bluuebunny/crossref_metadata_2025 huggingface dataset:
Sensing Iron: Reciprocal Regulation of Hepcidin mRNA by Soluble and Cell-Associated Hemojuvelin.
Authors: Lin Lan , Goldberg Y. Paul , Ganz Tomas
Published: April 2020

Human genetic studies identified HJV (also called HFE2) as the major cause for juvenile hemochromatosis (JH). Patients with HJV hemochromatosis have low urinary levels of hepcidin, the principal iron-regulatory hormone secreted by the liver. We attempted to establish the specific roles of HJV in iron metabolism, especially its relationship with hepcidin. Translation of the genomic sequence indicated a C-terminal GPI anchor for the protein product of HJV, hemojuvelin. This suggested that hemojuvelin may have either a soluble or a cell-associated form. In human hepatoma cell line Hep3B, knockdown of cellular HJV by siRNA decreased hepcidin expression, independently of the IL-6 pathway. Intriguingly, the addition of recombinant soluble hemojuvelin (rs-hemojuvelin) also suppressed hepcidin expression in primary human hepatocytes, in a log-linear dose-dependent manner, suggesting competition between soluble and cell-associated forms of hemojuvelin. Soluble hemojuvelin was found in human sera at concentrations similar to those required to suppress hepcidin mRNA in vitro. In cells engineered to express hemojuvelin, soluble hemojuvelin release was progressively inhibited by increasing iron or holotransferrin concentrations. Our study suggests that soluble and cell-associated hemojuvelin reciprocally regulate hepcidin mRNA levels, and that hemojuvelin may serve as a molecular messenger for iron homeostasis. Even in hepatocytes stimulated with IL-6, we observed strong suppression of hepcidin mRNA by rs-hemojuvelin. If rs-hemojuvelin or its active fragments also suppress hepcidin production in vivo, they could be used to alleviate anemia of inflammation.
High Response and Complete Remission Rates in Relapsed/Refractory Multiple Myeloma Treated with Bortezomib, Thalidomide, Dexamethasone and Zoledronic Acid (VTD-Z) Combination Therapy.
Authors: Teoh Gerrard , Tan Daryl , Hwang William , Koh Liang Piu , Chuah Charles , Ng Heng Joo
Published: April 2020

Purpose: Bortezomib (VELCADETM) is a novel proteasome inhibitor that has shown tremendous clinical efficacy either as a single agent or in combination with other cytotoxic agents in relapsed/refractory multiple myeloma (MM). In this study, we combined bortezomib (V) with thalidomide (Thal), dexamethasone (Dex) and zoledronic acid (Zol) to determine the efficacy and toxicity of this regimen (VTD-Z) in patients with relapsed/refractory MM.

Methods: Patients were treated for 2 to 11 three-weekly cycles of VTD-Z comprising: V 1.3 mg/m2 on days 1, 4, 8 and 11; Thal 50 mg ON; Dex 20 mg OM on days 1 to 4, 8 to 11, 15 to 18; and Zol 4 mg on day 1. Patients did not receive any prophylaxis for venous thrombosis. Response was graded according to Bladès criteria.

Results: Twelve patients (2 males, 10 females; median age 63.3 years) with relapsed/refractory MM were studied. Complex karyotypes were present in all (10 out of 10) patients who had karyotype studies performed at the time of diagnosis. Four patients had deletion chromosome 13 (del(13)). The overall response rate (RR) was 91.7% (11 out of 12), of which 50.0% (6) achieved complete remission (CR), 25.0% (3) achieved near-CRs (nCR), 16.7% (3) were partial responders (PR). There were no minimal responders (MR) and 1 (8.3%) non-responder (NR). Two patients who achieved CR had del(13). Transient grade 3 thrombocytopenia was the only severe adverse event and this was observed in 4 (33.3%) patients. Grade 2 peripheral neuropathy was observed in 4 (25.0%) of patients. None of the patients developed deep vein thrombosis or pulmonary embolism. The single NR was the only death that occurred during the period of study.

Conclusions: Our study demonstrates that the VTD-Z regimen is exceptionally effective and safe in patients with relapsed/refractory MM. The high CR/near-CR rates (total of 75.0%), attained after relatively few cycles of VTD-Z, suggest that these three drugs could be at least additive, if not, even synergistic. Longer term follow-up will be required to determine the durability of these responses.
Thalidomide Sensitizes 32D cl 3 Hematopoietic Progenitor Cells to Ionizing Irradiation.
Authors: Greenberger Emily E. , Epperly Michael W. , Franicola Darcy , Zhang Xichen , Jacobs Samuel , Greenberger Joel S.
Published: April 2020

Thalidomide (TL) has been used in the treatment of multiple myeloma and due to its anti-angiogenic effects has been postulated to be a potential radiosensitizer of tumors in vivo. We first investigated the effect of TL on growth of IL-3 dependent murine hematopoietic 32D cl 3 cells in semisolid methylcellulose containing medium also supplemented with TL ranging from 0 to 200 μM. The cells were incubated at 37°C for 7 days at which time colonies of greater than 50 cells were counted. TL increased cell colony formation 2-fold over the dose range of 10 to 100 μM. In serially decreasing concentrations of IL-3, TL (50 μM) was growth stimulatory of 32D cl 3 colony formation in 15% IL-3 and 10% IL-3 conditioned medium (146.2 ± 7.2 and 89.3 ± 2.4 colonies/500 cells plated in TL vs. 110.8 ± 11.4 and 58.0 ± 2.1 without TL, p = 0.0304 or 0.0006). To determine whether thalidomide was a radiosensitizer, cells were placed in: 0, 50, or 150 μM TL in three protocols:

1 hour before irradiation; 1 hour before irradiation plus also in methylcellulose following irradiation; or were placed in TL containing methylcellulose only following irradiation.

Cells were irradiated to doses ranging from 0 to 800 cGy and were plated in methylcellulose containing medium. Seven days later, colonies of greater than 50 cells were counted and analyzed by single hit, multi-target or linear quadratic models. Cells grown in 50 μM TL which stimulated cell growth resulted in an increased radiation resistance compared to control irradiated cells (D0 = 1.87 ± 0.02 vs. 1.30 ± 0.09 Gy, respectively, p = 0.0366). However, 150 μM TL (which did not stimulate cell growth) increased radiation sensitivity compared to control irradiated cells (D0 = 1.22 ± 0.5 vs. 1.64 ± 0.11 Gy, respectively, p = 0.0245). To determine whether thalidomide radiosensitized tumor cells in the therapeutic 150 μM range, SSC-VII murine squamous cell carcinoma cells were grown in TL at concentrations ranging from 0 to 200 μM and were assayed for colony formation. In contrast, SSC-VII cells showed no detectable stimulation or inhibition of cell growth for unirradiated cells in any concentration of TL. However, at 150 μM TL, SSC-VII cells were radiosensitive when incubated in TL before irradiation compared to control irradiated cells (D0 = 2.02 vs. 2.55 Gy, respectively). These results demonstrate that thalidomide may be a clinically valuable radiosensitizer.
Interim Analysis of a Phase II Trial of Bortezomib in Patients with Multiple Myeloma Who Failed To Achieve a Complete Response to Autologous Peripheral Blood Stem Cell Transplantation.
Authors: Rifkin Robert M. , Beveridge Roy A. , Brunvand Mark W. , Spitzer Gary , Kristi Boehm A. , Rauch Mary Ann , Zhan Feng , Asmar Lina
Published: April 2020

The majority of patients (pts) with multiple myeloma (MM) fail to achieve a complete response (CR) with standard induction therapy (ie, VAD, DVD, Thal-Dex). Bortezomib has been shown to induce a CR in ~10% of subjects with refractory/relapsed MM. This study was conducted to assess the safety and efficacy of bortezomib in converting pts, who failed to achieve a CR to high-dose chemotherapy, to complete responders.

A total of 68 pts are targeted for enrollment on the current study protocol. To date, 20 pts have enrolled and are the subject of this planned safety analysis. As of this submission, 18 pts have been treated and 16 pts have either completed or discontinued treatment. All pts have received at least one dose of bortezomib according to the following schedule. Bortezomib was administered at a dose of 1.3 mg/m2 via IV push on Days 1, 4, 8, and 11 every 21-days for 4 cycles. Patients who failed to demonstrate disease progression were eligible for 4 additional cycles of therapy administered according to the above schedule. The demographic data of the first 20 subjects enrolled are summarized in the table below.

Sixteen of 20 pts enrolled have now completed the study. Patients received a median of 4 cycles (range, 3–8). Reasons for discontinuation included: adverse events (n=3 neuropathy (± fatigue) and n=1 fatigue with constipation) and other (n=1 each, progressive disease, carpal tunnel, deemed ineligible). Six patients had dose(s) delayed and/or reduced due to adverse events (peripheral neuropathy; fatigue; neutropenia and herpes zoster; fatigue with neuropathy; nausea, vomiting, diarrhea, and sinus infection; hypertension, and fatigue with constipation).

Treatment-related Grade 3–4 toxicities have been infrequent and include: asthenia, peripheral neuropathy, and thrombocytopenia (11% each) and hypertension, abdominal pain, and neutropenia (5.6% each). Grade 4 toxicity was limited to 2 pts with thrombocytopenia. No life threatening infections or reactivation of herpes zoster have been observed. Bortezomib when administered in the post-transplant setting appears to be safe and well tolerated. Neuropathy and thrombocytopenia appear to be major factors in patient discontinuation. Following review of this planned interim analysis for safety, the study continues to accrue toward completion.

Patient characteristics Variable %Male:%Female 25:75 Mean age (range) 53 years (41–66) Number and percentage (%) of patients Myeloma Isotype IgA Kappa 3 (21.5) IgA Lambda 1 ( 7) IgG Kambda 4 (29) IgG Lambda 1 ( 7) Kappa light chain 3 (21.5) Lambda light chain 2 (14) Prior Chemotherapy 17 (85) Prior Radiation 5 (25) Type of Transplant Single 12 (60) Tandem 6 (30) Not Available 2 (10)
The Effects of Zoledronic Acid Dose and Infusion Rate on Pharmacokinetics (PK), Pharmacodynamics (PD), and Renal Function in Patients with Multiple Myeloma.
Authors: Berenson James R. , Major Pierre , Swift Regina A. , Vescio Robert , Rivera Christina , Bilic Sanela , Gauron Sonia , Denouel Jannick , Schran Horst
Published: April 2020

BACKGROUND: Zoledronic acid has demonstrated efficacy and safety in the treatment of bone lesions in patients with multiple myeloma. However, bisphosphonates are known to have dose-dependent and infusion rate-dependent effects on renal function and patients with multiple myeloma may have disease-associated compromise of renal function. The pharmacokinetics (PK), pharmacodynamics (PD), and safety of monthly intravenous (IV) infusions of zoledronic acid (4, 8, and 12 mg) were investigated in a subset of patients with multiple myeloma as part of an open-label, phase I, multiple-dose study. Doses higher than the approved 4-mg dose were infused over 1 or 2 hours based on an infusion-rate model that predicted a Cmax similar to that of the approved regimen (4 mg via 15-minute IV infusion). Moreover, levels of tumor markers were measured to assess potential clinical antitumor activity at various dose regimens.

METHODS: Patients received a total of 6 infusions of zoledronic acid in 28-day cycles: 4 mg via 15-minute infusion on day 1; 8 mg via 60-minute infusion on days 29 and 57; 12 mg via 120-minute infusion on days 85 and 113; and 4 mg via 15-minute infusion on day 141.

RESULTS: Ten patients with multiple myeloma were enrolled, and 7 completed all 6 infusions. Median age was 58 years, ECOG performance status was ≤ 1 in 9 patients and 2 in 1 patient; 80% had received prior antineoplastic therapy, and 100% had received prior radiation and/or surgery. Three patients discontinued because of abnormal laboratory values (n = 2) or withdrawal of consent (n = 1). The most common AEs were fatigue (60%), myalgia (50%), arthralgia (40%), and nausea (40%). Increased serum creatinine > 0.3 mg/dL over baseline prompted a dose delay/reduction in 1 patient and discontinuation of treatment in 2 patients. In multiple myeloma patients, mean Cmax (± SD) was 329 (± 89) ng/mL after the first 4-mg dose infused over 15 minutes (1st infusion), 407 (± 105) ng/mL after the 2nd 8-mg dose infused over 60 minutes (3rd infusion), and 385 (± 114) ng/mL after the 2nd 12-mg dose infused over 120 minutes (5th infusion). Increasing doses produced proportional increases in drug exposure (AUC). Decreases from baseline were observed in serum markers of bone resorption; however, there were no consistent or meaningful changes in tumor marker levels (eg, serum IL-6).

CONCLUSIONS: This study indicates that by prolonging infusion times to 1 hour and 2 hours using 8-mg and 12-mg doses of zoledronic acid, respectively, Cmax can be maintained and is similar to that obtained with the approved 4-mg dose infused over 15 minutes.
Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.
Authors: Lu Lulu , Song Yongping , Ma Baogen , Zhu Xiongpeng , Wei Xudong , Chen Zhizhe
Published: April 2020

Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft.

Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls.

Results:

The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01).

Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.
Highly Reproducible Engraftment of Chronic Lymphocytic Leukemia (B-CLL) Cells in NOD/SCID Mice.
Authors: Ebeling Peter , Duerig Jan , Grabellus Florian , Duehrsen Ulrich , Seeber Siegfried , Flasshove Michael , Moritz Thomas
Published: April 2020

In contrast to normal hematopoiesis and acute leukemias, research in CLL still is hampered by the lack of a reliable in vivo model for primary B-CLL. We here report highly reproducible engraftment of B-CLL cells, when 1x10^8 MNC derived from the peripheral blood of CLL patients were transplanted i.v and i.p. into NOD/SCID mice. So far, 14 different CLL samples were investigated in 41 mice. At weeks 4, 8 or 12 mice were sacrificed and bone marrow (BM), spleen, and peritoneal fluid (PF) were analyzed by FACS for human CD19/CD5/CD23/CD45 (B-CLL) cells and CD45/CD3/CD5 (T) cells. Additionally, HE- and immunostaining was performed on spleen sections.

Analysis at week 4 revealed engraftment in NOD/SCID mice for 13/14 samples (spleen: 13/14, BM: 4/14, PF: 12/14). B-CLL cells were observed predominantly in the spleen (8.9±2.4% or 9.1±4.4x10^5 cells) and PF (19.0±4.4% or 3.4±1.8x10^5 cells) with much lower engraftment in BM (0.6±0.3% or 0.1±0.1x10^5 cells). Detection of B-CLL cells in peripheral blood could be obtained in 3/14 experiments. Also substantial engraftment of human T-cells was observed in 13/14 experiments (spleen: 13/14, BM: 8/14, PF: 11/14). T-cells engraftment was highest in the spleen (23.8±9.8% or 28.7±13.1x10^5 cells) and somewhat lower in PF (16.4±8.2% or 3.0±1.6x10^5 cells) and BM (7.3±3.8% or 2.9±1.1x10^5 cells). Subpopulation analysis revealed a CD4+ phenotype in 65, 59 and 72 % of T-cells within spleen, PF and BM, respectively. Noteworthy, immunohistological analysis of HE stained spleen sections of engrafted animals revealed a pseudofollicular infiltration with human CD45LCA+ cells along splenic arterioles. Within these pseudofollicles human B-CLL but also CD3+ T-cells were detected. Contribution of B-CLL and T-cells to individual follicles was highly variable ranging from 5–95% for both cell types. When engraftment was analysed separately for the i.p and the i.v. route, engraftment of transplanted cells in PF seemed to be depended on the i.p. route whereas splenic engraftment was obtained following i.v. as well as i.p. injection. Sustained B-CLL engraftment was seen after 8 weeks (spleen: 3.1±1.4% or 7.3±3.1x10^5 total cells; PF: 57.6±23.3% or 1.0±0.5x10^5 cells; n=3 mice) and 12 weeks (spleen: 1.4±1.3% or 0.3±0.3x10^5 cells; PF: 10.2±7.3% or 0.5±0.5x10^5 cells; n=2 mice).

Thus, we have shown efficient engraftment of human B-CLL cells in the spleen and PF of NOD/SCID mice. This in vivo model should significantly help to understand B-CLL biology and to test novel therapeutic approaches. The observed pseudofolicular pattern of splenic infiltration supports the theory of T-cells creating a “microenvironment” sustaining the growth of the leukemic B cell clone.
Rapid Helper T-Cell Recovery at 3 Months Correlates to Successful Transplant Outcomes after Allogeneic Stem Cell Transplantation.
Authors: Moon Joon Ho , Baek Jin Ho , Kim Dong Hwan , Sohn Sang Kyun , Kim Jong Gwang , Lee Kyu Bo , Suh Jang Soo
Published: April 2020

Background: The current study attempted to evaluate the role of a simple quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes, including overall survival (OS), non-relapse mortality (NRM), and opportunistic infections, after allogeneic stem cell transplantation (SCT).

Methods: A total of 69 patients receiving an allogeneic SCT were included. The disease entities were as follows: AML 42, ALL 5, CML 15, NHL 5, and high-risk MDS 2. The peripheral lymphocyte subset counts, such as CD3+ T-cells, CD3+4+ helper T-cells, CD3+8+ cytotoxic T-cells, CD19+ B-cells, and CD56+ natural killer (NK) cells, were measured 3, 6, and 12 months post-transplant.

Results: The CD19+ B-cell reconstitution was slow, while a rapid CD56+ NK cell recovery was noted. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (p<0.0001), NRM (p=0.0007), and opportunistic infections (p=0.0108) when stratifying patients with cut-off value of 200×106/L CD4+ helper T-cells. A rapid CD4+ helper T-cell recovery was also independently associated with a higher CD4+ helper T-cell transplant dose (p=0.006) and donor type (p<0.001) in a regression analysis. An early CD4+ helper T-cell recovery at 3 months was associated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate survival analysis, a combination of a higher CD34+ cell dose and rapid recovery of CD4+ helper T-cells at 3 months was found to a have favorable prognosis in terms of OS (p=0.001, hazard ratio [HR] 3.653) and NRM (p=0.005, HR 4.836), yet not relapse.

Conclusion: A rapid recovery of the CD4+ helper T-cell count above 200×106/L at 3 months seemed to correlate with a faster immune reconstitution and predict a successful transplant outcome.

Figure. The overall survival according to the helper T-cell counts at 3 months (A) and the difference of total T-(B) and helper T-cell (C) immune reconstitution within 1-year post-transplant according to helper T-cell counts at 3 months Figure. The overall survival according to the helper T-cell counts at 3 months (A) and the difference of total T-(B) and helper T-cell (C) immune reconstitution within 1-year post-transplant according to helper T-cell counts at 3 months
Variable Number of Tandem Repeat (VNTR) Disparity between Donor and Recipient Has Potential To Predict Outcomes of HLA-Identical Allogeneic Stem Cell Transplantation.
Authors: Kim Dong Hwan , Jung Hee Doo , Lee Nan Young , Moon Joon Ho , Sohn Sang Kyun , Suh Jang Soo , Lee Kyu Bo
Published: April 2020

Background: Detecting the variable number of tandem repeats (VNTRs) between the recipient and the donor has already been adopted to monitor the degree of chimerism after allogeneic stem cell transplantation (SCT). In allogeneic SCT, besides MHC-disparity, the disparity of various polymorphous proteins encoded by several genes may play a critical role in the pathogenesis of graft-versus-host disease (GVHD). However, the biologic effect of VNTR disparity has scarcely been studied.

Materials & Methods: Eighty-four patients receiving an SCT from HLA-identical sibling (n=68) or unrelated donors (n=16) were analyzed. The enrolled diseases included AML 48, ALL 8, CML 15, NHL 10, and high-risk MDS 3. A PCR was performed to amplify 3 VNTR regions (D1S80, D1S111, and D17S5). The disparity was classified as fully matched, partially matched, or mismatched pairs.

Results: A strong correlation was observed between the D1S80 disparity and the transplant outcomes in terms of OS (p=0.0179) and NRM (p=0.0305), yet not for the D1S111 or D17S5 disparity. The fully matched D1S80 pairs showed a better OS (72% vs 38%) and lower NRM (17% vs 50%) compared to the partially matched or mismatched pairs. In multivariate analyses, a fully matched D1S80 pair was found to be an independent favorable prognostic factor for OS (p=0.03) and NRM (p=0.05). In addition, D1S80 disparity was significantly associated with the occurrence of gut chronic GVHD (p=0.05).

Conclusion: The present data suggest that disparities in D1S80 - located in chromosome 1 - seemed to be associated with an increased incidence of gut chronic GVHD and NRM.

Figure. Overall survial (A) and non-relapse mortality (B) according to D1S80 VNTR disparity Figure. Overall survial (A) and non-relapse mortality (B) according to D1S80 VNTR disparity
Impact of Activating Killer Immunoglobulin-Receptor Genotype of the Donor on Outcome of Unrelated Donor - Hematopoietic Stem Cell Transplantation.
Authors: Giebel Sebastian , Nowak Izabela , Kruzel Tomasz , Wojnar Jerzy , Markiewicz Miroslaw , Dziaczkowska Joanna , Wylezol Iwona , Krawczyk-Kulis Malgorzata , Bloch Renata , Czerw Tomasz , Karolczyk Agnieszka , Kusnierczyk Piotr , Holowiecki Jerzy
Published: April 2020

Function of NK cells is regulated by a balance between inhibitory and activating signals transmitted through killer immunoglobulin-like receptors (KIR). The goal of the present analysis was to evaluate the impact of donor activating KIR genotype (KIR2DS1 and KIR2DS2 loci) on outcome of unrelated donor-hematopoietic stem cell transplantation (URD-HSCT). Fourty-two URD-HSCT recipients with haematological malignancies, aged 28 (14–48) years (male/female - 26/16) were included in the analysis. The conditioning regimen was myeloablative and based on chemotherapy alone (n=33) or total body irradiation (n=9). Graft-vs-host disease (GVHD) prophylaxis consisted of cyclosporin, methotrexate, and pre-transplant anti-thymocyte globulin. Patients were grouped according to their donors’ activating KIR genotype including two loci: KIR2DS1 and KIR2DS2.

The overall survival at 2.5 years with respect of the donors’ activating KIR genotype was as follows: KIR2DS1(−)DS2(−) (n=15) − 82% (+/−12%), KIR2DS1(+)DS2(−) (n=9) − 78% (+/−14%), KIR2DS1(−)DS2(+)− (n=7) 86% (+/−13%), KIR2DS1(+)DS2(+) (n=11) − 0%. The OS rate differed significantly between KIR2DS1(+)DS2(+) group and the remaining patients: 0% vs. 82% (+/−7%), p=0.03. The difference resulted mainly from higher cumulative incidence of non-relapse mortality in the KIR2DS1(+)DS2(+) group: 100% vs. 18% (+/−8%), p=0.098. The reasons of death in patients with KIR2DS1(+)DS2(+) donors were chronic GVHD (n=4) and acute GVHD (n=1). We conclude that the concomitance of both KIR2DS1 and KIR2DS2 in the donor is associated with high risk of mortality following URD-HSCT, resulting mainly from the incidence of severe GVHD. Whether this effect is associated with the activity of NK cells or KIR-bearing T lymphocytes requires further investigation.
Higher Interleukin-10 (IL-10) and Lower Interferon-Gamma (IFN-γ), Tumor Necrosis Factor Alpha (TNF-α), IL-6, IL-8 Serum Levels in Patients Undergoing Reduced Intensity Versus Conventional Intensity Conditioning Hematopoietic Cell Transplantation (HCT).
Authors: Stradmann H. , Hegenbart Ute , Bruegel M. , Al-Ali Haifa K. , Niederwieser Dietger W.
Published: April 2020

Endogenous cytokines play an essential role in allogeneic HCT not only for hematopoietic reconstitution but also for the most frequent complications of HCT such as graft-versus-host disease (GvHD). Cytokines have important immune modulatory effects and are therefore important determinants for outcome. No report, however, exists to date on the role of cytokines in HCT with reduced intensity conditioning (RIC), which are associated with less severe GVHD and reduced transplant related mortality. Patients transplanted at the University of Leipzig between October 2000 and November 2001 with an autologous (n=10) or allogeneic (n=38) related or unrelated graft were included in this analysis. The median age of patients was 49 (range 21–69) years. Preparative regimens for the allogeneic HCT consisted of conventional (n=20) or RIC (n=18) conditioning.

Cyclophosphamide (60mg/kg for 2 consecutive days) was given either with busulfan (4 mg/kg per day for 4 days; n=9) or fractioned total body irradiation (TBI 12 Gy; n=11) for conventional HCT and cyclosporine in combination with metotrexate was used as GvHD prophylaxis. In contrast, patients with the reduced intensity treatment received low dose TBI (2 Gy) in combination with fludarabine (30 mg/m2 per day for 3 consecutive days) followed by cyclosporine and mycophenolat mofetil. Serum was collected three times weekly and was frozen at −70° until measurement. Cytokine-serum-levels were analyzed with a Multiplex Cytokine Assay (Bio-Plex, Bio-Rad Laboratories, Hercules, CA).

Endogenous IFN-γ and TNF-α showed similar fluctuations after RIC and non-RIC HCT with peak levels between day 14/17. However, serum levels of non-RIC patients increased faster than serum levels of RIC-patients. Higher IL-4 serum levels were detected in the RIC patients as compared to the non-RIC patients especially in the later course of HCT. In contrast, higher IL-6 and IL-8 values with peaks at d 6/9 in both groups were observed in the patients with conventional HCT. Similarly a difference for IL-13 levels was detected with higher values for the conventional group. The most prominent difference was observed in the endogenous IL-10 production with significant higher IL-10 levels in RIC compared to non-RIC patients.

We concluded that serum levels of IL-4 and IL-10 were higher and IFN-γ, TNF-α, IL-6, IL-8 and IL-13 lower in the RIC compared to the non-RIC group. These results are in line with previous work on cytokines after conventional preparative regimen and might provide an explanation for the lower transplant related mortality of RIC regimens.
Effects of Apoptosis in Human Activated Peripheral T-Lymphocytes by Immunosuppressive Drugs.
Authors: Zhu Xiaoli , Huang He
Published: April 2020

Immunosuppressive drugs are widely used for the prevention and treatment of graft rejection, graft-versus-host disease (GVHD) and autoimmune disorders. The major effect of immunosuppressive treatment is to prevent the activation-induced proliferation of T cells essential for the immune response. Nevertheless, the role of immunosuppressive drugs in regulating apoptosis in activated lymphocytes has not been clearly elucidated. For further investgation, we compared commonly used immunosuppressive drug mycophenolic acid (MPA) with cyclosporin A (CsA) for the influence of apoptosis in activated lymphocytes. Human Peripheral T-lymphocytes were activated by phytohemagglutinin (PHA). Cell morphology, DNA fragmentation, percentage of annexin V positive cells were measured by microscopic, electrophoretic and flow cytometric techniques respectively. Characteristic apoptotic morphology and DNA Ladder of activated T-lymphocytes were found in MPA treated group, but missing in CsA group. Treated with 0.1mmol/L ~10mmol/L MPA for 24h~48h, the Annexin V positive percentage of activated T-lymphocytes increased significantly(P<0.05), and with dose-dependant(r=0.752, P<0.001)and time-dependant(r=0.583, P=0.001)manner. Treated with 0.1mmol/L ~1mmol/L CsA for 24h~48h, the Annexin V positive percentage of activated T-lymphocytes decreased significantly(P<0.05). In conclusion, we found clinical relevant doses of MPA induced apoptosis in activated peripheral T-lymphocytes, whereas, CsA inhibited apoptosis. These results suggest that immunosuppressive drugs may exert immunomodulatory effects in transplantation in part by regulating apoptosis of activated lymphocytes. They can be classified according to their action on apoptosis-induction and inhibition and it would favour a rational combination therapy.
A SYBR Green - Based Real Time PCR Method for Detection of Haemopoietic Chimerism in Allogeneic Stem Cell Transplant Recipients.
Authors: Bai Lijun , Deng Yi-Mo , Dodds Anthony J. , Milliken Sam , Moore John , Ma David D.F.
Published: April 2020

Aim Detection of donor cells (chimerism) in patients post allogeneic stem cell transplantation is for monitoring engraftment, early detection of graft rejection and disease relapse. Current methods include cytogenetics, blood grouping and DNA microsatellite test. Our aim is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using SYBR green for detecting chimerism in allogeneic haemopoietic stem cell transplant recipients.

Methods Twelve specific nucleotide polymorphisms (NPs) on 11 different chromosomes (including X, Y) were selected. Specific primers and fluorogenic probes were used for Q-PCR analysis. These NPs were screened in pre-transplantation donor and recipient pairs for informative markers. One informative marker was then used for Q-PCR to detect donor cell chimerism post transplantation using either SYBR green or TaqMan probe. Quantitation of donor cell percentage was calculated using a standard amplification curve generated from artificially mixed donor/recipient chimeric DNA samples made with 10 serial dilutions (0.01% ~ 100%), low, medium and high controls were included.

