CD34-positive Cells Research Articles

Autologous stem cell transplantation in tandem with Anti-CD30 CAR T-cell infusion in relapsed/refractory CD30+ lymphoma

BackgroundLong-term outcome is unfavourable for relapsed/refractory (r/r) lymphoma patients who are resistant to salvage chemotherapy, even after subsequent autologous stem-cell transplantation (ASCT). Although anti-CD30 chimeric antigen receptor (CAR30) T-cell therapy induces high response rates in these patients, the duration of response is relatively limited.MethodsThis open-label, single-center and single-arm pilot study investigated the safety and efficacy of ASCT in tandem with CAR30 T-cell infusion in r/r CD30+ lymphoma. The primary endpoint was safety and key secondary endpoint was overall response rate, overall survival, progression-free survival, and duration of response.ResultsFive classical Hodgkin lymphoma (cHL) patients and 1 anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) patient were enrolled. The median age was 24 years. No patient had prior ASCT. Three patients (50.0%) relapsed for ≥ 2 times and 3 patients (50.0%) had primary refractory diseases. All had a Deauville score of 4 or 5, and 5 patients (83.3%) had a stable or progressive disease (SD/PD) at enrollment. All patients received myeloablative chemotherapy and infused CD34-positive hematopoietic stem cells (HSCs) and CAR30 T cells in tandem, with a median dose of 3.9 × 106/kg and 7.6 × 106/kg, respectively. Five paitents presented with cytokine release syndrome (CRS), all of which were grade 1. No neurotoxicity was observed. All patients had successful HSCs engraftment and reached an objective response, including 5 (4 cHL and 1 ALCL, 83.3%) with a complete response (CR) and 1 with a partial response (PR). With a median follow-up of 20.4 (range, 12.1–34.4) months, all remained alive and maintained their responses.ConclusionOur work demonstrates the combined administration of ASCT and CAR30 T-cell therapy is well-tolerate and highly effective in r/r cHL and ALCL, even in PET-positive or chemorefractory patients who are expected to have inferior outcome after ASCT, although further large-scaled validation in prospective clinical trial is warranted.Trial registration The trial was registered with the Chinese Clinical Trial Registry (ChiCTR, number ChiCTR2100053662).

In Vitro and In Vivo Characterization of 89Zirconium-Labeled Lintuzumab Molecule.

Objective: Positron emission tomography (PET) imaging is a powerful non-invasive method to determine the in vivo behavior of biomolecules. Determining biodistribution and pharmacokinetic (PK) properties of targeted therapeutics can enable a better understanding of in vivo drug mechanisms such as tumor uptake, off target accumulation and clearance. Zirconium-89 (89Zr) is a readily available tetravalent PET-enabling radiometal that has been used to evaluate the biodistribution and PK of monoclonal antibodies. In the current study, we performed in vitro and in vivo characterization of 89Zr-lintuzumab, a radiolabeled anti-CD33 antibody, as a model to evaluate the in vivo binding properties in preclinical models of AML. Methods: Lintuzumab was conjugated to p-SCN-Bn-deferoxamine (DFO) and labeled with 89Zr using a 5:1 µCi:µg specific activity at 37 °C for 1h. The biological activity of 89Zr-lintuzumab was evaluated in a panel of CD33 positive cells using flow cytometry. Fox Chase SCID mice were injected with 2 × 106 OCI-AML3 cells into the right flank. After 12 days, a cohort of mice (n = 4) were injected with 89Zr-lintuzumab via tail vein. PET/CT scans of mice were acquired on days 1, 2, 3 and 7 post 89Zr-lintuzumab injection. To demonstrate 89Zr-lintuzumab specific binding to CD33 expressing tumors in vivo, a blocking study was performed. This cohort of mice (n = 4) was injected with native lintuzumab and 24 h later 89Zr-lintuzumab was administered. This group was imaged 3 and 7 days after injection of 89Zr-lintuzumab. A full ex vivo biodistribution study on both cohorts was performed on day 7. The results from the PET image and ex vivo biodistribution studies were compared. Results: Lintuzumab was successfully radiolabeled with 89Zr resulting in a 99% radiochemical yield. The 89Zr-lintuzumab radioconjugate specifically binds CD33 positive cells in a similar manner to native lintuzumab as observed by flow cytometry. PET imaging revealed high accumulation of 89Zr-lintuzumab in OCI-AML3 tumors within 24h post-injection of the radioconjugate. The 89Zr-lintuzumab high tumor uptake remains for up to 7 days. Tumor analysis of the PET data using volume of interest (VOI) showed significant blocking of 89Zr-lintuzumab in the group pre-treated with native lintuzumab (pre-blocked group), thus indicating specific targeting of CD33 on OCI-AML3 cells in vivo. The tumor uptake findings from the PET imaging study are in agreement with those from the ex vivo biodistribution results. Conclusions: PET imaging of 89Zr-lintuzumab shows high specific uptake in CD33 positive human OCI-AML3 tumors. The results from the image study agree with the observations from the ex vivo biodistribution study. Our findings collectively suggest that PET imaging using 89Zr-lintuzumab could be a powerful drug development tool to evaluate binding properties of anti-CD33 monoclonal antibodies in preclinical cancer models.

