Normal Peripheral Blood Cells Research Articles

6985 Intravenous Immunoglobulin induced Type 1 diabetes remission

Abstract Disclosure: N. Shahid: None. D. Newbern: None. Introduction: Intravenous immunoglobulin [IVIG] is an immunomodulatory agent used for treatment of autoimmune and inflammatory conditions. However, it is not indicated in patients with type 1 diabetes [T1D] as its efficacy has not been established. Clinical Case: A 9-year-old previously healthy boy presented to the Emergency Department with several days of unexplained bruising and mucosal bleeding with no prior history of trauma, bleeding disorders or other systemic symptoms. Physical examination showed generalized petechiae, ecchymosis and purpura. Vitals were normal including weight and BMI. Initial labs confirmed Immune thrombocytopenic purpura [ITP] as his platelets were less than 1K/µL with otherwise normal peripheral blood cell counts and smear. Concomitantly, labs showed significant hyperglycemia [260mg/dL] with glycosuria and trace ketonuria. Glycated hemoglobin [Hba1c] was elevated at 10.5% and ultimately Pancreatic autoantibodies [GAD65, Zinc transporter-8 and islet cell antibody] came back positive confirming T1D. Other autoimmune disorders such as adrenal insufficiency, thyroiditis, and celiac disease were ruled out. For the treatment of ITP, he the patient received 1 dose of IVIG 1g/kg and responded well with a rise in platelets to 6K/µL. The morning following the infusion, due to fasting hyperglycemia, the patient was started on basal-bolus insulin regimen. By 2nd day post IVIG, he had hypoglycemia, so insulin was discontinued with normalization of blood sugars. He is now 7 months post IVIG with no recurrent episodes of thrombocytopenia. He also remains off insulin with Hba1c in the 5.9-6.5% range. He eats a regular diet with avoidance of simple carbohydrates. He wears onwears a a continuous glucose monitor, which shows that blood sugars are in range 93% of the time He remains euglycemic overnight with transient self-resolving mild post-prandial hyperglycemia. Pancreatic autoantibodies have been reassessed and remain positive Conclusions: This case demonstrates that IVIG used for the treatment of ITP has induced a state of remission in our patient with newly diagnosed with T1D. IVIG is not a standard of treatment for T1D, however, when given for other autoantibody- or T-cell-mediated autoimmune conditions, it may reduce the insulin need in patients with known diabetes or induce remission in patients with newly diagnosed diabetes.

Citral in lemon myrtle, lemongrass, litsea, and melissa essential oils suppress the growth and invasion of breast cancer cells

ObjectiveAlthough cancer therapy suppresses recurrence and prolongs life, it may be accompanied by strong side effects; thus, there is a strong demand for the development effective treatments with fewer side effects. Cancer therapy using plant-derived essential oils is attracting attention as one promising method. This study investigated the antitumor effects of essential oil volatiles on breast cancer cells and identifies four essential oils that display antitumor activity.MethodsBreast cancer cells were cultured in a 96-well plate, then one of twenty essential oils was added dropwise to the central well. The plate was incubated at 37 °C for 48 h and the effect of the volatile components of each essential oil on the surrounding breast cancer cell growth ability was examined using an MTT assay. Gas chromatography was used to investigate the concentration of the transpiration components that may affect cancer cells.ResultsOf the 20 essential oils, Lemongrass, Lemon myrtle, Litsea, and Melissa displayed strong anti-tumor effects. These essential oils inhibited the growth of nearby breast cancer cells, even when diluted more than 500-fold. The transpiration component of lemon Myrtle showed the strongest antitumor effect, but was the least cytotoxic to mononuclear cells in normal peripheral blood (PBMC). Each of these essential oils contained a very large amount of citral. The IC50 against breast cancer cells when citral was volatilized from each essential oil was 1.67 µL/mL for geranial and 1.31 µL/mL for neral. Volatilized citral alone showed strong anti-proliferation and infiltration-inhibiting effects.ConclusionThe transpiration components of Lemongrass, Lemon myrtle, Litsea, and Melissa are thought to inhibit breast cancer cell proliferation due to their high levels of citral.

Automatic classification of 10 blood cell subtypes using transfer learning via pre-trained convolutional neural networks

Human blood is primarily composed of plasma, red blood cells, white blood cells, and platelets. It plays a vital role in transporting oxygen and nutrients to all organs, and stores essential health-related data about the human body. Blood cells are utilized to defend the body against infections and disease. Hence, analysis of blood permits physicians to assess an individual's physiological condition. Blood cells are sub-classified into eight groups: Neutrophils, eosinophils, basophils, lymphocytes, monocytes, immature granulocytes (promyelocytes, myelocytes, and metamyelocytes), erythroblasts, and platelets (thrombocytes) on the basis of their nucleus, shape and cytoplasm. Traditionally, pathologists and hematologists have identified and examined these via microscopy prior to manual classification, with this manual approach being slow and prone to human error. Therefore, it is essential to automate this process. In the current study, transfer learning with a series of pre-trained Convolutional Neural Network (CNN) models—VGG16, VGG19, ResNet-50, ResNet-101, ResNet-152, InceptionV3, MobileNetV2 and DenseNet-201 was applied to the normal peripheral blood cells dataset (PBC). The overall accuracy achieved with individual CNNs ranged from 91.4 % to 94.7 %. Based on these pre-trained architectures, a CNN-based architecture has been developed to automatically classify all ten blood cell types. The proposed transfer learning CNN model was tested on blood cell images from the PBC, Kaggle and LISC datasets. Achieved accuracy was 99.91 %, 99.68 % and 98.79 %, respectively, across these three datasets. The presented CNN architecture outperforms all previous results reported in the scientific/medical literature with a high capacity for framework generalization in future applications of blood cell classification.

Clinical features of 19 children infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 in Hangzhou, China

To study the clinical features of children infected with the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The medical data of 19 children who were diagnosed with SARS-CoV-2 Omicron variant infection from January 28 to March 3, 2022 in Hangzhou were retrospectively reviewed. Among the 19 children, there were 7 boys (37%) and 12 girls (63%), and their age ranged from 6 months to 16 years, with a median age of 2 years and 1 month. Most of these children were infants and young children (aged ≤3 years, accounting for 53%). Among these children, 11 (58%) were unvaccinated with SARS-CoV-2 vaccine and 8 (42%) were vaccinated with SARS-CoV-2 vaccine, and 3 children (16%) had a history of underlying diseases. All 19 children had a clear history of close contact with persons infected with SARS-CoV-2, and 10 children (53%) were involved in the cluster outbreak in a maternal and infant care center. In terms of clinical classification, 13 children (68%) had mild coronavirus disease 2019 (COVID-19) and 6 (32%) had common COVID-19, with no severe cases of COVID-19. The most common clinical symptoms were cough (100%) and fever (63%). The children with a normal peripheral white blood cell count accounted for 84%, and those with a normal lymphocyte count accounted for 68%. There were no significant abnormalities in platelet count, procalcitonin, liver function parameters (alanine aminotransferase and aspartate aminotransferase), and renal function parameters (creatinine and urea). Six children (32%) had obvious signs of pneumonia on chest CT. All 19 children were given symptomatic treatment, and 12 children (63%) were given aerosol inhalation of interferon α. All children were cured and discharged. Children infected with Omicron variant strains are more common in infants and young children, with mild symptoms and good prognosis. Most of the children have a history of close contact with persons infected with SARS-CoV-2, and epidemic prevention and control should be strengthened in places with many infants and children, such as maternal and infant care centers.

A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy

To provide a research basis for a safe and effective cell therapy for β-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression of β-globin. The human β-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB, and the transcription and translation of β-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line. Furthermore, the erythroid differentiation of CD34+ cells which were transduced lentiviral virus carrying human β-globin from normal human umbilical cord blood cells and peripheral blood cells of patients with β-thalassemia major were confirmed by colony formation assay, cell smear assay and flow cytometry. The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test. The human β-globin was expressed stably in the MEL cells, and CD34+ cells from health umbilical cord blood as well as PBMC from patient with β-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells. The β-globin expression and differentiation in CD34+ cells were demonstrated successfully in the NSG mouse for about 35 months after post-transplant. Stable β-globin expression through the optimization of HS4 from CD34+ in the third generation lentiviral vector is safe and effective for patients with severe β-thalassemia and other β-globin abnormal diseases.

A case study on Ayurvedic management of Immune Thrombocytopenic Purpura w.s.r to Tiryaka Raktapitta

Immune Thrombocytopenic Purpura is an autoimmune disorder which is characterized by destruction and decrease in the number of platelets count due to the presence of autoantibodies directed against platelet. There are two types of Idiopathic thrombocytopenic purpura i.e. acute and chronic forms which are characterized by mucocutaneous bleeding such as oral mucosa, heavy menstrual bleeding or gastrointestinal bleeding and low platelet count with normal peripheral blood cells and smear. Clinical features of ITP are ecchymosis and petechiae or thrombocytopenia incidentally found on a routine CBC. Bleeding disorders are explained in classics under the heading of Raktapitta. ITP can be correlated with Tiryaka Raktapitta as in this disorder. It is tridoshaja vayadhi and its manifestation will be subcutaneous bleeding. In this study, a 45 years old female patient had complaints of bleeding from gums and heavy menstrual bleeding for 4 years was treated with Guduchi Kwatha and Aamlaki churna, Shatavari churna, Vata churna, Nimba Arka, fresh Papaya patra swarasa for 6 months. Significant improvement was observed in patient condition and blood reports and platelet count is stable without any allopathic medications.

A large dataset of white blood cells containing cell locations and types, along with segmented nuclei and cytoplasm

Accurate and early detection of anomalies in peripheral white blood cells plays a crucial role in the evaluation of well-being in individuals and the diagnosis and prognosis of hematologic diseases. For example, some blood disorders and immune system-related diseases are diagnosed by the differential count of white blood cells, which is one of the common laboratory tests. Data is one of the most important ingredients in the development and testing of many commercial and successful automatic or semi-automatic systems. To this end, this study introduces a free access dataset of normal peripheral white blood cells called Raabin-WBC containing about 40,000 images of white blood cells and color spots. For ensuring the validity of the data, a significant number of cells were labeled by two experts. Also, the ground truths of the nuclei and cytoplasm are extracted for 1145 selected cells. To provide the necessary diversity, various smears have been imaged, and two different cameras and two different microscopes were used. We did some preliminary deep learning experiments on Raabin-WBC to demonstrate how the generalization power of machine learning methods, especially deep neural networks, can be affected by the mentioned diversity. Raabin-WBC as a public data in the field of health can be used for the model development and testing in different machine learning tasks including classification, detection, segmentation, and localization.

MT2-MMP is differentially expressed in multiple myeloma cells and mediates their growth and progression

ObjectiveMembrane type-matrix metalloproteinases (MT-MMPs) are known as key regulators of cancer progression/metastasis. However, their roles in the growth and progression of multiple myeloma (MM) have not been yet elucidated. Methods and materialsThe expression of 6 MT-MMPs in MM, B cell lines, and normal peripheral blood (PB) cells were measured by RT-PCR, qRT-PCR, flow cytometry, western blotting, and immunocytochemistry. B lymphocytes, CD19−/CD138−, and CD19−/CD138+ cells, known as malignant plasma cells (MPC), were sorted from bone marrow (BM) aspirations of 10 MM patients, and MT2-MMP expression was examined in these cells using qRT-PCR, flow cytometry and immunohistochemistry, and western blotting. Moreover, the expression of MT2-MMP in BM biopsies from 13 normal individuals and 14 MM patients was analyzed by immunohistochemistry. MT2-MMP was also knocked down in U266 cells using siRNA technology and the adhesion, invasion, migration abilities, and cell proliferation were determined and compared with scrambled ones in both in vitro and in vivo studies. ResultsOur results showed that MT2-MMP expression is significantly higher in MM cell lines and MPC cells than B cell lines and other PB- or BM-derived cells. MT2-MMP is expressed in BM biopsies from all 14 patients with MM, and 67.85% ± 32.38 of BM cells were positive for MT2-MMP. In contrast, only 0.38 ± 0.76 of BM biopsies from normal individuals were positive for MT2-MMP. Importantly, MT2-MMP was expressed in all the patients' BM biopsies at the diagnosis, but not in the remission phase. MT2-MMP siRNA significantly decreased adhesion, invasion, migration, and 3D cell proliferation of U266 cells. Moreover, in the xenographic model, MT2-MMP siRNA prevented the growth and development of plasmacytoma. Taken together, these data demonstrate that MT2-MMP is strongly expressed in MM cells and plays important role in the growth and progression of these cells, suggesting that MT2-MMP is an appropriate biomarker in diagnosis and therapeutic interventions of MM.

Immune thrombocytopenic purpura in an elderly male

Idiopathic Thrombocytopenic Purpura (ITP) is an acquired disorder in which there is immune mediated destruction of platelets and also inhibition of platelet release from the megakaryocytes.[1] The features are by mucocutaneous bleeding and a low platelet count with an otherwise normal peripheral blood cells and smear. ITP is a heterogeneous disease with varying severity and clinical course. It is mainly a disorder of children. In adults the peak prevalence is between 20-50 years.[2] The clinical features in adults are different from those seen in children. ITP in persons above 60 years is being increasingly recognized in literature. ITP in elderly differs in its presentation, course and severity compared to its manifestations in younger adults. The response to treatment is different and the presence of co-morbid conditions such as diabetes, hypertension and ischemic heart disease needs to be kept in mind in the management algorithm.[3] A case of ITP in elderly is presented herewith with a brief review of literature.

Eco-friendly synthesis of copper nanoparticles using Mentha pulegium leaf extract: characterisation, antibacterial and cytotoxic activities

ABSTRACT Green copper nanoparticles (M-Cu NPs) were successfully synthesised by using the extract of Mentha pulegium plant, a flowering plant in the family of Lamiaceae. The synthesised M-Cu NPs were characterized by advanced technical tools. The maximum absorbance, λmax was found to be 754 nm for M-Cu NPs. The morphological analysis substantiated the spherical shape for NPs with particle size range of 21‒48 nm. The plant extract and synthesised Cu NPs exhibited potential antibacterial activity against the bacterial pathogens, Staphylococcus aureus, Bacillus cereus, Escherichia coli and Klebsiella pneumonia. The maximum zone of inhibition of 12 mm was recorded against B. cereus bacteria. The cell cytotoxicity of M-Cu NPs on normal human lymphocytes was assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT). M-Cu NPs demonstrated a significant antibacterial activity against S. aureus and B. cereus and almost 98% of normal human peripheral blood cells were found to survive up to 25 μg/mL of M-Cu NPs.

IQGAP1: A Potential Therapeutic Target in Acute Myelogenous Leukemia (AML)

Introduction: While AML is phenotypically diverse, genome-wide sequencing studies indicate that the median number of non-synonymous mutations is eight (Science 339,1546, 2013) and that the same pathways are affected in tumors with distinct genetic alterations (Nature 453,1112-1116, 2008). These insights provided the impetus to immunoaffinity purify shared antigenic moieties in human AML and examine their prevalence as well as function.Methods: We employed a previously described method wherein immunization of rabbits with normal human peripheral blood cells whose surface charge had been modified by incubation with fluorodinitrobenzene (FDNB) elicited an antibody response that cross reacted against a broad range of human leukemias (Nature 232,197, 1971). Replicating the described methods WBCs from healthy volunteer donors were suspended at 1x107 cells per ml of PBS and mixed with equal volume of 0.86 mM FDNB for 12 to 15 minutes at room temperature. Three rabbits were immunized with FDNB-treated cells (experimental rabbits). A control rabbit was immunized with sham-treated cells (control rabbit). After complement inactivation, immune sera were absorbed against WBCs from healthy donors. Absorbed immune sera were tested in clinical AML samples by Western blotting. Immunoprecipitation of antigens from whole cell lysates of clinical AML samples was performed using IgG adsorbed on protein A/G Agarose beads. Liquid chromatography and mass spectrometry of the immunoprecipitated material was performed. Fold change in IQGAP1 expression in normal vs AML bone marrow was determined by qRT-PCR and from raw data from Gene Expression Omnibus at the NCBI. IQGAP1 expression was evaluated by immunohistochemistry in cell lines, bone marrow biopsies from AML patients and normal healthy volunteers. IQGAP1 expression was modulated by RNAi and the effect on proliferation and colony formation was measured in cell lines.Results: Western blotting of whole cell lysates of clinical AML samples revealed bands that were recognized by immune serum from experimental rabbits but not the control rabbit. Immuno-precipitation of antigens from whole cell lysates of clinical AML samples revealed the scaffolding protein IQGAP1 as being differentially recognized in independent experiments. We focused on IQGAP1 because it regulates multiple cellular functions including signaling pathways involved in proliferation and transformation and stimulates cell motility and invasion. It is overexpressed in colon cancer, pancreatic cancer, hepatocellular carcinoma, glioma, and other cancers. (Cell Signaling 21,1471-1678, 2009). Western blots of human AML samples probed with anti-IQGAP1 antibody revealed the predicted 190 kDa band, see figure. There was 3 to 9-fold greater expression of IQGAP1 expression in AML versus normal bone marrow observed by qRT-PCR. Analysis of raw data from Gene Expression Omnibus at the NCBI revealed fold change of -3.22636, p-value 2.62 x 10e-7 between normal bone marrow versus AML. Preliminary IHC studies showed strong membranous staining in three leukemia cell lines and bone marrow biopsies from primary AML bone marrow samples but weak partial staining in normal bone marrow. Knocking down expression of IQGAP1 in K562, MV4-11 and THP1 cell lines resulted in significant decrease in proliferation and colony formation as compared to effects of scramble control. Mean and SD of colonies per 2000 cells plated were for K562: naïve 311+/-46, IQGAP1 knockdown 37+/-12, scramble control 127+/-12, MV4-11: naïve 156+/37, IQGAP1 knockdown 55+/- 8, scramble control 147+/-16, and THP1 naïve 232+/-45, IQGAP1 knockdown 17+/-2.9, scramble control 150+/-24.Conclusions: 1. The scaffolding protein IQGAP1is overexpressed in AML compared to normal bone marrow. 2. Knocking down expression of IQGAP1 in AML cell lines resulted in significant decrease in proliferation and colony formation providing proof of concept that IQGAP1 is a potential therapeutic target in AML. [Display omitted] DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Novel Human Anti-HLA-Bw4 and B61 Monoclonal Antibodies Kill Malignant B Cells Via CDC/ADCC While Sparing Normal Peripheral Blood Cells

Introduction: The development of monoclonal antibodies (mAbs), such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab), has revolutionized the treatment of lymphoid malignancies. The potential efficacy of anti-HLA mAbs in treating hematological malignancies has been reported in several studies. Previous attempts to develop mAbs specific for some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of HLA molecule polymorphisms. To overcome this problem, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process takes approximately 1 month to produce human anti-HLA mAbs to treat myeloma and lymphoma.Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells were cultured in RPMI-1640 + 10% FCS containing a cytokine cocktail. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. Microarray chips were coated with a PBS-containing purified HLA antigen (10 μg/ml). After removing the antigen solution, 100 μl of the CD138+ cell suspension was added to the chip. After washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells. Finally, antigen-specific antibody-secreting cells (ASCs) from individual microwells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA was amplified and transfected into HEK293 cells to obtain a supernatant containing complete antibody molecules (Zaimoku et al., Blood 2017). The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry. Next-generation sequencing (Adaptive Biotechnologies Corp) was used to quantify the ASC frequency in PB of donors. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of mAbs were assessed using conventional methods. The following cell lines and PB mononuclear cells (PBMCs) from 5 healthy individuals who possessed Bw4 and/or B61 were used for CDC/ADCC assays: 5 B-lymphoblastoid cell lines (B-LCLs), 11 myeloma cell lines (AM01, IM-9, KMS-12BM, KMS-18, KMS-28PE, KMS-34, LP-1, NCI929, OPM-2, RPMI8226, and U266), 4 mantle cell lymphoma cell lines (JVM, Jeko, Mino, and Z138), and 2 diffuse-large B cell lymphoma cell lines (KPUM-UH1 and KPUM-MS3). Daratumumab, elotuzumab, and anti-influenza virus human mAbs were used as control mAbs.Results and Discussion: Two novel human mAbs specific for HLA-Bw4 or HLA-B61 were successfully generated. The ASC frequency that produced anti-Bw4 and B61 mAbs in the cytokine-stimulated PBMCs derived from the donors were 9.0 × 10−5 and 1.4 × 10−5, respectively. Flow cytometry showed that mAbs bound to all normal PB cells and malignant lymphoid cells with corresponding alleles, but the expression levels of the HLA alleles were lower in normal PBMCs than in malignant cells (HLA-Bw4-mean florescence intensity (MFI), 1098 ± 246 vs. 6556 ± 4045, P =0.02). mAbs showed CDC /ADCC activities against all 5 B-LCLs, 6 of 9 myeloma, and 2 of 2 lymphoma cell lines with corresponding alleles but not against 2 myeloma and 4 lymphoma cell lines without corresponding alleles; they showed very weak CDC activities against PBMCs derived from 5 healthy donors who have corresponding alleles (CDC, 39 ± 16 vs. 4 ± 3%, P =0.001). Four myeloma cell lines (IM-9, KMS-28PE, KMS-34, and U266) that had low CD38/high CD59 expression and were resistant to daratumumab were killed by anti-Bw4 and/or HLA-B61 mAbs. In keeping with a current report showing low CDC activities of daratumumab on myeloma cells that highly express CD55/CD59 (Nijhof et al., Blood 2016), some myeloma cell lines highly expressing CD59 were resistant to CDC induced by anti-Bw4/HLA-B61 mAbs despite their high expression levels of HLA alleles (Figure 1AB). Elotuzumab showed obvious CDC and ADCC activities against 0 of 11 and 2 (KMS-34 and U266) of 11 myeloma cell lines, respectively.Conclusions: NovelmAbs specific for HLA-Bw4 or B61 showed in vitro preferential killing of B-cell malignancies while sparing normal PBMCs, suggesting their potential usefulness in treating malignant lymphoma and myeloma. [Display omitted] DisclosuresTanaka:HLA Laboratory: Employment. Kawai:Wakunaga Pharmaceutical Co., Ltd.: Employment.

Platelet-Targeted Gene Therapy of Hemophilia a Using Hematopoietic Stem Cells Derived from Genome-Edited Induced Pluripotent Stem Cells

Hemophilia A (HA), an X-linked recessive hereditary bleeding disorder, is caused by mutations in the F8 gene, which encodes the coagulation factor VIII (FVIII). Replacement therapy is the mainstay in clinic, but it can also cause inhibitory antibodies and is therefore restricted in wide use. Effective therapy of HA with inhibitors needs to be considered as a matter of priority. Evidence has been obtained that functional FVIII can be ectopically expressed and stored in platelets after transplantation of F8 gene modified-hematopoietic stem cells (HSCs), and the stored-FVIII can be released during the activation of platelets to exert therapeutic effect on HA with inhibitors. This unique strategy, however, requires enough sources of HSCs and a safe and effective way of HSCs modification. Induced pluripotent stem cells (iPSCs) are capable of self-renewing, large-scale expansion and differentiating into almost all types of cells and tissues. These features make iPSCs a promising path in the field of regenerative medicine and cell therapy. The recently developed techniques of genome editing, especially the CRISPR/Cas9 system, provide a powerful tool for gene therapy, owing to significantly reduced risk of insertional mutagenesis as compared to the random integration in retroviral-mediated gene therapy. Hence, we seek to explore the possibility of utilizing iPSCs and CRISPR/Cas9 in the platelet-targeted gene therapy of HA.Here, we established a HA mouse iPSCs (HA-iPSCs) derived from mouse-tail tip fibroblasts, and verified the pluripotency of these cells. On the other hand, we synthesized a codon optimized B domain-deleted FVIII construct (opBDD-F8) in order to enhance the expression efficiency of FVIII. Indeed, our in vitro expression data showed that opBDD-F8 achieved a nearly 4-fold increase of FVIII level as compared to BDD-F8, either under control of the platelet specific GPαIIb promoter or CMV promoter. This increased FVIII expression was further confirmed by hydrodynamic tail vein injection. Then, we inserted the GPαIIb promoter controlled opBDD-F8 (2bopF8) cassette into the Rosa26 locus of HA-iPSCs via CRISPR/Cas9-mediated homologous recombination, and obtained 2bopF8-modified HA-iPSCs (2bopF8-HAiPSCs). No obvious off-target mutation was found in the clones. To generate functional HSCs for transplantation, we performed an in vivo differentiation strategy. We injected 2bopF8-HAiPSCs with hHoxB4 retrovirus transduction into immunodeficient mice to create teratoma, from which we isolated HSCs. The cells were subsequently transplanted into lethally irradiated HA recipient mice. Hematopoiesis was recovered in the recipients, and normal peripheral blood cell numbers sustained for more than 40 weeks. FVIII was detected in platelets of the recipients, suggesting that the cells were originated from 2bopF8-HAiPSCs and FVIII could be expressed during thrombopoiesis. Bleeding phenotype was corrected in the recipients, demonstrating that FVIII released during platelet activation was functional in ensuring coagulation cascade. Furthermore, we did secondary HSC transplantation by using the recipients as the donors. FVIII activity could be detected in the platelets of the new recipients, and bleeding phenotype was corrected in these animals, indicating that long-term HSCs could be generated from 2bopF8-HAiPSCs. During the whole period of research, the recipients were all in good health without teratoma formation or other sickness. Furthermore, we successfully produced chimeric mice with the 2bopF8-HAiPSCs. FVIII expression was observed in the platelets and HSCs were isolated from the mice. Transplantation of these HSCs into lethally irradiated HA mice reconstituted hematopoiesis and generated platelets with FVIII activity. In these mice, coagulation deficiency was also corrected.Taken together, our proof of principle studies suggest that the genome-edited 2bopF8-HAiPSCs could differentiate towards HSCs with physiological functions, and the functional platelets could be derived from these HSCs. Meanwhile, FVIII could be expressed in platelets and abrogate bleeding diathesis of HA mice. Thus, iPSC-derived HSCs together with genome editing tools represent a potential route for platelet-targeted gene therapy that is worth further investigation. DisclosuresNo relevant conflicts of interest to declare.

Molecular insights into the pathogenesis of follicular lymphoma

: Follicular lymphoma (FL) represents a group of B-cell neoplasms derived from germinal center (GC) B cells. A better understanding of the pathogenic mechanisms of FL is important for developing innovative therapies. The most common form of FL is primarily nodal and associated with the t(14;18). Recent advances obtained via genomic profiling have provided unprecedented insights into the pathogenesis of FL. Conventional FL evolves through multiple independent or convergent genetic pathways. The classical pathogenesis of t(14;18)-positive FL is a multi-stage and multi-hit process escalating along accumulation of genetic and epigenetic alterations, which starts from FL-like B cells, through premalignant lesions, into full-blown malignancy. Early precursor lesions have been recognized in the form of FL-like B cells in normal peripheral blood, and both in-situ follicular neoplasia (ISFN) and duodenal-type FL. In comparison, t(14;18)-negative FL is much more heterogeneous at the molecular level, and the underlying mechanisms are less well understood. Some variants of FL, while lacking upregulation of BCL2, share a common mutational profile with conventional FL, including mutations in epigenetic modifiers. These cases also show some clinical overlap with BCL2-positive FL, including mainly nodal involvement. Other forms of FL show more profound differences, both clinically and biologically. These emerge more clearly as separate entities and include FL, grade 3B, testicular FL (TFL), pediatric-type FL (PTFL), and primary cutaneous follicle center lymphoma (PCFCL). Mutations in epigenetic regulators and 1p36/TNFRSF14 abnormalities are highly recurrent and are seen across different subtypes of FL. Genetic profiling has offered important new insights, and will continue to impact the diagnostic approach, with changes in future classification schemes.

Proteomic Studies of Primary Acute Myeloid Leukemia Cells Derived from Patients Before and during Disease-Stabilizing Treatment Based on All-Trans Retinoic Acid and Valproic Acid.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive hematological malignancy, and the only possibility of cure is intensive antileukemic treatment. Such treatment is only possible for relatively young and fit patients, but there is a large subset of elderly/unfit patients above 65–70 years of age who cannot receive intensive treatment. The low-toxicity combination of valproic acid (VP) plus all-trans retinoic acid (ATRA) has an AML-stabilizing effect for a subset of patients, even those with chemoresistant relapse. This response may be an indirect effect due to the modulation of the metabolic environment, but in this article, we describe that the in vivo treatment with ATRA/VP also has direct effects on the fundamental functions of human AML cells. Furthermore, new and less intensive antileukemic therapies are now available (i.e., venetoclax, azacitidine, decitabine), and the combination of these new agents with ATRA/VP may represent a new therapeutic strategy in AML.All-trans retinoic acid (ATRA) and valproic acid (VP) have been tried in the treatment of non-promyelocytic variants of acute myeloid leukemia (AML). Non-randomized studies suggest that the two drugs can stabilize AML and improve normal peripheral blood cell counts. In this context, we used a proteomic/phosphoproteomic strategy to investigate the in vivo effects of ATRA/VP on human AML cells. Before starting the combined treatment, AML responders showed increased levels of several proteins, especially those involved in neutrophil degranulation/differentiation, M phase regulation and the interconversion of nucleotide di- and triphosphates (i.e., DNA synthesis and binding). Several among the differentially regulated phosphorylation sites reflected differences in the regulation of RNA metabolism and apoptotic events at the same time point. These effects were mainly caused by increased cyclin dependent kinase 1 and 2 (CDK1/2), LIM domain kinase 1 and 2 (LIMK1/2), mitogen-activated protein kinase 7 (MAPK7) and protein kinase C delta (PRKCD) activity in responder cells. An extensive effect of in vivo treatment with ATRA/VP was the altered level and phosphorylation of proteins involved in the regulation of transcription/translation/RNA metabolism, especially in non-responders, but the regulation of cell metabolism, immune system and cytoskeletal functions were also affected. Our analysis of serial samples during the first week of treatment suggest that proteomic and phosphoproteomic profiling can be used for the early identification of responders to ATRA/VP-based treatment.

Inhibition of Autophagy Enhances the Antitumor Effect of Thioridazine in Acute Lymphoblastic Leukemia Cells.

Acute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells that affects children and adults. Despite the high cure rates, drug resistance still remains a significant clinical problem, which stimulates the development of new therapeutic strategies and drugs to improve the disease outcome. Antipsychotic phenothiazines have emerged as potential candidates to be repositioned as antitumor drugs. It was previously shown that the anti-histaminic phenothiazine derivative promethazine induced autophagy-associated cell death in chronic myeloid leukemia cells, although autophagy can act as a “double-edged sword” contributing to cell survival or cell death. Here we evaluated the role of autophagy in thioridazine (TR)-induced cell death in the human ALL model. TR induced apoptosis in ALL Jurkat cells and it was not cytotoxic to normal peripheral mononuclear blood cells. TR promoted the activation of caspase-8 and -3, which was associated with increased NOXA/MCL-1 ratio and autophagy triggering. AMPK/PI3K/AKT/mTOR and MAPK/ERK pathways are involved in TR-induced cell death. The inhibition of the autophagic process enhanced the cytotoxicity of TR in Jurkat cells, highlighting autophagy as a targetable process for drug development purposes in ALL.

Myeloid-like γδ T cell subset in the immune response to an experimental Rift Valley fever vaccine in sheep

γδ T cells are a numerically significant subset of immune cells in ruminants, where they may comprise up to 70 % of all peripheral blood mononuclear cells (PBMCs) in young animals and 25 % in adults. These cells can be activated through traditional TCR-dependent mechanisms, or alternatively in a TCR-independent manner by pattern recognition receptors and have been shown to uptake antigen, as well as process and present it to αβ T cells. We have identified a novel CD11b+ subset of γδ T cells in normal sheep peripheral blood. An increase in the frequency of these cells in sheep peripheral blood in response to immunization with an experimental recombinant subunit Rift Valley fever (RVF) vaccine was observed. However, injection of the vaccine adjuvant ISA-25VG alone without the recombinant RVF virus antigens demonstrated the same effect, pointing to an antigen-independent innate immune function of CD11b+ γδ T cells in response to the adjuvant. In vitro studies showed repeatable increases of CD11b-, CD14-, CD86-, CD40-, CD72-, and IFNγ- expressing γδ T cells in PBMCs after 24 h of incubation in the absence of a mitogen. Moreover, the majority of these myeloid-like γδ T cells were demonstrated to process exogenous antigen even in the absence of mitogen. ConA activation increased CD25- and MHCII- expression in γδ T cells, but not the myeloid associated receptors CD14 or CD11b or co-stimulatory molecules such as CD86 and CD40. Considering the role of CD11b and CD14 in the activation of innate immunity, we hypothesize that this subpopulation of sheep γδ T cells may function as innate antigen presenting and pro-inflammatory cells during immune responses. The results presented here also suggest that stress molecules and/or damage-associated molecular patterns may be involved in triggering antigen presenting and pro-inflammatory functions of γδ T cells, given their appearance in vitro in the absence of specific stimulation. Taken together, these data suggest that the early appearance of γδ T cells following adjuvant administration and their possible role in early activation of αβ T cell subsets may non-specifically contribute to augmented innate immunity and may promote strong initiation of the adaptive immune response to vaccines in general.

Epidemiological and Clinical Findings of Short-Term Recurrence of Severe Acute Respiratory Syndrome Coronavirus 2 Ribonucleic Acid Polymerase Chain Reaction Positivity in 1282 Discharged Coronavirus Disease 2019 Cases: A Multicenter, Retrospective, Observational Study.

BackgroundShort-term recurrence of positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) polymerase chain reaction (PCR) in discharged coronavirus disease 2019 (COVID-19) patients attracts the public’s concern. This study aimed to determine the clinical and epidemiological results of such patients.MethodsThis retrospective study was conducted on 32 designated hospitals for COVID-19 patients discharged from January 14 to March 10, 2020. After 28-day followed-up, patients who tested positive again for SARS-CoV-2 RNA and confirmed by reverse-transcriptase polymerase chain reaction were re-admitted to hospital for further treatments. All of the close contacts of patients who tested positive again were asked to self-segregate for 14 days. Data of epidemiology, symptoms, laboratory tests, and treatments were analyzed in those patients, and their close contacts were investigated.ResultsOf 1282 discharged patients, 189 (14.74%) tested positive again for SARS-CoV-2 RNA during 28-day follow-up. The median time from discharge to the next positive test was 8 days (interquartile range [IQR], 5–13). Patients in the group that tested positive again were younger (34 vs 45 years, P < .001) with a higher proportion of moderate symptoms (95.77% vs 84.35%, P < .001) in the first hospitalization than in the negative group. During the second hospitalization, all patients who tested positive again showed normal peripheral white blood cells and lymphocytes and no new symptoms of COVID-19; 78.31% further improved on chest computed tomography scan compared with the first discharge, yet 25.93% accepted antiviral therapy. The median time of re-positive to negative test was 8 days (IQR, 4–15). None of the close contacts developed COVID-19.ConclusionsOur data suggest that the short-term recurrence of positive SARS-CoV-2 RNA in discharged patients is not a relapse of COVID-19, and the risk of onward transmission is very low. This provides important information for managing COVID-19 patients.

WNT7A Expression is Downregulated in T Lymphocytes after T-Cell Receptor Activation Due to Histone Modifications and in T-ALL by DNA Methylation.

WNT signaling pathway regulates several processes involved in the homeostasis of normal cells. Its dysregulation is associated with pathological outcomes like cancer. We previously demonstrated that downregulation of WNT7A correlates with higher proliferation rates in acute lymphoblastic leukemia. However, the regulation of this gene in pathological and normal conditions remains unexplored. In this work, we aimed to analyze the transcriptional regulation of WNT7A in leukemic cells and in normal T lymphocytes after a proliferative stimulus. WNT7A expression was measured in blood cells and in T lymphocytes after phytohemagglutinin-L (PHA-L) treatment or T-cell receptor (TCR) activation by qPCR and Western blot. Promoter methylation was assessed using methylation-sensitive restriction enzymes, and histone modifications were determined by chromatin immunoprecipitation and qPCR. In T-cell acute lymphoblastic leukemia (T-ALL), WNT7A expression is silenced through DNA methylation of CpG island in the promoter region. In normal peripheral blood cells, WNT7A is mainly expressed by monocytes and T lymphocytes. TCR activation induces the downregulation of WNT7A in normal T lymphocytes by changes in histone methylation marks (H3K4me2/3) and histone deacetylases. A proliferative stimulus mediated by IL-2 keeps WNT7A expression at low levels but in the absence of IL-2, the expression of this gene tends to be restored. Furthermore, after TCR activation and WNT7A downregulation, target genes associated with the WNT canonical pathway were upregulated indicating an independent activity of WNT7A from the WNT canonical pathway. WNT7A expression is silenced by long-term DNA methylation in T-ALL-derived cells and downregulated by histone modifications after TCR activation in normal T lymphocytes.