Lymphatic Organs Research Articles

Whole-Body Diffusion-Weighted Magnetic Resonance Imaging and Positron Emission Tomography Combined with Computed Tomography in the Staging of Lymphomas

Objective – to compare the diagnostic efficiency of whole-body diffusion-weighted magnetic resonance imaging (DW-MRI) and positron emission tomography combined with computed tomography (PET/CT) in the staging of lymphomas.Material and methods. The prospective study enrolled 92 adult patients with Hodgkin's lymphoma or non-Hodgkin's lymphoma. Before treatment, all the patients underwent whole-body DW-MRI and PET/CT. The techniques were compared for their efficiency in diagnosing damage to lymph nodes (LNs) and organs and in determining the stage of lymphoma.Results. The sensitivity, specificity, and accuracy of DW-MRI in diagnosing a lesion in the enlarged LNs were 98.2%, 99.9%, and 99.3%, respectively; those of PET/CT were 99.4%, 100.0%, and 99.8%. ROC analysis showed that the efficiency of the techniques in diagnosing a lesion in the enlarged LNs did not differ (p>0.06). The sensitivity of DW-MRI and PET/CT in diagnosing a lesion in the unenlarged LNs was 77.8% and 88.1%, respectively (p<0.001). The sensitivity, specificity, and accuracy of DW-MRI in diagnosing lung damage were 73.3%, 98.7%, 94.6%; those of PET/CT were 86.7%, 98.7%, and 96.7%; those for the spleen were 54.8%, 98.3%, 83.3% and 100.0%, 100.0%, 100.0%; those for bone marrow (BM) were 87.1%, 96.4%, 93.0% and 64.5%, 87.3%, 79.1%,; those for all organs were 72.9%, 98.1%, 91.4% and 80.0%, 96.6%, 92.2%. The ROC analysis indicated that the efficiency of the techniques in diagnosing a lesion in the lung did not differ (p>0.3), that of PET/CT for the spleen was higher (p <0.0001); that of DW-MRI for BM was higher (p<0.0008); that for all organs did not differ (p>0.35). DW-MRI and PET/CT could determine the correct stage of lymphoma in the same number of patients (86%). The new lymphoma staging algorithm involving whole-body DW-MRI in all patients, PET/CT, and BM biopsies according to the established indications made it possible to determine the correct stage of lymphoma in 95% of patients.Conclusion. Whole-body DW-MRI has comparable efficiency to PET/CT in the staging of lymphoma. The new lymphoma staging algorithm based on whole-body DW-MRI is superior to PET/CT and PET/CT with BM biopsy.

Immunomodulatory Activity Test of Syrup Dosage Form of Combination Phyllantus niruri Linn. And Sterculia quadrifida R.Br. Extract

Meniran (Phyllantus niruri Linn.) is used generally as an immunomodulator and hepatoprotector. Empirically, bark of faloak (Sterculiaqudrifida R.Br.) is used as a medicinal plant to cure diseases such as hepatitis, gastroentritis, and diabetes. The combination of meniran extract and bark of faloak extract has the potential as a natural immunomodulatory agents. Immunomodulators are compositions of either recombinant, synthetic, or natural, which can restore the immune system. Therefore it is necessary to optimize the optimal formula in obtaining syrup preparations which have optimal immunomodulatory activity. The aim of this study was to find out the optimal syrup combination of meniran extract and faloak bark extract to produce optimal response values ​​in the in vitro immunomodulatory activity test with TNF-α and NF-κB parameters, and immunomodulatory activity tests in vivo with activity parameters macrophages. The extract was obtained through reflux extraction. The results of the extract were formulated and approved to determine their ability to induce TNF-α and NF-κB, and in the test of mice balb/b (Mus musculus L.) orally for 21 days. The lymph organs were isolated, rinsed with an RPMI-1640 solution, and analyzed using ELISA reader 450 nm. The determination of optimal formulas is done by using Rstudio v.3.5.3 software with ANOVA test parametric test and using Design Expert v.11 software using the Simplex Lattice Design (SLD) method. The results showed that the formula for the preparation of combination syrup to produce results induced TNF-α and NF-κB, and macrophage activity, namely the formula using meniran and faloak 0.96993: 0.0300704.

The study biochemical indicates of blood rats at conditions acute hypoxia

In the experimental simulation hypoxia, the liver and rhinitis coincide with the discontinuation of rheological organs of the bones and organ lymph, which has diminished in shorter duration of the constriction, increasing the viscosity and thrombogenic processes in the blood and lymph. Unilateral hypoxic hypoxia (1.5 hrs) on liver tissue, decreased blood pressure, decreased intoxication levels in beds at 8%. %. The concentration of the total protein concentration and the elevation of the activation ALT and AST were investigated by the changes in the osmotic resistance of the erythrocytes and of urine. It has been established that experimental live animals with hypoxia, lymphatic aching, loss of general protein, high levels of alimentary tract and ALT, AST, abnormalities of rheological properties, and lymph nodes and morphophysiological condition of the lymphatic system.

Microangiopathic hemolytic anemia/Acquired Thrombotic Thrombocytopenic Purpura as a first presentation of pancreatic cancer

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threatening hematologic disorder. It is mainly characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA), fever, renal impairment and neurological abnormality. Plasmapheresis and steroids are the standard of care. MAHA/TTP can be the initial presentation of solid organ malignancies especially gastrointestinal tumors. We report a 56-year-old female patient who presented with progressive back pain, tiredness, easy bruising, fever and weight loss. Laboratory results showed anemia, thrombocytopenia, and schistocytes in the peripheral smear. An initial diagnosis of thrombotic thrombocytopenic purpura (TTP) was made on the basis of clinical presentation and lab findings. She was treated with corticosteroids and plasma exchange but with no major response. CT abdomen and PET CT were suggestive of pancreatic carcinoma with extensive lymph nodal, organs and bone metastases. Supraclavicular lymph node biopsy was compatible with metastatic adenocarcinoma. As a result, the diagnosis of pancreatic cancer was established and the decision was for palliative treatment. This case highlights the need to consider malignancy in patients with MAHA/TTP especially if it does not respond to plasmapheresis. Hence, the treatment of MAHA/TTP could be directed to the underlying malignancy if available.

Relative Hypoxia in Immunized Mice Spleen Macrophages as Indicated by Hypoxia Inducible Factors, Cytoglobin and Peroxisome Proliferator Activated Receptor Gamma Coactivator (PGC)‑1α

Introduction: Spleen is the largest lymphatic organ in the body that is responsible for initiating an immune response to antigens, consistof macrophages and lymphocytes. Macrophages are cells that engulf foreign bodies by migration, phagocytosis, O 2 burst, lysosomes fusion, synthesis and secretion of various cytokines which would require enormous amounts of energy. Therefore, macrophages needs of O 2 to increase resulting in hypoxia relative. To overcome this situation, the cell is equipped with a special mechanism which is under the control of HIF. They are highly dependent to O 2 existence to produce energy. This process is important for biochemical adaptation in homeostasis. Macrophages expressing Cygb for supplying O 2 and PGC‑1 for mitochondrial biogenesis in producing energy. This study aims to determine whether the spleen macrophages in mice being immunized intraperitoneally with SRBC increase the expression of HIF, Cygb and PGC‑1α. Method: 24 male BALB/c mice, aged 2 months were immunized by injecting 0.2 ml SRBC 2%. Macrophages were taken from spleen of mice. Expression of mRNA and protein levels of HIF‑1α, HIF‑2α, Cygb, PGC‑1α in macrophages were measured by real time RT‑PCR and ELISA, respectively. Level of O 2 burst were measured with WST Salts. Observations were made at 24, 48, and 72 hours post‑immunization. Results: The ability to oxidize antigen levels (O 2 burst) found higher at 24 hours after immunization. The levels of protein and mRNA expression of HIF‑1α and HIF‑2α showed the highest increase at 24 and 48 hours after immunization. The levels of Cygb protein increased at 48 hours group after immunization while mRNA expression increased at 24 hours after immunization. PGC‑1α protein levels decreased in 24 hours after immunization and then increased gradually. Conclusion: This study shows the expression pattern of several proteins in spleen macrophages of mice that was immunized

Clinicoepidemiological profile of extranodal lymphoma: The experience of a tertiary care center in India

Introduction: Lymphoma is a neoplastic proliferation of lymphoid cells at various stages of differentiation and affects lymph nodes, with infiltration into the bone marrow, spleen, and thymus, which form the primary lymphatic organs. Extranodal lymphoma, by definition, involves sites other than lymph nodes, spleen, thymus, and the pharyngeal lymphatic ring. Extranodal involvement is less common with Hodgkin lymphoma (HL) than with non-Hodgkin lymphoma (NHL). Materials and Methods: A single-center retrospective observational study was conducted over the period of 2017–2019. Age, gender, histologic type, location, type of clinical presentation, histologic diagnosis, and presence of specific symptoms were recorded, as were the specialty of the physician initially consulted and of the physician taking the diagnostic sample. Results: Twenty-seven cases of extranodal lymphoma were diagnosed: 12 (44.4%) were male and 15 (56.6%) were female patients. The median age for males was 49.6 years and for females was 45.7 years. In this study, we had varied presentations of the lymphoma with involvement of various structures. We had few patients with very rare site of involvement like an elderly female patient presented with firm swelling over the right forearm which on excision turned out to myeloid sarcoma. Similarly, an elderly woman had presented with pyrexia of unknown origin that was later diagnosed as having primary bone Hodgkin lymphoma (HL) which is a very rare diagnosis and rarely has been described in world literature. The most common site of extranodal lymphoma was gastrointestinal tract (7 out of 27 patients; 25.9%); the other sites reported were testis (14.8%), breast (7.4%) thyroid, and ovary. Few rare sites reported were bone, central nervous system, and mediastinum. We report an extremely rare patient who had presented with anterior chest wall swelling, and on evaluation was detected to have primary mediastinal B cell lymphoma. Conclusion: Extranodal lymphoma is the rare presentation of NHL and extremely rare for HL. As it is a very rare disease, there are very limited studies available for its staging and management. We have presented a case series of extranodal lymphoma with few very rare presentations.

Development of a RACE-based RNA-Seq approach to characterize the T-cell receptor repertoire of porcine γδ T cells

Recent data suggest that porcine γδ T cells exhibit a similar degree of functional plasticity as human and murine γδ T cells. Due to the high frequency of TCR-γδ+ cells in blood and secondary lymphatic organs, the pig is an attractive model to study these cells, especially their combined features of the innate and the adaptive immune system. Using a 5’ RACE-like approach, we translated a human/murine NGS library preparation strategy to capture full-length V-(D)-J TRG and TRD clonotypes in swine. After oligo(dT) primed conversion of input RNA, the cDNA population was enriched for full-length V(D)J TCR transcripts with porcine-specific primers including Illumina adaptor sequences as overhangs for Illumina MiSeq analysis. After quality control and processing by FastQC and ea-utils, porcine TRG and TRD sequences were mapped against the human IMGT reference directory. Porcine blood-derived CD2+ and CD2‾ TCR-γδ+ cells exhibited two distinct clonotypes Vγ11JγP1 (74.6%) and Vγ10JγP1 (57.7%), respectively. Despite the high TCR-δ diversity among CD2+ cells (39 clonotypes), both subsets shared the same abundant Vδ1DδxJδ4 clonotype at approximately identically frequencies (CD2+: 31.2%; CD2‾: 37.0%). The flexible nature of this approach will facilitate the assessment of organ-specific phenotypes of γδ T cell subsets alongside with their respective TCR diversity at single cell resolution.

Therapeutic effects of an azaphenothiazine derivative in mouse experimental colitis.

Phenothiazines represent a class of compounds of potential therapeutic utility. In this report we evaluated therapeutic value of an azaphenothiazine derivative, 6-acetylaminobutyl-9-chloroquino[3,2-b]benzo[1,4]thiazine (QBT), given intragastrically, in the model of dextran sodium sulfate-induced colitis in C57BL/6 mice using 5-aminosalicylic acid (5-ASA) as a reference drug. Colitis symptoms such as body weight loss, diarrhea and hematochezia (blood in stool) were observed and registered and disease activity index (DAI) was calculated. In addition, weight and cell numbers in the lymphatic organs and histological parameters of the colon wall were analyzed. The effects of QBT on viability of colon epithelial cell lines were also determined. We showed that weight and cell number of draining mesenteric lymph nodes were lower in mice treated with QBT in comparison to their control counterparts. The number of thymocytes, drastically reduced in control mice, was elevated in mice treated with the compounds with a significant effect of 5-ASA. In addition, an abnormal composition of blood cell types was partially corrected in these groups. Histological analysis of the colon revealed that the pathological changes were partially normalized by QBT and even to a higher degree by 5-ASA. In conclusion we demonstrated a therapeutic efficacy of the compound in amelioration of local and systemic pathological changes associated with chemically-induced colitis in mice. A possible mechanism of action of the compound is discussed.

Smeared Multiscale Finite Element Models for Mass Transport and Electrophysiology Coupled to Muscle Mechanics

Mass transport represents the most fundamental process in living organisms. It includes delivery of nutrients, oxygen, drugs, and other substances from the vascular system to tissue and transport of waste and other products from cells back to vascular and lymphatic network and organs. Furthermore, movement is achieved by mechanical forces generated by muscles in coordination with the nervous system. The signals coming from the brain, which have the character of electrical waves, produce activation within muscle cells. Therefore, from a physics perspective, there exist a number of physical fields within the body, such as velocities of transport, pressures, concentrations of substances, and electrical potential, which is directly coupled to biochemical processes of transforming the chemical into mechanical energy and further internal forces for motion. The overall problems of mass transport and electrophysiology coupled to mechanics can be investigated theoretically by developing appropriate computational models. Due to the enormous complexity of the biological system, it would be almost impossible to establish a detailed computational model for the physical fields related to mass transport, electrophysiology, and coupled fields. To make computational models feasible for applications, we here summarize a concept of smeared physical fields, with coupling among them, and muscle mechanics, which includes dependence on the electrical potential. Accuracy of the smeared computational models, also with coupling to muscle mechanics, is illustrated with simple example, while their applicability is demonstrated on a liver model with tumors present. The last example shows that the introduced methodology is applicable to large biological systems.

Expresión de ARNm de IL1β, TNF-α, TLR2, TLR4 y TLR9 en ovinos inmunizados con antígenos de Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis is the etiological agent of Caseous Lymphadenitis in small ruminants and causes granulomas in lymph nodes and organs. The role of innate immune mechanisms involved in the pathogenesis of this bacterium has not yet been completely elucidated and must be understood for the rational development of vaccine candidates. This study evaluated IL1β, TNF-α, TLR2, TLR4 and TLR9 mRNA expression in sheep infected with a low virulence strain (T1) and immunized with secreted antigens of T1 and later challenged with a virulent strain of C. pseudotuberculosis. Five sheep were inoculated with a dose of 109 CFU of T1 (a low virulence strain of C. pseudotuberculosis), five were immunized with 250 µg of secreted antigens in chemically defined medium (CDM) and three animals were used in the control group in which they received only 0.09% saline solution. All animals were challenged with a dose of 2x105 CFU of a virulent C. pseudotuberculosis strain, and subsequently necropsied. Control group animals after challenge were used to compose the infected animal group. The results showed that there was slightly higher expression of TNF-α, TLR2, TLR4 and TLR9 mRNA in animals from group T1, which did not develop lesions, however, it was not statistically significant. All groups influenced in the expression of IL1β, TNF-α, TLR2, TLR4 and TLR9 mRNA.

Distinct Features of Canine Non-conventional CD4-CD8α- Double-Negative TCRαβ+ vs. TCRγδ+ T Cells.

The role of conventional TCRαβ+CD4+ or TCRαβ+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαβ or TCRγδ but lacking CD4 and CD8α expression [i.e., CD4−CD8α− double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCRγδ. In contrast, TCRγδ+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCRαβ+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCRαβ+CD4+ sp, and almost half of the frequency of TCRαβ+CD8α+ sp T cells (i.e., ~15% of all TCRαβ+ T cells). Among TCRαβ+CD4−CD8α− dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCRαβ+CD4−CD8α− dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαβ+CD4−CD8α− dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαβ+CD4−CD8α− dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3−TCRαβ+CD4−CD8α− dn T cells was even higher as compared with TCRαβ+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCRγδ+CD4−CD8α− dn T cells also expressed substantial proportions of GATA-3. In addition, TCRαβ+CD4−CD8α− dn T cells produced IFN-γ and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCRαβ+CD4−CD8α− dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-γ or IL-17A and (ii) a small TCRγδ+CD4−CD8α− dn T cell subset also expressing GATA-3 without production of IFN-γ or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

CLL-Derived Exosomes Function As Trojan Horses That Induce SMAD6-Dependent Apoptosis of Normal B-Cells

Studies from recent years unraveled the role of monocytes and T-cells in the pathogenesis of chronic lymphocytic leukemia (CLL). The role of other immune cells in the pathobiology of CLL is less known. Specifically, whether B-cells, the normal counterpart of CLL cells play a role in CLL is unknown. Nevertheless, since both CLL cells and wild type B-cells reside in lymphatic organs and travel in blood, they either share or compete over common environmental resources. According to the cell competition theory, a sensing mechanism measures the relative fitness of a cell and ensures the elimination of cells deemed to be less fit then their neighbors. Since constitutive activation of intracellular pathways protect CLL cells from apoptosis, the cell competition theory predicts that compared with normal B-cells these cells are sensed as "super fit" and B-cells, the less fit counterparts, are eliminated. Yet, what delivers this massage across a population of cells is unknown. Exosomes are nanosized particles that are secreted by various types of cells. Exosomes carry a cargo of proteins and different types of RNA. They travel in body fluids and are taken up by cells in their vicinity. Since cancer cells including CLL cells secrete exosomes, we have formulated our hypothesis, namely, that exosomes derived from CLL cells are the vehicles that carry a death massage to wild type B-cells. To test this hypothesis, we isolated CLL cells from 3 previously untreated patients with CLL. We then grew these cells in exosome free media for 72 hours and harvested the exosomes by ultracentrifugation. We used NanoSight tracking analysis, Western immunoblotting for CD63, a common exosomal marker, and electron microscopy imaging studies to ensure that our pellet include the typical 100nm exosomal particles. Subsequently, we subjected normal B-cells derived from healthy volunteers to CLL derived exosomes stained by FM-143 dye. Using flow cytometry we found that exosomes are taken up by normal B-cells in a dose- and time- dependent manner. Double staining of the recipient B-cells to Annexin/PI revealed that exosomes induce apoptosis of these cells in a dose- and time- dependent manner. We then used RNA-seq to trace the changes in the molecular makeup of B-cells after exosomal uptake?? they took up exosomes. We found 24 transcripts that were differentially expressed (11 that were upregulated and 13 that were downregulated). We then verified the array results by quantitative real-time PCR for four of these genes. Among the top transcripts that were upregulated in exosome-positive B-cells is SMAD6. Because the upregulation of the SMAD family members including SMAD6 is associated with the induction of apoptosis in various malignant and non-malignant cells we wondered whether the upregulation of SMAD6 also induces apoptosis in normal B-cells. To test this, we transfected normal B-cells with SMAD6 containing vector and verified by RT-PCR that level of SMAD6 transcript were upregulated and by Western immunoblotting that levels of SMAD6 protein are upregulated as well. As expected, the rate of apoptosis was higher, and the rates of viable cells and proliferating cells were significantly lower in SMAD6-transfected B-cells. Taken together, we show here that CLL cells secrete exosomes that function as "Trojan horses". Once they are taken up by normal B-cells they induce SMAD6-dependent apoptosis. In this way the neoplastic cells may actively eliminate their competitors and take over the common environmental resources. Disclosures No relevant conflicts of interest to declare.

Examining the Role of CD30 in an Anaplastic Large Cell Lymphoma Mouse Model

Anaplastic large cell lymphoma (ALCL) represents a heterogeneous group of T-cell non-Hodgkin lymphoma (NHL) mainly affecting children and young adults. About 70% of systemic ALCLs are associated with a characteristic chromosomal translocation, t(2;5)(p23;35) which fuses the anaplastic lymphoma kinase (ALK) gene on chromosome 2 with the nucleophosmin (NPM) gene on chromosome 5, resulting in the NPM-ALK fusion gene, its over-expression and constitutive kinase activity. Immunophenotypic characterization of human ALCL revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on disease development remains unclear and the relationship between NPM-ALK and CD30 has been investigated mostly using cell line models. Syngeneic mouse models of cancer can serve as useful models since the tumors develop in situ where the contribution made by the immune system and the extracellular matrix can be investigated. Here, we focus on the involvement of CD30 in a retroviral murine bone marrow transplantation model of ALCL. In this model, the BM of Lck-Cre-transgenic mice is infected with a MSCV-Stop-NPM-ALK-IRES-EGFP vector leading to expression of NPM-ALK in early T-cells. With a latency of 3-4 months, mice develop lymphomas and die from neoplastic T-cell-infiltration of BM and lymphatic organs. To investigate the impact of abrogation of CD30 signals on the development of NPM-ALK+ ALCL in our model, CD30 knockout mice were crossed with Lck-Cre mice. Both Lck-Cre NPM-ALK CD30 wt and Lck-Cre NPM-ALK CD30 ko recipients develop a human ALCL-like lymphoma with a pure T-cell phenotype characterized by Thy1.2+ cells infiltrating the thymus, lymph nodes, spleen and BM. First results from Lck-Cre NPM-ALK CD30 ko transplanted mice showed impaired disease induction and prolonged survival compared to CD30 wt animals. Moreover, secondary transplantation of NPM-ALK thymic lymphomas led to distinct deceleration of disease development upon CD30 deletion. Microarray analyses have shed some light on the mechanisms underlying the delayed lymphoma progression of CD30 deleted tumors with an upregulation of inflammatory pathways and proteins that are master players in inflammation and immune responses. Further characterization of the role of CD30-mediated immune response in disease progression using this mouse model and immunocompromised mice is ongoing. An improved understanding of how the immune system affects tumor progression will extend the rationale in translational strategies to use immunotherapies for patients with T-NHLs. Disclosures No relevant conflicts of interest to declare.

A Case of Acute Lymphocytic Leukaemia with t(3;13) and Central Nervous System Leukemia after Allogenic Cord Blood Transplantation

Background: Acute lymphoblastic leukemia (ALL) is a neoplastic cancer characterized by clonal expansion of leukemic cells in lymph organs and bone marrow. Lots of kinds of different chromosomal translocation can be found in those leukemic cells. However, the role of the abnormal chromosomals and genes in leukemogenesis is not yet fully understood. Identifying new chromosomal translocation can facilitate a better understanding of pathogenesis of this disease. Case presentation: We report a rare case of acute lymphocytic leukaemia with t(3;13)(q29,q21). The patient was diagnosed pre-B-ALL with no abnormal chromosomal or gene fusion and achieved complete remission (CR) after induction chemotherapy. Ten months later, she relapsed in the consolidation with cytogenetics test showing 46,XX,t(3;13)(q29,q21). Given no CR after 2 chemotherapy regimens, she received salvage cord blood transplantation. Regular intrathecal methotrexate was applied to prevent central nervous system leukemia. Good graft-versus-leukemia (GVL) effect was induced by daily injection of low dose of IL-2 two months post transplantation. Minimal residual disease (MRD) negativity was maintained until central nervous system leukemia was found 8 months after transplantation. A whole exome sequencing was performed. Nine driver mutation genes and seven tumor genes were found. Conclusions: We highly suspect that the relapse in central nervous system after transplantation is associated with the rare chromosomal translocation. Disclosures No relevant conflicts of interest to declare.

Expansion of T Helper Cell Subsets in Chronic Lymphocytic Leukemia Cell Co-Cultures with Nurselike Cells

Background: In secondary lymphatic organs (SLO), chronic lymphocytic leukemia (CLL) cells form characteristic pseudo-follicles, also called proliferation centers, in which proliferating CLL cells are in intimate contact with CD4+ T helper cells. Despite evidence for a co-evolution of CLL cells and counterpart T helper cells, the prevalence and dynamics of T cell subsets, such as T follicular helper cells (TFH), T regulatory helper cells (TRegs) and IL-17-producing T helper cells (Th17) has not been characterized in detail. The CLL nurselike cell (NLC) model recapitulates key cellular and molecular interactions between CLL cells and the microenvironment in SLO, based on functional and gene expression studies, and represents a valid in vitro model for the SLO microenvironment in CLL. To gain insight into the potential role of T cells in CLL SLO, we utilized the NLC model to characterize T helper cell subsets and their dynamics in NLC co-cultures. Methods and Results: First, we quantified CD4+ and CD8+ T cell subsets in fresh peripheral blood mononuclear cell (PBMC) samples from 14 patients and then placed 1x107 PBMCs/ml in culture to establish NLC. We rechecked the relative proportion of CD4+ and CD8+ T cells, as well as absolute T cell counts, and their viability after 3, 6, 9, 12 and 14 days in NLC co-culture conditions. Interestingly, throughout the 14 days of culture, the number of T helper cells remained stable when compared to baseline samples. Next, we characterized T helper cell subsets in 25 different CLL samples, comparing CD4+ T cell subsets in freshly isolated CLL PBMC with matched samples harvested after 14 days of NLC culture. Samples were stained with subset-specific fluorescence-labeled antibody combinations, and analyzed by flow cytometry. NLC co-culture resulted in a significant expansion of TFH and TReg cells. TFH absolute cell counts assessed by flow cytometry using counting beads, increased from 9.4±2.2/μl at baseline to 29.0±5.9/μl in NLC co-cultures (n=14, p=0.001) and TReg from 17.0±3.3/μl to 51.0±15.0/μl (n=14, p=0.027). In contrast, Th17 absolute cell counts declined after 14 days of culture from 113.0±21.0/μl to 68.0±13.0/μl (n=14, p=0.001). Moreover, TH2 cells declined from 42.0±7.4/μl to 30.0±8.2/μl (n=14, p=0.005). Next, we analyzed for changes in TFH subsets (TFH1, TFH2 and TFH17). When compared to TFH cells from CLL PBMC, NLC culture resulted in a relative increase in TFH2 from 17.0±2.4% to 26.0±1.7% (n=25, p=0.013), and in TFH17 from 12.0±1.6% to 21.0±2.7% (n=25, p=0.006) In contrast, TFH1 frequency decreased after 14 days of culture (54.0±3.5% versus 34.0±2.9%, n=25, p=0.001). T follicular regulatory (TFr) cells also increase under co-culture conditions from undetectable to 4.8±1.9% (n=25, p=0.025)(Figure1). Looking at the maturity of CD4+ cells we noted a relative increase of central memory cells from 42.0±3.2% to 50.0±3.2% (n=25, p=0.023), whereas effector memory cells decreased from 34.0±3.4% to 26.0±2.7% (n=25, p=0.009), the fraction of naïve CD4+ cells remained unchanged. Next, we assessed the activation status of co-inhibitory receptors on CD3+CD4+ cells. We found a significant relative increase in CD4+ T cells expressing PD1 and CTLA-4 after 14 days of culture. Additionally, the TFH and TReg subsets demonstrated a significantly higher CTLA-4 expression after culture while TReg cells also addressed an increase in PD1 frequency. Furthermore, we observed a significantly higher CD28 expression on TFH and TReg subsets after 14 days of co-culture. Following, to address the residence and migration capacity of T cells in NLC co-cultures, we analyzed the CD69 and CD62L expression. Our results revealed significantly higher expression of CD69 in NLC co-cultures, whereas CD62L levels remained unchanged. Conclusion: The expansion of TFH and TReg cells in NLC co-cultures suggests the selection and clonal expansion of T cells that may support and engage in crosstalk with CLL cells. Additionally, the expansion of TFr and TFH17 cells could reflect a milieu of immune tolerance establishing in NLC co-cultures. Ongoing TCR sequencing of serial CD4+ T cell samples (baseline versus 14 days under NLC conditions) will characterize changes of clonal architecture in the T helper cell compartment, and will further improve our understanding of co-evolution of CLL and T helper cells. Disclosures Wierda: Gilead Sciences: Research Funding; Juno Therapeutics: Research Funding; KITE pharma: Research Funding; Sunesis: Research Funding; Miragen: Research Funding; Janssen: Research Funding; Xencor: Research Funding; Acerta Pharma Inc: Research Funding; Pharmacyclics LLC: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Cyclcel: Research Funding; Loxo Oncology Inc.: Research Funding. Burger:Aptose Biosciences, Inc: Research Funding; BeiGene: Research Funding; Gilead Sciences: Research Funding; AstraZeneca: Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Pharmacyclics, an AbbVie company: Research Funding.