CXCR4, CXCR5 and CD44 May Be Involved in Homing of Lymphoma Cells into the Eye in a Patient Derived Xenograft Homing Mouse Model for Primary Vitreoretinal Lymphoma.

Background: Primary vitreoretinal lymphoma (PVRL), a rare malignancy of the eye, is strongly related to primary central nervous system lymphoma (PCNSL). We hypothesized that lymphoma cells disseminate to the CNS and eye tissue via distinct homing receptors. The objective of this study was to test expression of CXCR4, CXCR5, CXCR7 and CD44 homing receptors on CD20 positive B-lymphoma cells on enucleated eyes using a PCNSL xenograft mouse model. Methods: We used indirect immunofluorescence double staining for CD20/CXCR4, CD20/CXCR5, CD20/CXCR7 and CD20/CD44 on enucleated eyes of a PCNSL xenograft mouse model with PVRL phenotype (PCNSL group) in comparison to a secondary CNS lymphoma xenograft mouse model (SCNSL group). Lymphoma infiltration was evaluated with an immunoreactive score (IRS). Results: 11/13 paired eyes of the PCNSL but none of the SCNSL group were infiltrated by CD20-positive cells. Particularly the choroid and to a lesser extent the retina of the PCNSL group were infiltrated by CD20+/CXCR4+, CD20+/CXCR5+, few CD20+/CD44+ but no CD20+/CXCR7+ cells. Expression of CXCR4 (p = 0.0205), CXCR5 (p = 0.0004) and CD44 (p < 0.0001) was significantly increased in the PCNSL compared to the SCNSL group. Conclusions: CD20+ PCNSL lymphoma cells infiltrating the eye co-express distinct homing receptors such as CXCR4 and CXCR5 in a PVRL homing mouse model. These receptors may be involved in PVRL homing into the eye.

Management of wounds with exposed bone structures using an induced-membrane followed by polymethyl methacrylate and second-stage skin grafting in the elderly with a 3-year follow-up.

The treatment of traumatic wounds with exposed bone or tendons is often challenging. An induced membrane (IM) is used to reconstruct bone defects, as it provides an effective and sufficient blood supply for bone and soft-tissue reconstruction. This study explored a novel two-stage strategy for wound management, consisting of initial wound coverage with polymethyl methacrylate (PMMA) and an autologous split-thickness skin graft under the IM. Fifty inpatients were enrolled from December 2016 to December 2019. Each patient underwent reconstruction according to a two-stage process. In the first stage, the defect area was thoroughly debrided, and the freshly treated wound was then covered using PMMA cement. After 4-6 weeks, during the second stage, the PMMA cement was removed to reveal an IM covering the exposed bone and tendon. An autologous split-thickness skin graft was then performed. Haematoxylin and eosin (H&E) staining and immunohistochemical analysis of vascular endothelial growth factor (VEGF), CD31 and CD34 were used to evaluate the IM and compare it with the normal periosteal membrane (PM). The psychological status and the Lower Extremity Function Scale (LEFS) as well as any complications were recorded at follow-up. We found that all skin grafts survived and evidenced no necrosis or infection. H&E staining revealed vascularised tissue in the IM, and immunohistochemistry showed a larger number of VEGF-, CD31- and CD34-positive cells in the IM than in the normal PM. The duration of healing in the group was 5.40 ± 1.32 months with a mean number of debridement procedures of 1.92 ± 0.60. There were two patients with reulceration in the group. The self-rating anxiety scale scores ranged from 35 to 60 (mean 48.02 ± 8.12). Postoperatively, the LEFS score was 50.10 ± 9.77. Finally, our strategy for the management of a non-healing wound in the lower extremities, consisting of an IM in combination with skin grafting, was effective, especially in cases in which bony structures were exposed in the elderly. The morbidity rate was low.

Skin fibrosis associated with keloid, scleroderma and Jorge Lobo's disease (lacaziosis): An immuno-histochemical study.

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α-smooth muscle actin (α-SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF-β. Higher expression of vimentin in comparison to α-SMA in all three lesion types was found. CD31- and CD34-positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16-positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α-actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105-positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206-positive cells in the fibrotic process. TGF-β was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial-to-mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role.

16 (PB002) - Predicting response to naratuximab emtansine, an anti-CD37 antibody-drug conjugate (ADC), in combination with rituximab in Diffuse Large B Cell Lymphoma (DLBCL), by analyzing the spatial arrangement of CD37 and CD20 positive cells using deep learning

Background: DLBCL is the most common type of Non-Hodgkin’s lymphoma, accounting for 30–40% of cases. Despite improvements in survival with standard of care treatment, up to 40% of patients have relapsed and/or refractory (R/R) disease. A phase 2 study (NCT02564744) evaluated the efficacy of naratuximab emtansine, an anti-CD37 ADC, in combination with rituximab, in 80 patients with R/R DLBCL. We performed an exploratory analysis of the study to find pathology-based biomarkers predictive of response.

S3576 Collagenous Gastritis: A Rare Cause of Anemia

Introduction: A relatively common cause of gastrointestinal symptoms including abdominal pain and diarrhea is microscopic colitis. Much less commonly seen is the related disorder collagenous gastritis, which relies on pathology and high clinical suspicion for diagnosis. We report on a case of a patient with this rare finding. Case Description/Methods: A 32-year-old male with a past medical history of achalasia status post myotomy presented with symptomatic normocytic anemia. His initial work up was significant for a hemoglobin of 5g/dL with reported use of non-steroidal anti-inflammatory drugs (NSAID). An esophagogastroduodenoscopy (EGD) showed atypical gastropathy with gross cobblestoning in the gastric body. Pathology showed chronic gastritis with focal gastric intestinal metaplasia, normal duodenal mucosal biopsies, and celiac disease serology was negative. Repeat EGD with gastric mapping demonstrated chronic gastritis, no intestinal metaplasia, negative H. pylori. Trichome stain highlighted increased subepithelial collagen layer which was confirmed by a gastrointestinal pathologist, consistent with collagenous gastritis. Colonoscopy was normal; colon biopsies were not taken as patient was asymptomatic. He was started on iron supplementation and remains well. (Figure) Discussion: Collagenous enteritides are a class of gastrointestinal disorders encompassing collagenous colitis, sprue, and gastritis. While collagenous colitis is relatively common, collagenous gastritis remains very rare. Typical symptoms include abdominal pain, nausea, diarrhea, and anemia. The most common endoscopic findings include gastric nodularity in the gastric body, erosions, and erythema. The diagnosis depends on pathologic findings of subepithelial infiltration with chronic inflammatory cells in the lamina propria, as well as deposition of collagen bands greater than 10 microns in thickness. The pathogenesis remains unclear, but medications like NSAIDs as well as smoking have been implicated. It is also associated with several autoimmune diseases including Sjogren’s and ulcerative colitis, causing postulation that there may be overexpression of HLA DR by epithelial cells and CD25 positive cells. There is currently no established therapy, and management is usually supportive care. Our case highlights the importance of a high level of suspicion for this rare disease, and pursuit of accurate diagnosis in the face of clinical perplexity. Likely etiology in our case appears to NSAIDs use which is known trigger for collagenous colitis.Figure 1.: Endoscopic view of the gastric body in collagenous gastritis.

