoclonal patients. The heavy/light chain assay was able to quantify the monoclonal component migrating in SPE b region: this occurred in 10% of our IgG and 50% of our IgA patients. Conclusions: The concordance scores indicate that heavy/light chain and Ig’Tot assays show differences at high monoclonal component values. The heavy/light chain ratio, serum immunofixation electrophoresis, and free light chain ratio showed partial concordance. Our study confirmed that, in the context of heavy/light chain assay, heavy/light chain Ig’k and Ig’ absolute values and heavy/light chain ratio are both important tools to monitor the presence of monoclonal component that are difficult to be identified in SPE

Comparison of separation methods for immunomodulatory extracellular vesicles from helminths

Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone

Change of Accent as an Atypical Onset of non Fluent Primary Progressive Aphasia

Language disorders can be the first symptom of many neurodegenerative diseases, including Alzheimer's disease (AD) and primary progressive aphasia (PPA). The main variants of PPA are: the non-fluent/agrammatic variant, the semantic variant and the logopenic variant

Immunoglobulin free light chains and GAGs mediate multiple myeloma extracellular vesicles uptake and secondary NfkB nuclear traslocation

Multiplemyeloma(MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasmacells in the bone marrow. Monoclonal plasmacells often secrete high amounts of immunoglobulin free light chains(FLCs)that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles(EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS)serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs withanti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished afteranti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure.The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular redistribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches

Immunoglobulin free light chains and GAGs mediate multiple myeloma extracellular vesicles uptake and secondary NfkB nuclear traslocation

Multiplemyeloma(MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasmacells in the bone marrow. Monoclonal plasmacells often secrete high amounts of immunoglobulin free light chains(FLCs)that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles(EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS)serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs withanti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished afteranti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure.The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular redistribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches