Total Number Of Mononuclear Cells Research Articles

Clinical Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation Using Busulfan/Cyclophosphamide and Melphalan or Thiotepa As Conditioning Regimen in 37 Patients with Acute Megakaryoblastic Leukemia

Background Acute megakaryoblastic leukemia (AMKL) is a rare and heterogeneous subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of childhood AML and about 1% of adult AML. Patients with non-Down's syndrome AMKL (non-DS-AMKL) are often associated with a poor prognosis with a 3-year overall survival (OS) rate of less than 40%. Currently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative therapy for AMKL. However, the long-term survival post-transplant needs to be improved. Here we conducted a single-center study to evaluate the long-term efficacy and safety of allo-HSCT in 37 non-DS-AMKL patients using an innovative conditioning regimen of Busulfan/Cyclophosphamide (Bu/Cy) and Melphalan (MEL) or Thiotepa (TT). Methods From August 2015 to July 2023, a total of 37 non-DS-AMKL patients (M/F 21:16) were treated consecutively in our hospital (Hebei Yanda Lu Daopei Hospital). The median age was 2 years (range 1-9 years). Chromosomal abnormalities were present in 24 cases, with fusion genes as follows CBFA2T3/GLIS2 (5 cases) 13.5%, NUP98-KDM5A (2 cases) 5.4%, MLL-AF10 (3 cases) 8.1%, MLL-AF9 (1 case) 2.7%. Gene mutations: WT1 (10 cases) 27%, JAK2 (7 cases) 18.9%, EVI1 (6 cases) 16.2%, TET2 (4 cases) 10.8%, ASXL1 (4 cases) 10.8%, PTPN11 (4 cases) 10.8%, NRAS (3 cases) 8.1%, GATA1 (2 cases) 5.4%. Before transplantation, in bone marrow (BM), 29 patients were in complete remission (CR), 3 were in partial remission (PR) and the rest 5 were in non-remission (NR). Thirty-three patients received the haploidentical transplantation (donors from parents n=31, siblings n=2) including 1 with the 3 rd transplant, and 4 received matched-unrelated donor transplantation. The stem cell sources were from BM and peripheral blood stem cells (PBSCs) among the 33 related donors, and PBSCs for the 4 unrelated donors. Patients received a median cell number of total mononuclear cells 20.15×10 8/kg (10.7-32.85), CD34+ cells 12×10 6/kg(4.05-27.14) and CD3+ cells 4.66×10 8/kg(1.81-19.38). The conditioning regimens administered were modified Bu/Cy (fludarabine 30 mg/m 2/d×5d; cytarabine 2g/m 2/d×5d; Bu 3.2 mg/kg/d×4d, Cy 1.8g/m 2/d×2d; etoposide 100mg/m 2/d×2d; antithymocyte globulin 1.5mg/kg/d×5d) in 2 patients, Bu/Cy+MEL (MEL 55mg/m 2/d×2d) in 31 patients, and Bu/Cy+TT (TT 2.5mg/kg/d×2d) in the rest 4 patients. Cyclosporine A (CSA)+ mycophenolate mofetil (MMF)+ short methotrexate (sMTX) were used as graft-versus-host disease (GVHD) prophylaxis. Results The median follow-up time was 24 months (2-84 months), and all 37 cases survived the graft, with a median leukocyte engraftment of +13 days (9-21 days), and a median platelet engraftment of +9 days (4-38 days). BM was assessed one month after transplantation, with all grafts fully donor-type. The 5-year OS was 54.1% (Figure 1). There was a significantly higher OS in the CR group (5-year OS 69%) than the PR or NR groups (P<0.001) (Figure 2). Among the CR patients, no significant difference was observed in the 3 different condition regimen groups with modified BU/CY group OS of 50%, BU/CY+MEL group OS of 69.6% and BU/CY+TT group OS of 75% (P=0.869) (Figure 3). After transplantation, 17 patients died including 11 from relapse, 4 from GVHD, 1 from gastrointestinal bleeding and 1from cerebral hemorrhage. The cumulative recurrence rate (CIR) was 29.7%, among which the CIR of the CR group was 17.2%, the PR group of 66.7% and the NR group of 80% (P<0.001). Post-transplant, the incidence of acute GVHD (aGVHD) was 35.13% (Grade II-IV), and severe aGVHD (Grade III-IV) incidence was 18.91%. And 51.35% of patients developed chronic GVHD. Other complication incidences were cystitis at 16.21%, cytomegalovirus at 5.94%, Epstein-Barr viremia at 13.5% and infections at 21.62%. Conclusions Our study indicates that the use of modified Bu/Cy +MEL or +TT conditioning regimens in allo-HSCT procedures presents a feasible and effective approach, demonstrating an improvement in overall survival for non-Down Syndrome AMKL patients. Crucially, it was observed that achieving CR prior to transplantation significantly contributes to better outcomes. Therefore, it is strongly recommended that allo-HSCT is performed in CR status. However, to corroborate these findings, it is essential to conduct long-term, large-scale clinical research across multiple centers.

Brain-death donors as an alternative source of human stromal mesenchymal cells for cell-based therapy

Background & Aim In the last decades, human mesenchymal stromal cells (hMSC) have been widely investigated, due to their capacity to extensively proliferate in vitro while maintaining their differentiation potential into several mesodermal lineages. Besides, they have low immunogenicity, immunomodulatory effects, ability to migrate and graft at inflammation sites and secrete bioactive molecules. These properties are of great interest for applications in cell therapy and regenerative medicine and hMSCs are currently being evaluated in several clinical trials. Various sources of hMSCs have been studied, being bone marrow (BM) the most commonly used for autologous or allogeneic clinical applications. In the latter case, one of the difficulties is the scarce availability of living donors of BM. In contrast, the alternative use of deceased donors as an allogeneic source has been little explored. The aim of our study was to evaluate the relative potential of BM from brain-death donors (BDD) as a source of hMSCs for cell therapy, obtained from two different sites: femur (F) and iliac crest (IC). Methods, Results & Conclusion Adequate procedures were developed for BM collection and processing, as well as for in vitro culture, immunophenotyping and differentiation. Fourteen donor-matched BM samples (IC and F) were processed from seven deceased donors (Table 1). Our results show that it is possible to obtain viable hMSC from both sites. Although the total number of mononuclear cells (MNC) per gram of BM appears to be greater in the CI than in F, no statistically significant differences were observed between both sources. Neither the total content of fibroblastoid colony-forming units (CFU-F) per gram of BM nor the amount of CFU-F per MNC differ significantly. The comparative analysis of MSC yields showed that there were no significant differences in MSC obtained per gram of IC-BM or F-BM during the first three passages of cell culture. Furthermore, there were no statistical differences in cell doubling time and population duplications. Cultured cells from both sites exhibited the ability to adhere to plastic and express CD105, CD73, CD90 but not CD45, CD34, CD14 markers by flow cytometry analysis. Trilineage differentiation was also achieve, meeting the ISCT minimum criteria to define hMSC. These results show the feasibility to obtain hMSC from two sites in BDD, and underscores the potential of this new source as a suitable allogeneic source of hMSC for cell-based therapies.

Plasminogen and the Plasminogen receptor, Plg‐RKT, regulate efferocytosis and macrophage reprogramming

Inflammation resolution is an active process that functions to restore tissue homeostasis. Phagocytic clearance of apoptotic leukocytes (efferocytosis) at sites of inflammation plays an important role in inflammation resolution by inducing remarkable macrophage phenotypic plasticity. The M1/M2 nomenclature provides a basic classification system to distinguish proinflammatory (M1) from anti‐inflammatory/resolving macrophages (M2/Mres). Plasminogen (Plg) is synthesized by the liver and is the proenzyme of the broad‐spectrum serine protease, plasmin (Pla), which is the major enzyme responsible for fibrinolysis. The participation of the Plg system in the productive phase of inflammation is well known, but the involvement of the Plg system in the resolving phase of inflammation is just emerging. Here we investigated the effects of deletion of Plg and the Plg receptor, Plg‐RKT, separately, in the context of resolution of inflammation in vivo. Plg−/− and Plg‐RKT −/− mice and their respective wild type littermates were challenged by intrapleural injection of LPS (250ng/mouse, i.pl.) or PBS and cells present in the pleural cavity were harvested at 4, 8, 24 and 48 hr and processed for total and differential leukocyte counts and flow cytometric analyses. In this model of pleurisy there is a time‐dependent influx of leukocytes into the pleural cavity. The number of neutrophils and M1 macrophages peaks at 8 hr, and decreases at 48 hr. There is a significant increase in mononuclear cell recruitment into the pleural cavity that coincides with the resolution phase of inflammation and with prevalence of M2 and Mres macrophages at 48hr. In the current study, in the setting of LPS‐induced inflammation, increased numbers of M1 macrophages were present in Plg−/− mice 8h after LPS challenge without changes in number of M2 or Mres macrophages, as compared to Plg+/+ littermates [Plg+/+ = 155 ± 39 and Plg−/− = 301 ± 57 × 103 M1 macrophages/cavity (P < 0.05, n=6)]. There were no genotype‐dependent effects on the number of neutrophils recruited to the pleural cavity at all time points evaluated. At 48 hr the total number of mononuclear cells recruited to the pleural cavity was significantly decreased in both Plg−/− and Plg‐RKT −/− mice, relative to their respective wild type littermate controls [Plg+/+ = 3,432 ± 335 and Plg−/− = 2,377 ± 242 (P < 0.01, n=5); Plg‐RKT +/+ = 2,780 ± 341 and Plg‐RKT −/− = 1,725 ± 95 × 103 mononuclear cells/cavity (P < 0.05, n=6)]. Additionally, the frequency of the M2 marker, CD206, and Annexin A1 (both important for clearance of apoptotic cells) was decreased in Plg−/− macrophages 48 hr after LPS injection. To explore the roles of Plg and Plg‐RKT in efferocytosis, mice were injected with zymosan intraperitoneally, followed by an intraperitoneal injection of apoptotic human neutrophils labeled with carboxyfluorescein succinimidyl ester (CFSE). Significantly decreased efferocytosis of apoptotic neutrophils by Plg−/− macrophages and by Plg‐RKT−/− macrophages, compared with their wild type littermate controls was observed [Plg+/+ = 20.16 ± 3.57 and Plg−/− = 9.04 ± 3.50 (P < 0.05, n=5); Plg‐RKT +/+ = 18.11 ± 4.57 and Plg‐RKT−/− = 5.80 ± 2.90 frequency of total cells/cavity (P < 0.05, n=6)]. These results suggest that Plg regulates macrophage polarization and efferocytosis of apoptotic cells, thereby contributing to the resolution of inflammation and that these effects are mediated by the Plg receptor, Plg‐RKT.Support or Funding InformationCAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), National Institutes of Health (grant HL 081046 to L.A.M.) and by Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs (to R.J.P.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