Genomic Characterization of a Pediatric Cohort with Non-Malignant Lymphoproliferative Disorders

Introduction: Pediatric non-malignant lymphoproliferative disorders (LPDs) are a clinically and genetically heterogeneous. While transient lymphadenopathy is extremely common and rarely dangerous, long-standing immune dysregulation and lymphoproliferation in children may be life-threatening. Data to guide evaluation and treatment of children with benign LPD are lacking. The primary objective of this study was to define the genomic spectrum and clinical characteristics of a cohort of children with nonmalignant LPD. Identification of the underlying pathogenic mechanisms may facilitate timely interventions and potentially guide optimal therapeutic strategies. Method s: Patients at Texas Children's Hospital and collaborating referral centers who met criteria for non-malignant LPD were offered participation in this study, approved by the Baylor College of Medicine Institutional Review Board. LPD was defined as persistent lymphadenopathy, lymph organ involvement, or lymphocytic infiltration for more than 3 months, with or without chronic or significant EBV infection. Chronic or significant EBV infection was defined as recurrent or persistent EBV viremia more than 3 months, invasive EBV disease, or EBV PCR copies >100,000. All subjects and/or family members provided written informed consent to have their clinical and genetic information published. Genetic testing was performed clinically or through research-based whole-exome sequencing (WES). Results : Fifty-one subjects from 47 different families with non-malignant LPD were identified. Median age at disease presentation was 3.3 years (range 3.9 weeks - 21 years) with nearly equal proportions of males (n = 26) and females (n = 25). Almost half of subjects were Hispanic (49%), and 29% were non-Hispanic white. Fifteen subjects (29%) met HLH-2004 diagnostic criteria for HLH. Twenty-one patients had EBV-associated lymphoproliferative disorders (EBV-LPD) and 6 of the 51 ultimately developed malignancy. Clinical genetic testing was performed in 29 patients, and research-based WES was performed in 44 patients. Potential disease-causing genetic defects were identified in 62% of families. Of these pathogenic variants, targeted therapies may be effective for treatment in at least 10 of the conditions (17 subjects, 33%). Furthermore, genetic results supported potential for curative HSCT in 35% of the patients. Mechanistically, all of the LPD-associated genes could be placed into 1 of 3 categories: 1) defective control of lymphocyte activity; 2) impaired lymphocyte activation, cytoskeletal organization, and apoptosis; and 3) dysregulated inflammation. Ten-year survival for the entire cohort was 72.4% with a median 5.6 years of follow-up (range 0.10 - 26.6). Patients without evidence of a genetic explanation had a lower ten-year survival compared to those patients for whom a genetic explanation was identified (48% versus 82%, respectively, p=0.03). When both EBV-LPD and genetic explanation were considered, the ten-year survival estimate for those with EBV-associated disease and no genetic explanation was significantly worse than those with EBV-associated disease and a genetic explanation (17% vs. 90%; p =0.002). Patients without EBV-associated disease were at lower risk of death than those with EBV-LPD. Evaluating outcomes associated with maximum treatment received, ten-year survival was lowest (25% survival) among those who underwent HSCT. Conclusion s: Pediatric non-malignant LPD represents a group of conditions with high risk of death. WES identified actionable mutations in the majority of LPD cases in this cohort. Early identification of these mutations can guide therapy by confirming diagnosis, revealing molecular targets and/or supporting definitive therapy with stem cell transplant. Disclosures Forbes: Takeda: Consultancy. Jolles:CSL Behring: Consultancy, Honoraria, Research Funding, Speakers Bureau; LFB: Consultancy, Honoraria, Research Funding, Speakers Bureau; UCB Pharma: Consultancy, Other: Drug Safety Committee; Shire/Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharming: Consultancy, Honoraria, Research Funding, Speakers Bureau; Octapharma: Consultancy, Honoraria, Research Funding, Speakers Bureau. Heslop:Marker Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allovir: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cell Medica: Research Funding. Rouce:Novartis: Consultancy, Honoraria; Tessa Therapeutics: Research Funding; Kite, a Gilead Company: Consultancy, Honoraria.

Is the Recommendation of a Pelvic Lymphadenectomy in Conjunction with Radical Prostatectomy in Prostate Cancer Patients Justified? Report from a Multidisciplinary Expert Panel Meeting.

Pelvic lymphadenectomy (pLA) in prostate cancer (PCa) is one of the most common uro-oncologic surgical procedures. An increased complication rate is accompanied by unproven oncologic benefit. Extent of pLA and mechanisms of metastasis are discussed controversially. We aimed to explore evidence and knowledge gaps in pLA and mechanisms of metastasis in PCa and to develop further steps to clarify oncologic benefits through an expert panel. A multidisciplinary expert meeting was initiated, compiling available facts on pLA and mechanisms of metastasis in PCa. Questions and hypotheses were formulated. The resulting protocol was modeled on priority and consistency in four anonymized voting rounds using the Delphi method (March 2018-June 2018). The oncologic benefit of pLA in PCa is still unclear. Results of randomized trials (RCTs) are pending. Extent and techniques of pLA are differently applied and inconsistently recommended by the guidelines as well as the indication for pLA. Different growth rates for the primaries and metastases and different survival curves for lymph node and organ metastasis at diagnosis argue against metastasis originating from positive nodes. However, results from clinical and basic research support this opportunity in PCa. The RCTs required to clarify the estimated low oncologic benefit of pLA prove to be difficult because of the great effort (e.g., high case number). Establishing a network of treatment centers for implementation of high-quality cohort studies could be an alternative approach. Future studies with larger panels and international participants based on the presented feasibility should be launched to set this process in motion. Until valid data are available, benefits and harms of pLA should be weighted under consideration of low-invasive techniques (e.g., sentinel pLA).

