Introduction: Previous studies have shown that cPC can be detected in PB by conventional flow cytometry (FC) in around 70% of multiple myeloma (MM) and 37% of monoclonal gammopathy of undetermined significance (MGUS) patients at diagnosis. Its presence in MGUS has been associated with a higher risk of malignant transformation. We here investigated the utility and sensitivity of the EuroFlow-IMF NGF-MM minimal residual disease (MRD) approach for detecting circulating cPC in PB of patients with PCN. Methods: A total of 137 samples (including 71 PB and 66 bone marrow -BM- paired samples) from 71 newly-diagnosed PCN patients (37 MGUS; 21 MM; 5 SMM and 8 solitary plasmacytomas -SP-), plus 6 PB samples from healthy controls, were studied. Samples were processed following the EuroFlow Bulk Lysis Standard Operating Protocol (SOP) and stained with the EuroFlow-IMF MM MRD panel (Tube 1:CD138BV421/CD27BV510/CD38FITC/CD56PE/CD45PerCP-Cy5.5/CD19PE-Cy7/CD117APC/CD81APC-C750, and; Tube 2: identical to Tube 1 except for CyKappaAPC/CyLambdaAPC-C750). A median of 10.6 x106 events (range: 1.7 x106 - 15.7x106) were measured for PB samples using a FACSCanto II (BD Biosciences, San Jose, USA) instrument. Data were analyzed using the Infinicyt software (version 1.8.0RC6; Cytognos SL, Salamanca, Spain). Risk stratification of MGUS patients was established by the Mayo Clinic index. ROC analysis was used to define a cut-off to distinguish between MM and MGUS cases according to the percentage and absolute number of circulating PB cPC. Results: Overall, cPC were detected in the PB of all MM and SMM cases studied (100%) and more than half of MGUS patients (60%; p=0.005), while constantly absent in the eight patients with SP. Upon classifying MGUS patients according to the Mayo Clinic Index (n=32), positive PB samples were found in 25%, 62% and 73% of cases with scores of 0, 1 and 2, respectively. Median (range) percentage and absolute cPC numbers (per µL) were of 13 to 16 and 10 to 200 times lower (p<0.0001) in MGUS -0.0002% (<0.0001%-0.05450%) and 0.011 cPC/µL (range: <0.0001 cPC/µL -3.2 cPC/µL)- than in SMM -0.0026% (0.00020%-0.23%) and 0.14 cPC/µL (range: 0.022 cPC/µL - 14.58 cPC/µL) and MM -0.0033% (0.00064%-1.05%) and 2.01 cPC/µL (range: 0.043 cPC/µL -103.8 cPC/µL)-, respectively. Interestingly, a clear relationship was found between the presence of circulating cPC in PB of both MGUS, SMM and MM cases, and BM involvement by >60% of cPCs within the PC BM compartment (R2 = 0.75; n=66). The cut-off obtained to distinguish between MM and MGUS cases according to the percentage and absolute number of cPCs circulating in PB was of 0.0009% and 0.055 cPC/µL with a sensitivity of 93% and 86%, and a specificity of 75% and 75% for relative and absolute numbers, respectively. Conclusions: The EuroFlow-IMF NGF-MM MRD panel and approach are well-suited for high sensitive detection of circulating cPC in the PB of virtually every newly-diagnosed MM and SMM patient and the majority of MGUS cases, particularly among MGUS at higher risk of malignant progression; interestingly in both patients groups, the presence of PB involvement and its levels were closely associated with the degree of involvement of the BM PC compartment by cPC. * Both authors have contributed similarly to this work and they should both be considered as first author. Disclosures Paiva: EngMab AG: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Millenium: Consultancy; Binding Site: Consultancy; Sanofi: Consultancy; BD Bioscience: Consultancy; Onyx: Consultancy. Puig:Janssen: Consultancy; The Binding Site: Consultancy. Mateos:Celgene: Consultancy, Honoraria; Takeda: Consultancy; Onyx: Consultancy; Janssen-Cilag: Consultancy, Honoraria. Durie:Celgene: Consultancy; Onyx: Consultancy; Takeda: Consultancy; Johnson and Johnson: Consultancy. van Dongen:BD Biosciences (cont'd): Other: Laboratory Services in the field of technical validation of EuroFlow-OneFlow antibody tubes in dried format. The Laboratory Services are provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL; Cytognos: Patents & Royalties: Licensing of IP on Infinicyt software, Patents on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies, Patents on MRD diagnostics, and Patents on PID diagnostics. ; Cytognos (continued): Patents & Royalties: Royalty income for EuroFlow Consortium. The Infinicyt software is provided to all EuroFlow members free-of-charge.Licensing of Patent on detection of IgE+ B-cells in allergic diseases. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL; InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.. Royalty income for EuroClonality-BIOMED-2 Consortium ; Immunostep: Patents & Royalties: Licensing of IP and Patents on immunobead-based dection of fusion proteins in acute leukemias and other tumors. Royalties for Dept. of Immunology, Erasmus MC and for EuroFlow Consortium ; BD Biosciences: Other: Educational Services: Educational Lectures and Educational Workshops (+ related travelling costs). The lectures and workshops fully focus on the scientific achievements of the EuroFlow Consortium (No advertisement of products of BD Biosciences). , Patents & Royalties: Licensing of IP and Patent on EuroFlow-based flowcytometric Diagnosis and Classification of hematological malignancies; Royalty income for EuroFlow Consortium.; Roche: Consultancy, Other: Laboratory Services in the field of MRD diagnostics, provided by the Laboratory of Medical Immunology, Dept. of Immunology, Erasmus MC, Rotterdam, NL.. San Miguel:Janssen-Cilag: Honoraria; Onyx: Honoraria; Millennium: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria.

Human subcutaneous dirofilariosis has several clinical presentations. Many cases present as subcutaneous nodules, as a consequence of a local inflammatory reaction that encapsulates and destroys the worms. In addition, there are cases in which migrating worms located in the ocular area remain unencapsulated. In the present work, the levels of two pro-inflammatory eicosanoids, thromboxane B2 (TxB2) and leukotriene B4 (LTB4) are analysed by commercial Enzime-Linked immunosorbent assay (ELISA) in serum samples from 43 individuals, 28 diagnosed as having subcutaneous dirofilariasis presenting a subcutaneous nodule, five diagnosed as having dirofilariasis, in which the worms remained unencapsulated in the periphery of the eye, and ten healthy individuals living in a non-endemic area, used as controls. The worms were surgically removed, identifying Dirofilaria repens as the causative agent in all cases, by Polymerase Chain Reaction (PCR). Individuals with nodules showed significantly higher levels of TxB2 and LTB4 than healthy controls, whereas significant differences in LTB4 levels were observed between individuals with unencapsulated worms and healthy controls. It is speculated that the absence of LTB4 may contribute to the fact that worms remain unencapsulated as a part of immune evasion mechanisms.

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Background: Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease (Dirofilaria immitis) causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction and hypoxia. The aim of this study was to analyze whether the excretory/secretory antigen of adult worms interacts and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Methods: Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEGF, as well as DiES+VEGF and Cut+VEGF were used. The production of VEGF-A and other proangiogenic [soluble VEGFR-2 (sVEGFR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEGFR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. Results: DiES+VEGF and Cut+VEGF significantly increased the production of VEGF and VEGFR2, and only Cut+VEGF significantly increased the production of VEGFR1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEGF produced a significant increase in viable cell number and cell migration, as well as in the organization and number of cell connections. Conclusions: Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.