Acquisition and Expansion of Adult Rat Bone Marrow Multipotent Mesenchymal Stromal Cells

Abstract This study was initiated in order to test a mini-invasive method of mesenchymal stem/progenitor cells (MS/PCs) isolation from a rat bone marrow (BM), and subsequently their expansion, differentiation, and evaluation of their immunophenotypic characteristics; and later their preservation as donor cells in an optimal condition for potential autotransplantation. The study group comprised of 6 adult male Sprague-Dawley (S-D) rats, weighing 480—690 g. The rats were anaesthetised by isoflurane with room air in a Plexiglas box and maintained by inhalation of a mixture of isoflurane and O2. Their femurs were surgically exposed and their diaphyses double-trephined. Then BM cells were flushed out by saline with heparin and aspirated into a syringe with a solution of DMEM (Dulbecco’s modified eagle’s medium) and heparin. The mononuclear cells from the BM were isolated by centrifugation and expanded in a standard culture medium supplemented with ES-FBS (es-cell-qualified foetal bovine serum), L-glutamine and rh LIF (recombinant human leukemia inhibitory factor). Following 14 days of passaging cultures, the cells were split into 2 equal parts. The first culture continued with the original medium. The second culture received additional supplementation with a human FGFβ (fibroblast growth factor beta) and EGF (epidermal growth factor). The populations of these cells were analysed by light-microscopy, then the mean fluorescence intensities (MFIs) of CD90 and Nestin were evaluated by a tricolour flow cytometry using monoclonal antibodies. The type of general anaesthesia used proved to be appropriate for the surgical phase of the experiments. All rats survived the harvesting of the BM without complications. The total number of mononuclear cells was 1.5—4.0 × 106 per sample and the proportion of CD90/Nestin expressing cells was < 1 %. Following 14 days of expansion, the cells became larger, adherent, with fibrillary morphology; the proportion of cells expressing CD90/Nestin increased to almost 25 %, i. e. they earned basic phenotypic characteristics of MSCs. Throughout the further cultivation a gradual decrease of the CD90/Nestin expression occurred. This suggested that the suitability of rat bone marrow derived MS/PCs for replacement therapy would probably be the highest between days 12—15 of cultivation and then would diminish.

Vaccine draining lymph nodes are a source of antigen-specific B cells

PurposeOur research is focused on using vaccine draining lymph nodes as a source of immune cells to better understand the immune response and to attempt to generate new anti-cancer reagents. Following a vaccine, harvesting the lymph node can only be done once. We endeavored to determine the range of times that B cells secreting anti-KLH antibodies were present in the node of KLH-vaccinated mice. ResultsFollowing vaccination the total number of mononuclear cells (MNCs) increased in the vaccine-draining lymph node (VDN). The percentage of MNCs that were B cells nearly doubled. B cells recovered from the node that secreted anti-KLH antibodies were evident by day 7. The number continued to increase and then slowly decreased over the observed time range to 28days after vaccination. The VDN, compared to the spleen, the bone marrow and the nonVDN, contained a higher percentage of B cells that secreted anti-KLH antibodies. ConclusionsAfter a vaccine, there is a multi-week window of time when an increasing number of B cells are present in a VDN that secrete anti-KLH antibodies. These results support using the VDN as a source for B cells that secrete anti-vaccine antibodies.

Subtyping of B-Cell Malignancies By B-Cell Subset Associated Gene Signatures (BAGS), Generated from Human Primary and Secondary Lymphoid Organs.

BackgroundToday's diagnostic tests for B-cell malignancies reflect the criteria of the updated WHO classification based on cellular biomarkers and clinicopathologic heterogeneity. To that end, we proposed a new biological subtyping of malignancies by B-cell subset associated gene signatures (BAGS), generated from the normal B-cell hierarchy in primary and secondary lymphoid organs. Our concept is that BAGS can be used for identification of the "cell of origin" subtyping in selected B-cell malignancies (1), as recently shown for diffuse large B-cell lymphoma (DLBCL) based on B-cell sorting of normal tonsil tissue (2), and currently investigated in Multiple myeloma (MM).Material and MethodsWe used multiparametric flow cytometry (MFC) phenotyping by a multicolor single tube Euro flow standard approach (CD3/CD10/CD19/CD20/CD27/CD34/CD38/CD45) to identify and sort distinct B-cell subset of 20,000-50,000 cells from normal sternal bone marrow for microarray analysis. Isolated mRNA from sorted subsets of PreB-I, PreB-II, immature (I), naïve (N), memory (M) and plasma cell (PC) was amplified and hybridized to Affymetrix Gene Chip Human Exon 1.0 ST array to generate gene expression profiles (GEP).Construction of the bone marrrow specific BAGS was based on median-centered probe sets from the GEP data using regularized multinomial regression with 5-6 discrete outcomes representing B-cell subtypes using the software R.The classification system was validated using online available GEP data from tumor biopsy samples of newly diagnosed MM patients. Each patient tumor GEP data underwent BAGS assignment according to the highest predicted probability score above 0.15 or was otherwise unclassified. To compensate for cohort-wise technical batch effects, each cohort was median centered and adjusted probe set-wise to have same variance as the normal bone marrow specific data.ResultsIn bone marrow from sternum splits, the CD45+ leukocyte population accounted for a median of 55% (range 39-63%) of total number of mononuclear cells aspirated and the B-cell compartment was enumerated as the CD45+/CD19+/CD3- compartment of median 14% (3-27%). Among these 0.7% (0.2-2.4%) were PreBI, and 28% (6-38%) were PreBII subsets. A median of 4% (2-6%) were I cells and 29% (16%-39%) were N cells. The post-germinal cells comprised a median of 5 % (2-10%) M subsets and 19 % (13-30%) PC subsets.Following FACS and GEP of these minor subsets we validated the quality of each subset by expression of the used CD genes and expected transcription factors (TF) as illustrated in Figure 1A-D and published for DLBCL (2).The resultant tumor assignments exhibited BAGS subtyping frequencies of patient cases in available cohorts of MM (N=1,126) as shown in Table 1 and in DLBCL (N= 1,139) as published (2). The percentage of patients in each subtype was not significantly different between data set of MM, as in DLBCL (2)ConclusionWe have validated datasets of distinct normal B-cell subsets and generated bone marrow and tonsil specific BAGS to be used for subtyping of multiple myeloma (2) and diffuse large B cell lymphomas (3), with impact on staging and prognosis.

Effects of T cell-specific Blimp-1 on the number and function of lymphocytes in mice

Objective To investigate the effects of B lymphocyte-induced maturation protein-1 (Blimp-1) on the number and function of splenic lymphocytes. Methods The mice with defective Blimp-1 in T cells were generated by cross-breeding B6.Blimp-1flox/flox mice with B6.Lck-Cre mice. The mononuclear lymphocytes isolated from spleen of T cell conditional Blimp-1 knockout (Blimp-1CKO) mice and wild type (WT) C57/B6 mice were comparatively analyzed. Alterations of CD4+ T and CD8+ T cell subsets, the secretion of cytokines as well as the expression of C-C chemokine receptor type 7 (CCR7) and Sphingosine-1-phosphate receptor 1 (S1P1) in mice from the two groups were analyzed by flow cytometry. The changes of CD19+ B cell subsets were also detected. Results Compared with WT mice, the total numbers of mononuclear cells, T and B lymphocytes were all significantly increased in Blimp-1CKO mice (P<0.05). The absolute numbers of CD4+ T, CD8+ T and CD19+ CD5+ CD1d+ B cells in mice form Blimp-1CKO group were higher than those of the control group (P<0.05), however, no significant differences with the percentages of these cell populations were observed between two groups. Higher numbers and percentages of CD19+ CD5+ B cells were detected in mice from Blimp-1CKO group (P<0.01). The Blimp-1CKO mice showed increased secretion of IFN-γ, TNF-α, IL-17 and IL-2, but decreased expression of CCR7 on CD8+ T cells as compared with WT mice (P<0.05). No significant differences with the changes of S1P1 were found between the two groups. Conclusion Blimp-1 played an important role in the maintenance of number, phenotype and function of T cells. Furthermore, not only T cells but also B cell subsets in mice were affected by the deletion of Blimp-1 in T cells. Key words: B lymphocyte-induced maturation protein-1; T lymphocyte; B lymphocyte

Characteristics of Myeloid Differentiation and Maturation Pathway Derived from Human Hematopoietic Stem Cells Exposed to Different Linear Energy Transfer Radiation Types

Exposure of hematopoietic stem/progenitor cells (HSPCs) to ionizing radiation causes a marked suppression of mature functional blood cell production in a linear energy transfer (LET)- and/or dose-dependent manner. However, little information about LET effects on the proliferation and differentiation of HSPCs has been reported. With the aim of characterizing the effects of different types of LET radiations on human myeloid hematopoiesis, in vitro hematopoiesis in Human CD34+ cells exposed to carbon-ion beams or X-rays was compared. Highly purified CD34+ cells exposed to each form of radiation were plated onto semi-solid culture for a myeloid progenitor assay. The surviving fractions of total myeloid progenitors, colony-forming cells (CFC), exposed to carbon-ion beams were significantly lower than of those exposed to X-rays, indicating that CFCs are more sensitive to carbon-ion beams (D 0 = 0.65) than to X-rays (D 0 = 1.07). Similar sensitivities were observed in granulocyte-macrophage and erythroid progenitors, respectively. However, the sensitivities of mixed-type progenitors to both radiation types were similar.In liquid culture for 14 days, no significant difference in total numbers of mononuclear cells was observed between non-irradiated control culture and cells exposed to 0.5 Gy X-rays, whereas 0.5 Gy carbon-ion beams suppressed cell proliferation to 4.9% of the control, a level similar to that for cells exposed to 1.5 Gy X-rays. Cell surface antigens associated with terminal maturation, such as CD13, CD14, and CD15, on harvest from the culture of X-ray-exposed cells were almost the same as those from the non-irradiated control culture. X-rays increased the CD235a+ erythroid-related fraction, whereas carbon-ion beams increased the CD34+CD38− primitive cell fraction and the CD13+CD14+/−CD15− fraction. These results suggest that carbon-ion beams inflict severe damage on the clonal growth of myeloid HSPCs, although the intensity of cell surface antigen expression by mature myeloid cells derived from HSPCs exposed to each type of radiation was similar to that by controls.

Differential Lumbar Spinal Cord Responses among Wild Type, CD4 Knockout, and CD40 Knockout Mice in Spinal Nerve L5 Transection-Induced Neuropathic Pain

BackgroundOur previous studies have indicated that both lumbar spinal cord-infiltrating CD4+ T cells and microglial CD40 contribute to the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain spinal nerve L5 transection (L5Tx). To further delineate the CD4 and CD40-mediated mechanisms involved in the development of L5Tx-induced neuropathic pain behaviors, we examined the lumbar spinal cord mononuclear cells of wild type (WT) BALB/c, BALB/c-CD4 knockout (KO), and BALB/c-CD40 KO mice via flow cytometry.ResultsIn WT mice, L5Tx induced significant but transient (at day 3 and/or day 7) increases of the total numbers of mononuclear cells, microglial cells (CD45loCD11b+), and infiltrating leukocytes (CD45hi) in the ipsilateral side of the spinal cord. In CD4 KO mice, significant elevation of microglia was detected only on day 7 post-L5Tx, while no significant increase in infiltrating leukocytes post-L5Tx was observed. CD40 KO mice did not exhibit any of the changes observed in WT mice. Furthermore, neutralizing CD40 antibody treatment indicated an early involvement of CD40 signaling in the development of L5Tx-induced mechanical hypersensitivity.ConclusionsAltogether, data indicate that both CD4 and CD40 play a role in L5Tx-induced leukocyte infiltration into the lumbar spinal cord but have differential contributions to spinal cord microglial activation following peripheral nerve injury.

MRI of the proximal femur predicts marrow cellularity and the number of mesenchymal stem cells

To determine whether the marrow conversion index (MCI) in MRI is related to the total number of mononuclear and mesenchymal stem cells (MSCs) in proximal femoral metaphysis of patients with hip osteoarthritis. Thirty-two hips of 32 consecutive patients who underwent total hip arthroplasty (THA) for hip osteoarthritis were included in this study. MRI of the hip was performed preoperatively and MCI was subsequently calculated. Three-milliliter bone marrow samples were obtained from the proximal femur during THA and the number of total mononuclear cells was determined using a hemocytometer. Colony forming unit-fibroblasts (CFU-Fs) assays of MSCs were performed by transferring a total of 2 × 10(4) mononuclear cells to each of five 60-mm plates. One week later, the numbers of colonies were counted. The total number of mononuclear cells decreased with increasing MCI. Likewise, the prevalence and total number of CFU-Fs increased with increasing number of total mononuclear cells, and decreased with increasing MCI. Our results suggest that measurement of MCI in MRI can be an objective and noninvasive method to predict marrow cellularity and the number of MSCs in patients with hip osteoarthritis.

Use of day 4 CEP and baseline CXCR4 plus CEC as predictive markers for bevacizumab in mCRC.

3599 Background: Bevacizumab plus chemotherapy has become a standard therapy for metastatic colorectal cancer (mCRC). The purpose of this study is to examine the role of the circulating endothelial cell (CEC) subpopulations in bevacizumab. Methods: All patients were enrolled using institutional review board-approved protocols in the Cancer Institute Hospital and from whom written informed consent had been obtained. Eligibility criteria: PS (ECOG) of 0 to 2; histopathology of adenocarcinoma; adequate major organ functions. CEC subpopulations from whole blood drawn before treatment initiation (baseline), day 4, two weeks, and eight-twelve weeks after initiation of chemotherapy were isolated and enumerated using flow cytometry. The proportions of CECs or CEPs were calculated as percentages of the total number of mononuclear cells after an evaluation of at least 50,000 cellular events to detect CECs and CEPs (CD31 positive, CD34 highly positive, CD133 positive, and CD45 slightly positive fraction). To further characterize CEC subpopulations, four different cell surface markers including VEGFR1, VEGFR2, Tie-2, and CXCR4 were used for categorizing CECs. Results: From July 2007 to January 2009, 82 patients with measurable mCRC were enrolled into a prospective study. Combining bevacizumab with FOLFOX4 markedly decreased CEP levels on day 4 as compared with an FOLFOX alone (p = 0.03). The logistic regression interaction test revealed that bevacizumab affected the level of CEP of day 4 whereas it did not affect the other factors including adverse effects. The cases that have CEPs less than 0.04% of PBMC on day 4 were significantly associated with longer PFS and OS as compared with the cases of 0.04% or more CEPs (logrank test; p < 0.001, p = 0.02, respectively). In addition, less than 20% of CXCR4+ cells in CECs at baseline were associated with significant longer PFS and OS as compared with that of others (p = 0.006, p = 0.018, respectively). In contrast, there was no correlation between baseline VEGFR1, VEGFR2, or Tie-2 positive CEC levels and outcome. Conclusions: The levels of CEPs on day4 and the proportion of CXCR4+ CECs at the baseline were correlated with the prognosis of bevacizumab combination chemotherapy. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Chugai Pharma, Yakult Honsha Co., Ltd

Expression of IFN‐γ before and after treatment of oral lichen planus with 0.1% fluocinolone acetonide in orabase

Oral lichen planus (OLP) is a common chronic inflammatory mucosal disease in which T-cell-mediated immune responses are implicated in the pathogenesis. The purpose of this study was to investigate the effect of 0.1% fluocinolone acetonide in orabase (FAO) on the in situ expression of IFN-gamma in patients with OLP. Twenty OLP patients were enrolled in this study. Biopsy specimens and serum samples were obtained before and 1-month after the treatment with 0.1% FAO. In situ expression and serum levels of IFN-gamma were determined using immunohistochemistry and ELISA, respectively. The number of IFN-gamma-positive mononuclear cells in OLP lesions before the treatment was significantly higher as compared with that after the treatment. Similarly, the mean number of total mononuclear cells was clearly decreased after the treatment. However, the serum levels of IFN-gamma were not detectable. Our results suggest that IFN-gamma expression in OLP tissue may involve in the immunopathogenesis and the treatment with 0.1% FAO had an immunomodulating effect on the decrease of IFN-gamma.

Early-Life Psychological Stress Exacerbates Adult Mouse Asthma via the Hypothalamus–Pituitary–Adrenal Axis

Despite accumulating evidence that psychological stress has a short-lasting detrimental effect on asthma, little is known about the way stress in childhood predisposes to adult asthma. Using a communication box, we investigated the long-lasting effect of early psychological and physical stress on adult asthma in mice. Male BALB/c mice were exposed to either psychological stress or physical stress three times (every other day) during their fourth week of life. The mice were sensitized to ovalbumin at 8 and 10 weeks, and an ovalbumin airway challenge was conducted at the age of 11 weeks. Twenty-four hours after ovalbumin challenge, both psychological and physical stress-exposed mice exhibited a significant acceleration in the number of total mononuclear cells and eosinophils and airway hyperresponsiveness compared with control mice. No differences in serum anti-OVA-specific immunoglobulin E levels were found between stress-exposed and control animals after antigen sensitization. In the psychological stress group, but not in the physical stress group, an elevation of the serum corticosterone levels during ovalbumin challenge was significantly attenuated in comparison with the control group. Moreover, pretreatment with RU-486, a glucocorticoid receptor antagonist, before ovalbumin challenge completely inhibited a psychological stress-induced exacerbation of asthma. However, pretreatment with GR-82334, a neurokinin-1 receptor antagonist, failed to affect physical stress-induced augmentation of airway inflammation. Early psychological and physical stresses aggravated adult asthma via hyporesponsiveness of the hypothalamic-pituitary-adrenal axis during antigen challenge and via a pathway(s) distinct from the hypothalamic-pituitary-adrenal axis or neurokinin-1 receptors.

Increased expression of activation markers in renal cell carcinoma infiltrating lymphocytes.

As manifested by the presence of immune competent cells, failure to control the progression of renal cell carcinoma by a local immune response attests to impaired local cell mediated immunity. To test this hypothesis we compared the expression of T-cell activation markers in renal cell carcinoma infiltrating lymphocytes with the expression of activation markers of peripheral blood lymphocytes in the same patients. Tumor infiltrating lymphocytes were harvested from a patient with renal cell carcinoma undergoing radical nephrectomy. Peripheral blood was obtained before surgery. Tumor infiltrating and peripheral blood lymphocytes were incubated with monoclonal antibodies defining specific differentiation and activation markers on the cell surface, and analyzed by flow cytometry. Cell subsets are expressed as a fraction of the total number of mononuclear cells. The T-cell subset level was significantly higher in peripheral blood than in renal cell carcinoma tissue of the same patient. However, the level of activated T-cell subset expressing HLA-DR was significantly higher in renal cell carcinoma tissue than in peripheral blood. The levels of interleukin-2 receptor and transferrin receptors expressing T-cell subsets were also significantly higher in carcinoma tissue than in peripheral blood. Natural killer cells were found in significantly higher proportions in renal cell carcinoma than in peripheral blood. These results point to significant activation of T, B and natural killer tumor infiltrating lymphocytes. The inability of tumor infiltrating lymphocytes to mount an effective immune response to renal cell carcinoma may be secondary to the presence of suppressive factors in the tumor that prevent tumor infiltrating lymphocytes from transforming into effector cells. These factors may be particularly valuable for the further study of renal cell carcinoma-host interactivity.

Recombinant TAT-HOXB4 Protein Promotes Ex Vivo Expansion of Primitive Human Hematopoietic Cells.

The clinical application of therapeutic protocols depending on hematopoietic stem cell (HSC) transplantation for long term reconstitution with donor-derived HSCs, particularly in patients previously exposed to intensive radiation or chemo-therapy, or when grafts are purged of infiltrating malignant or alloreactive T cells, can be severely hampered by limited numbers of HSCs in the graft. In mouse bone marrow transplantation models, engineered overexpression of HOXB4 has been one of the most potent stimulator of HSC expansion identified to date. The simple addition of soluble recombinant TAT-HOXB4 protein was also recently reported to enable rapid in vitro expansion of mouse HSCs that retain their in vivo proliferation and differentiation capacity. To test the feasibility of using TAT-HOXB4 as a stimulator of human HSC expansion, we performed a series of experiments using CD34+ populations isolated from healthy volunteers. The CD34+ cell populations were cultured in X-Vivo medium supplemented with Stem Cell Factor (300 ng/mL) and G-CSF (50 ng/mL) in the presence or absence of TAT-HOXB4 protein (50 nmol/L) for 4 days. In response to TAT-HOXB4, total numbers of mononuclear cells demonstrated a modest but distinct 2-fold increase compared to controls. TAT-HOXB4 treatment had the largest proliferation enhancing effect on more primitive cell populations such as CFU-GEMM, BFU-E and BFU-Meg, whose numbers increased 26.5 ± 1.4 fold (mean±S.D.), 2.2 ± 0.7 fold and 2.1 ± 0.2 fold, respectively, over their input values, and 19.1 ± 1.3 fold, 2.7 ± 0.7 and 31 ± 3.4 fold, respectively, compared to growth factor only controls. In response to TAT-HOXB4, the total numbers of CD34+CD38-Lin- cells increased 2.1 ± 0.7 fold above their starting numbers compared to a 1.5 ± 0.5 fold loss of this population in control cultures. HSC numbers were enumerated at the beginning, and after a 4-day TAT-HOXB4 treatment period using a NOD/SCID repopulation assay. In response to 50 nM TAT-HOXB4, NOD/SCID repopulating cell (SRC) numbers increased ~2-fold over their input values, compared to a 9-fold loss in control cultures without TAT-HOXB4. These results show that recombinant TAT-HOXB4 protein has the capacity to rapidly induce ex vivo expansion of primitive human bone marrow populations, and suggest that optimization of treatment conditions will rapidly lead to clinically useful expansion of human HSCs.

Increased Expression of Activation Markers in Renal Cell Carcinoma Infiltrating Lymphocytes

As manifested by the presence of immune competent cells, failure to control the progression of renal cell carcinoma by a local immune response attests to impaired local cell mediated immunity. To test this hypothesis we compared the expression of T-cell activation markers in renal cell carcinoma infiltrating lymphocytes with the expression of activation markers of peripheral blood lymphocytes in the same patients. Tumor infiltrating lymphocytes were harvested from a patient with renal cell carcinoma undergoing radical nephrectomy. Peripheral blood was obtained before surgery. Tumor infiltrating and peripheral blood lymphocytes were incubated with monoclonal antibodies defining specific differentiation and activation markers on the cell surface, and analyzed by flow cytometry. Cell subsets are expressed as a fraction of the total number of mononuclear cells. The T-cell subset level was significantly higher in peripheral blood than in renal cell carcinoma tissue of the same patient. However, the level of activated T-cell subset expressing HLA-DR was significantly higher in renal cell carcinoma tissue than in peripheral blood. The levels of interleukin-2 receptor and transferrin receptors expressing T-cell subsets were also significantly higher in carcinoma tissue than in peripheral blood. Natural killer cells were found in significantly higher proportions in renal cell carcinoma than in peripheral blood. These results point to significant activation of T, B and natural killer tumor infiltrating lymphocytes. The inability of tumor infiltrating lymphocytes to mount an effective immune response to renal cell carcinoma may be secondary to the presence of suppressive factors in the tumor that prevent tumor infiltrating lymphocytes from transforming into effector cells. These factors may be particularly valuable for the further study of renal cell carcinoma-host interactivity.

Indirect demonstration of the lifetime function of human thymus.

The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent. Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker-positive CD2, CD4 and CD8 cells, percentages of double-positive CD4CD8 and CD2CD8 cells, double-negative CD4CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens. The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue. The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age. The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age-related decline of the total number of mononuclear cells (-0.024 on a semilogarithmic scale). The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4+ (P=0.017) and double-positive CD4+CD8+ (P=0.016) cells, which tended to decrease with age. The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age. We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life. With respect to the number of involved lymphoid cells, the function exponentially decreases with age.

Marrow sensitization to 5-fluorouracil using the ligands for Flt-3 and c-Kit

In vivo administration of c-kit ligand (KL) expands early hemopoietic progenitors and stem cells and sensitizes clonogenic progenitors to 5-FU-mediated cell death. Studies were performed to determine whether the in vivo administration of Flk-2/Flt-3 ligand (FL) is also capable of sensitizing progenitors to 5-FU. Mice were treated with FL (100 μg/kg every 12 hours for a total of 3 doses), KL (50 μg/kg, same schedule) or both, either alone or in combination with 5-FU (a single 125 mg/kg injection 3 hours before the last dose of cytokine). Femurs and spleens were harvested 48 hours following the last dose of cytokine, and the total numbers of mononuclear cells and colony forming unit cells (CFU-C) per femur and spleen were determined. Statistically significant increases in the number of CFU-C per femur were observed in response to FL, KL and FL+KL. In the spleen, statistically significant increases in CFU-C were observed only with the FL+KL combination. 5-FU alone produced marked reductions in CFU-C both in the femur and in the spleen. In the femur, 5-FU-mediated reductions in CFU-C were enhanced 3- to 30-fold in the presence of concomitant KL, FL or KL+FL administration. Surprisingly, the combination of KL+FL was no more effective in sensitizing marrow CFU-C to 5-FU than was KL alone, suggesting that CFU-C that are capable of surviving the KL/5-FU combination cannot be driven into cell cycle by FL. The effects of concomitant cytokine/5-FU administration in the spleen contrasted sharply with those observed in the femur, as FL, KL and FL+KL all failed to enhance 5-FU-mediated reductions in CFU-C. The ability of FL+KL to stimulate CFU-C expansion in the spleen combined with the inability of this cytokine combination to augment 5-FU-mediated progenitor toxicity in the spleen supports the contention that cytokine-mobilized progenitors are not in cycle. FL’s capacity to specifically sensitize marrow to the effects of cytotoxic drugs may have applications in bone marrow transplant conditioning regimens.

Erythroid burst-forming units (BFU-E) predict hematopoietic recovery after peripheral blood progenitor cell transplantation in patients with advanced breast cancer.

In 29 consecutive heavily pretreated stage IV breast cancer patients, we analyzed patient factors and in vitro characteristics of peripheral blood progenitor cell (PBPC) collections that might correlate with the speed of hematopoietic recovery after autologous transplantation. PBPC collections were assessed for total number of mononuclear cells infused/patient weight in kg and hematopoietic progenitor cell content using in vitro colony-forming assays. In these patients, who received PBPC as the sole hematopoietic support after myeloablative chemotherapy, the number of erythroid burst-forming units (BFU-E) infused correlated significantly with both time to neutrophil (P = 0.008) and platelet (P = 0.0001) recovery and was a better predictor of hematopoietic recovery than number of CFU-GM administered. By day 75 after transplantation, six patients with poor BFU-E yields failed to engraft platelets. Our data suggest that the number of BFU-E infused correlate with time to hematopoietic engraftment and may predict failure of platelet engraftment in heavily pre-treated patients.