Calcium-activated chloride channel regulator 1 (CLCA1): More than a regulator of chloride transport and mucus production

CLCA1 is a member of the CLCA (calcium-activated chloride channel regulator) family and plays an essential role in goblet cell mucus production from the respiratory tract epithelium. CLCA1 also regulates Ca2+-dependent Cl− transport that involves the channel protein transmembrane protein 16A (TMEM16A) and its accessary molecules. CLCA1 modulates epithelial cell chloride current and participates in the pathogenesis of mucus hypersecretory-associated respiratory and gastrointestinal diseases, including asthma, chronic obstructive pulmonary disease, cystic fibrosis, pneumonia, colon colitis, cystic fibrosis intestinal mucous disease, ulcerative colitis, and gastrointestinal parasitic infection. Most studies have been focused on the expression regulation of CLCA1 in human specimens. Limited studies used the CLCA1-deficient mice and CLCA1 blocking agents and yielded inconsistent conclusions regarding its role in these diseases. CLCA1 not only regulates mucin expression, but also participates in innate immune responses by binding to yet unidentified molecules on inflammatory cells for cytokine and chemokine production. CLCA1 also targets lymphatic endothelial cells and cancer cells by regulating lymphatic cell proliferation and lymphatic sinus growth in the lymphatic organs and controlling cancer cell differentiation, proliferation, and apoptosis, all which depend on the location of the lymphatic vessels, the type of cancers, the presence of Th2 cytokines, and possibly the availability and type of CLCA1-binding proteins. Here we summarize available studies related to these different activities of CLCA1 to assist our understanding of how this secreted modifier of calcium-activated chloride channels (CaCCs) affects mucus production and innate immunity during the pathogenesis of respiratory, gastrointestinal, and malignant diseases.

Bruton Tyrosine Kinase Inhibitors: Present and Future.

Bruton tyrosine kinase (BTK) is a nonreceptor tyrosine kinase that plays a central role in the signal transduction of the B-cell antigen receptor and other cell surface receptors, both in normal and malignant B lymphocytes. B-cell antigen receptor signaling is activated in secondary lymphatic organs and drives the proliferation of malignant B cells, including chronic lymphocytic leukemia (CLL) cells. During the last 10 years, BTK inhibitors (BTKis) are increasingly replacing chemotherapy-based regimen, especially in patients with CLL and mantle cell lymphoma (MCL). Bruton tyrosine kinase inhibitors are particularly active in patients with CLL and MCL, but also received approval for Waldenström macroglobulinemia, small lymphocytic lymphoma, marginal zone lymphoma, and chronic graft-versus-host disease. Current clinical practice is continuous long-term administration of BTKi, which can be complicated by adverse effects or the development of drug resistance. Alternatives to long-term use of BTKi are being developed, such as combination therapies, permitting for limited duration therapy. Second-generation BTKis are under development, which differ from ibrutinib, the first-in-class BTKi, in their specificity for BTK, and therefore may differentiate themselves from ibrutinib in terms of adverse effects or efficacy.

Clinical and Serological Evaluation of LINDA Virus Infections in Post-Weaning Piglets.

The novel pestivirus species known as lateral-shaking inducing neuro-degenerative agent (LINDA) virus emerged in 2015 in a piglet-producing farm in Austria. Affected piglets showed strong congenital tremor as a result of severe lesions in the central nervous system. Here, we report the results of a controlled animal infection experiment. Post-weaning piglets were infected with LINDA to determine the susceptibility of pigs, the clinical consequences of infection and the humoral immune response against LINDA. No clinically overt disease signs were observed in the piglets. Viremia was hardly detectable, but LINDA was present in the spleen and several lymphatic organs until the end of the experiment on day 28 post-infection. Oronasal virus shedding together with the infection of one sentinel animal provided additional evidence for the successful replication and spread of LINDA in the piglets. Starting on day 14 post-infection, all infected animals showed a strong humoral immune response with high titers of neutralizing antibodies against LINDA. No cross-neutralizing activity of these sera with other pestiviral species was observed. According to these data, following postnatal infection, LINDA is a rather benign virus that can be controlled by the pig’s immune system. However, further studies are needed to investigate the effects of LINDA on the fetus after intrauterine infection.