Effect of Replacement of Wharton Acellular Jelly With FBS on the Expression of Megakaryocyte Linear Markers in Hematopoietic Stem Cells CD34.

Animal environments for the growth of stem cells cause the transmission of some diseases and immune problems for the recipient. Accordingly, replacing these environments with healthy environments, at least with human resources, is essential. One of the media that can be used as an alternative to animal serums is Wharton acellular jelly (AWJ). Therefore, in this study, we intend to replace FBS with Wharton jelly and investigate its effect on the expression of megakaryocyte-related genes and markers in stem cells. In this study, cord blood-derived CD34 positive HSCs were cultured and expanded in the presence of cytokines including SCF, TPO, and FLT3-L. Then, the culture of expanded CD34 positive HSCs was performed in two groups: 1) IMDM culture medium containing 10% FBS and 100 ng / ml thrombopoietin cytokine 2) IMDM culture medium containing 10% AWJ, 100 ng / ml thrombopoietin cytokine. Finally, CD41 expressing cells were analyzed with the flow cytometry method. The genes related to megakaryocyte lineage including FLI1 and GATA2 were also evaluated using the RT-PCR technique. Results: The expression of CD41, a specific marker of megakaryocyte lineage in culture medium containing Wharton acellular jelly was increased compared to the FBS group. Additionally, the expression of GATA2 and FLI1 genes was significantly increased related to the control group. This study provided evidence of differentiation of CD34 positive hematopoietic stem cells from umbilical cord blood to megakaryocytes in a culture medium containing AWJ.<br />.

S3597 Extensive Gastric Mucormycosis in a Patient With Newly Diagnosed Diffuse Large B-Cell Lymphoma of the Stomach

Introduction: Non-Hodgkin’s lymphoma (NHL) affects the gastrointestinal tract in 10 to 15 percent of the cases. Mucormycosis in the gastrointestinal tract is rare and may cause severe infection in immunocompromised patients with hematological malignancy. We present a case of newly diagnosed diffuse large B-cell lymphoma (DLBCL) with malignant masses in the stomach and extensive ulcerations with mucormycosis. Case Description/Methods: A 62 year-old man with a history of hypertension presented with left abdominal pain and weight loss, associated with decreased appetite. Abdominal exam unremarkable, but there was a nontender left supraclavicular mass. Initial labs revealed mild microcytic anemia with elevated ferritin and C-reactive protein. Computed tomography showed multiple lymphadenopathy above and below the diaphragm with gastric wall thickening at the fundus. Upper endoscopy revealed multiple duodenal nodules, mucosal inflammation at the antrum and multiple masses with non-bleeding deep ulcers at the fundus (Figure). Biopsy revealed CD20 positive large atypical cells with flow cytometry confirming the diagnosis of DLBCL with germinal center type. Fluorescent in situ histochemistry (FISH) was negative for double-hit lymphoma. Biopsy of the ulcers also showed Mucorales identified by GMS and PAS-D stains. Patient was started on posaconazole prior to initiation of chemotherapy with R-CHOP. Discussion: The stomach is the most common gastrointestinal location of extranodal NHL. Diagnosis is confirmed by immunohistochemistry or flow cytometry. Germinal center B cell (GCB) is associated with a favorable prognosis. FISH is used to rule out double or triple-hit high-grade lymphoma with MYC and BCL2 and/or BCL6 rearrangements. Standard treatment consists of six cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) with 60 percent cure rate in patients with DLBCL. Mucormycosis is caused by mold found in decaying vegetation and soil. Immunocompromised hosts are at risk of potentially fatal angioinvasive infection with local ischemia, necrosis and deep ulcerations. Gastrointestinal mucormycosis occurs in only 7% of reported cases, most commonly affecting the stomach. Empiric treatment with polyene antifungal is necessary. Surgical resection may be needed if there is massive bleeding or impending perforation. Delaying treatment increases mortality by two-fold. Early diagnosis and treatment of mucormycosis is imperative for successful outcome.Figure 1.: Endoscopic findings in the stomach; a. 5-8 mm nodular masses at the duodenal bulb; b. Scattered mucosal inflammation at the gastric antrum; c, d & e. Malignant mass with ulceration at the gastric fundus.

AML-230 Predictive Value of CD34-Positive versus CD34-Negative Leukemic Stem Cells on Survival Outcome in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) have emerged as a potential factor contributing to an overall dismal outcome in acute myeloid leukemia (AML). In this study, using flowcytometric immunophenotyping (FCMI), we have demonstrated LSC identification and quantification in both the CD34+ and CD34-compartments to find its relevance in predicting treatment outcomes. Furthermore, we correlated the impact of LSC quantification at diagnosis as a predictor of long-term survival. This is a prospective analysis of patients diagnosed with AML (per standard WHO criteria). Tertiary care cancer center. A total of 161 patients with a diagnosis of non-acute promyelocytic leukemia (non-APL) AML patients diagnosed over a period of two years (January 2019 to December 2020) were evaluated. Bone marrow aspirates collected in EDTA were processed for FCMI using a stain-lyse-wash technique with a singletube, 10-color comprehensive antibody panel. LSCs were identified primarily in the CD34+CD38- compartment; however, in a substantial number of CD34- AML, LSCs were segregated from the CD34-CD38- compartment using CD117 as an alternate gating marker and CD123, CD33, and CD11b as LSC-specific markers in all cases. 1. CD34+ versus CD34-LSC and 2. Baseline LSC% as a predictor of survival. LSCs were identified in 82.6% (133/161) of patients. CD34+ LSCs and CD34- LSCs were isolated in 82.53% (104/126) and 82.85% (29/35) of cases, respectively. The median survival times (in weeks) in CD34- LSCs was higher than in CD34+ LSCs; EFS (54 vs. 47; P=0.27), RFS (79 vs. 48; P=0.14), and OS (121 vs. 71; P=0.41) were found to be comparable in both groups. Furthermore, the total study cohort was divided into three groups based on diagnostic LSC percentage, i.e., LSCneg (LSC% <0.004%), LSClow (LSC%=0.004%-0.892%), and LSChigh (LSC% >0.892%). The LSChigh group had significantly shorter OS, EFS, and RFS compared to the LSClow and LSCneg groups. This study confirms the presence of LSCs beyond the usual CD34+CD38- compartment; however, there is no difference in survival outcomes in both groups. Also, LSC quantification is a powerful and independent prognostic parameter in AML predicting long-term survival and risk of relapse.

Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1

To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β, colony forming ability of AE9a leukemia cells was determined by colony formation assay. In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1β:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1β in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1β, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue.

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated β-galactosidase activity (SA-βgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-βgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies.

Construction of tandem diabody (IL-6/CD20)-secreting human umbilical cord mesenchymal stem cells and its experimental treatment on diffuse large B cell lymphoma

BackgroundMore than 40% patients with diffuse large B cell lymphoma (DLBCL) experienced relapse or refractory (R/R) lymphoma after the standard first R-CHOP therapy. IL-6 was reportedly associated with chemotherapy resistance of rituximab. Further, mesenchymal stem cells (MSCs) are known as the potential cell vehicle for their tropism toward tumor. A MSCs-based tandem diabody for treating DLBCL is currently lacking.MethodsWe constructed a tandem diabody (Tandab(IL-6/CD20)) with modified umbilical cord MSCs (UCMSCs) and designed a cell-based Tandab releasing system. Western blot, qPCR and immunofluorescence were used to confirm the construction and expression of lentivirus-infected UCMSCs. The vitality, apoptosis and homing abilities of UCMSCs were examined via CCK-8 assay, apoptosis, wound healing and migration analysis. Cell binding assay was used to demonstrate the targeting property of Tandab binding to CD20-positive DLBCL cells. Furthermore, we evaluated the viability of SU-DHL-2 and SU-DHL-4 by using CCK-8 and EDU assay after the treatment of UCMSCs-Tandab(IL-6/CD20).ResultsTandab protein peaked at 6273 ± 487 pg/ml in the medium on day 7 after cell culture. The proliferation and homing ability of UCMSCs did not attenuate after genetically modification. Immunofluorescence images indicated the Tandab protein bound to the lymphoma cells. UCMSCs-Tandab(IL-6/CD20) inhibited the growth of SU-DHL-2 or SU-DHL-4 cells in vitro.ConclusionsUCMSCs-Tandab(IL-6/CD20), which bound with both tumor-associated surface antigens and pro-tumor cytokines in tumor microenvironment, might serve as a potential treatment for DLBCL, evidenced by inhibiting the growth of SU-DHL-2 or SU-DHL-4 cells.

Characterisation of canine CD34+/CD45 diminished cells by colony-forming unit assay and transcriptome analysis.

Haematopoietic stem and progenitor cells (HSPCs) are used for transplantation to reconstruct the haematopoietic pathways in humans receiving severe chemotherapy. However, the characteristics of canine HSPCs, such as specific surface antigens and gene expression profiles, are still unclear. This study aimed to characterise the haematopoietic ability and gene expression profiles of canine bone marrow HSPCs in healthy dogs. In this study, the CD34 positive (CD34+) cells were defined as classical HSPCs, CD34+/CD45 diminished (CD45dim) cells as more enriched HSPCs, and whole viable cells as controls. Haematopoietic abilities and gene expression profiles were evaluated using a colony-forming unit assay and RNA-sequencing analysis. Canine CD34+/CD45dim cells exhibited a significantly higher haematopoietic colony formation ability and expressed more similarity in the gene expression profiles to human and mouse HSPCs than those of the other cell fractions. Furthermore, the canine CD34+/CD45dim cells expressed candidate cell surface antigens necessary to define the canine haematopoietic hierarchy roadmap. These results indicate that the canine CD34+/CD45dim cells express the HSPC characteristics more than the other cell fractions, thereby suggesting that these cells have the potential to be used for studying haematopoietic stem cells in dogs.

Plerixafor use in New Zealand 2016-2019: an observational study.

Plerixafor is used to mobilise CD34-positive stem cells for autologous transplantation to treat haematological malignancy. Funded in New Zealand since 2016, plerixafor can be used 'pre-emptively' to salvage a failing first attempt or as a 'rescue' strategy involving re-mobilising after 4 weeks. The rate of failed mobilisation and plerixafor uptake in New Zealand is not known, while international practice varies widely. To establish success rates for conditioning regimes used in New Zealand. We reviewed 203 consecutive patients with myeloma (n = 122) or lymphoma (n = 81) undergoing stem cell mobilisation between 1 January 2016 and 5 August 2019 at Christchurch hospital. We recorded demographics, conditioning regimens, harvest outcome and apheresis duration. Successful harvest was defined as collection of >2 × 106 CD34+ cells/kg. Seventeen percent of patients received plerixafor. Harvest success rates for lymphoma and myeloma respectively were 77% and 86% with standard conditioning, 95% and 100% with 'pre-emptive' plerixafor and 71% and 89% with 'rescue' plerixafor. 'Pre-emptive' plerixafor was non-inferior to standard conditioning. Following local guidelines resulted in at least one successful harvest for 96% lymphoma and 99% myeloma patients. Plerixafor strategies in New Zealand allow successful stem cell mobilisation for ≥96% of patients. Further research is required to investigate whether increased use would be cost-effective through reduced chemotherapy and apheresis duration, and improved graft quality.

Bone marrow-confined IL-6 signaling mediates the progression of myelodysplastic syndromes to acute myeloid leukemia.

Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risk of progression to acute myeloid leukemia (AML). The mechanisms of transformation of MDS to AML are poorly understood, especially in relation to the aging microenvironment. We previously established an mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with oversecretion of proinflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12–14 months of age. These mice showed myeloblast replacement of fibrotic bone marrow and widespread leukemic infiltration. Strikingly, depletion of IL-6 in these mice largely rescued the leukemic transformation and markedly extended survival. Single-cell RNA sequencing analyses revealed that DKO leukemic mice had increased monocytic blasts that were reduced with IL-6 knockout. We further revealed that the levels of surface and soluble IL-6 receptor (IL-6R) in the bone marrow were significantly increased in high-risk MDS patients. Similarly, IL-6R was also highly expressed in older DKO mice. Blocking of IL-6 signaling significantly ameliorated AML progression in the DKO model and clonogenicity of CD34-positive cells from MDS patients. Our study establishes a mouse model of progression of age-related MDS to AML and indicates the clinical significance of targeting IL-6 signaling in treating high-risk MDS.

Multiple myeloma diagnosed by flow cytometry plotting of CD19-positive and negative plasma cells

Multiple myeloma diagnosed by flow cytometry plotting of CD19-positive and negative plasma cells

Secondary rituximab-associated versus primary immunodeficiencies: The enigmatic border.

Rituximab (RTX), a chimeric monoclonal antibody targeting CD20-positive cells, is a valuable treatment option for malignant and benign immune-related disorders. The rationale of targeting the CD20 antigen relies on depletion of both healthy and autoreactive/malignant CD20-espressing cells, but normal B-cell reconstitution is expected within months after treatment. Nevertheless, a number of recent studies have documented prolonged B-cell deficiency associated with new-onset hypogammaglobulinemia in patients receiving RTX. Awareness of post-RTX hypogammaglobulinemia has become wider among clinicians, with a growing number of reports about the increased incidence, especially in children. Although these patients were previously regarded as affected by secondary/iatrogenic immunodeficiency, atypical clinical and immunological manifestations (e.g., severe or opportunistic infections; prolonged B-cell aplasia) raise concerns of delayed manifestations of genetic immunological disorders that have been unveiled by B-cell perturbation. As more patients with undiagnosed primary immune deficiency receiving RTX have been identified, it remains the challenge in discerning those that might display a higher risk of persistent RTX-associated hypogammaglobulinemia and need a tailored immunology follow-up. In this review, we summarize the principal evidence regarding post-RTX hypogammaglobulinemia and provide a guideline for identifying patients at higher risk of RTX-associated hypogammaglobulinemia that could harbor an inborn error of immunity.

Fludarabine and antithymocyte globulin-based conditioning regimen combined with post-transplantation cyclophosphamide for haploidentical allogeneic hematopoietic stem cell transplantation in patients with high-risk acute myeloid leukemia and myelodysplastic syndrome

IntroductionRelapse and graft-versus-host disease (GVHD) are the important complications influencing mortality for patients with high-risk acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD prophylaxis based on post-transplant cyclophosphamide (PTCy) or antithymocyte globulin (ATG) is widely used in haploidentical HSCT (haplo-HSCT). ObjectiveWe developed a modified intensified conditioning regimen including fludarabine (Flu) and investigated the effect of ATG-PTCy combination on transplant outcomes in high-risk AML and MDS compared with those patients who received only ATG as GVHD prophylaxis. MethodsA total of 80 patients with high-risk AML and MDS were divided into two groups and assigned to one-to-one pairing. ResultsThe modified ATG-PTCy group had more infused mononuclear cells, CD34-positive cells and CD3-positive cells than those in the ATG group (P < 0.05). The amount of platelet transfusion was higher in the ATG group than the modified ATG-PTCy group [2 (range, 1–6) U vs 2 (range, 1–5) U, P = 0.005]. The median of platelet recovery was better in the modified ATG-PTCy group than in the ATG group (12 days vs 13 days,P = 0.041). The infection rates of bacteria, fungi and virus at 100 days after transplantation were similar in both groups. Compared with the ATG group, individuals who received the modified ATG-PTCy regimen had higher 2-year GVHD- and relapse-free survival(GRFS) [60.0% (95%CI, 44.9–75.1%) vs 34.8% (95%CI, 19.9–49.7%), P = 0.028]; lower 180-day incidence of II-IV acute GVHD (aGVHD) [15.0% (95%CI, 4.0–26.0%) vs 39.8% (95%CI, 23.9–55.7%), P = 0.029]; lower 1-year incidence of moderate to severe chronic GVHD (cGVHD) [2.9% (95%CI, 2.0–3.8%) vs 19.6% (95%CI, 5.3–33.9%), P = 0.039]; and without an increase in the 2-year cumulative incidence of relapse (CIR) [19.5% (95%CI, 6.6–32.4%) vs 30.4% (95%CI, 15.3–45.5%), P = 0.291]. ConclusionsHigh-dose stem cells can promote blood cell implantation. The modified ATG-PTCy combination was associated with decreased risk of aGVHD and cGVHD, no increased risk of recurrence, and improved GRFS. It represents an effective strategy for high risk AML and MDS.

Clozapine-specific proliferative response of peripheral blood-derived mononuclear cells in Japanese patients with clozapine-induced agranulocytosis.

Although clozapine-induced granulocytopenia (CIG) is less severe than clozapine-induced agranulocytosis (CIA), and some patients with CIG may not go on to develop serious complications, clozapine is discontinued in cases of both CIA and CIG. Understanding the pathogenic mechanisms of CIA/CIG could provide better management of clozapine therapy. Recently, as a mechanistic insight into adaptive immune systems, European groups reported clozapine-specific proliferative responses and clozapine-specific T cells using blood taken from patients with CIA and/or CIG. The aims of our study are to support this mechanistic evidence and to investigate the difference in the lymphocyte response to clozapine between patients with CIG and those with CIA. Lymphocyte stimulation tests (LSTs) were conducted using CD25-positive cell-depleted peripheral blood-derived mononuclear cells (PBMCs) isolated from blood of four Japanese patients with CIA, four patients with CIG, and nine clozapine-tolerant subjects. Three of four patients with CIA and one of four patients with CIG showed proliferative responses to clozapine with a stimulation index of greater than 2. In contrast, none of the nine clozapine-tolerant subjects showed any response to clozapine. Olanzapine did not stimulate PBMCs of patients with CIA, patients with CIG, or clozapine-tolerant subjects. Clozapine- and CIA-specific lymphocyte reactions in a Japanese population provided supportive evidence that the pathogenesis of CIA is based on adaptive immune reactions. In addition, patients with CIG who show a positive response to an LST may at the very least not be chosen for clozapine-rechallenge and further prospective studies are desirable to verify this hypothesis.