Results Thirty-seven donor/recipient pairs including 9 sex-mismatched allograft pre-transplantation DNA samples were examined. In all cases, it is possible to discriminate between recipient and donor genetic profiles. The detection limitation is 0.01%, which is more sensitive than the currently used Microsatellite and FISH methods. The results are highly reproducible with accurate quantitation. In 16 post transplant samples, donor-recipient chimerism were distinguishable using this method. Furthermore this new assay detected similar levels of chimerism compared with either FISH for sex-mismatch donor/recipient pairs or Microsatellite for sex match donor-recipient pairs. We also found that the SYBR green Q-PCR is less expensive and as accurate as the TaqMan probe Q-PCR.

Conclusion The new single platform Q-PCR method is capable of detecting haemopoietic chimerism after transplantation and has the potential to replace the three current methods (i.e. cytogenetics, blood grouping and microsatellite).
Donor-Specific Tolerance Is Induced after Nonmyeloablative Conditioning and Allogeneic Hematopoietic Cell Transplantation and May Persist after Graft Rejection.
Authors: Yunusov Murad Y. , Abrams Kraig , Kuhr Christian S. , Storb Rainer , Hwang Billanna , Sale George E. , Storer Barry , Georges George E. , Nash Richard A.
Published: April 2020

The association between stable hematopoietic chimerism and donor specific tolerance (DST) for solid organ grafts was demonstrated clinically and in different experimental models. However, the existing data also demonstrate that the establishment of hematopoietic chimerism does not always correlate with the development of DST, especially for skin grafts. As these issues could have major implications for organ transplantation in general, the present study was undertaken to assess DST in recipients with transient or stable hematopoietic cell (HC) chimerism established after nonmyeloablative conditioning regimens. In a canine model of allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning, DST to skin grafts was evaluated in DLA-identical recipients with stable mixed hematopoietic chimerism (MC) (n=11), or following transient HC engraftment (n=22). There was a significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with stable MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in 4 recipients with MC developed an inflammatory reaction without skin graft loss consistent with a chronic rejection process. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged in recipients after transient HC engraftment compared to normal recipients (P=0.002). However, a chronic inflammatory process without graft loss developed in all of the HC donor-derived skin grafts from this group. An increased time to rejection of the hematopoietic graft was significantly associated with an improved survival of the subsequent skin graft (P=0.02). The time to rejection of third-party (DLA-nonidentical) skin grafts (n=40) was the same in recipients with stable MC and previous HC graft as well as normals (median- 8 days). Autologous skin grafts (n=40) survived for the duration of follow-up without developing chronic inflammation. Extended HC donor-specific skin graft survival (>15 days) was predictive of subsequent stable engraftment at second HCT in recipients with previous graft rejection after first HCT. Five of 6 dogs with extended survival of the skin graft beyond day 15 became stable MC after second HCT compared to 0/9 dogs with skin grafts surviving less than 11 days (P=0.0008). In this model, DST to skin grafts in recipients with stable MC after nonmyeloablative HCT may not be complete, and chronic inflammation may develop without loss of that graft. Partial DST may persist after rejection of HC grafts established with nonmyeloablative conditioning. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.
Development of Clinical Scale Cytomegalovirus-Specific T Cells for Adoptive Immunotherapy Using pp65.
Authors: Diamanti Paraskevi , Garland Russell J. , Pamphilon Derwood H. , Steward Colin G. , Blair Allison
Published: April 2020

Cytomegalovirus (CMV) represents a major cause of morbidity in patients undergoing allogeneic stem cell transplantation (SCT). Although antiviral prophylaxis has contributed to the reduction of early CMV disease, recovery of CMV-specific cytotoxic lymphocytes is still necessary for the long-term control of CMV reactivation after SCT. It has been demonstrated that CMV-specific CD4+ and CD8+ cytotoxic T lymphocytes from the donor are important in resolving CMV infection and protecting recipients from developing CMV disease post SCT. Recently CMV recombinant protein pp65, an immunodominant target for CD4+ and CD8+ T cell responses, has been developed for the stimulation and isolation of donor CMV-specific T cells. The aim of this study was to investigate the potential of pp65 to generate CMV-specific CD8+ and CD4+ T cells from seropositive subjects for use as adoptive immunotherapy after SCT. Peripheral blood mononuclear cells (5x108 cells) were isolated from single unit CMV-positive buffy-coats and were stimulated with pp65 for 16 hours. CMV-specific T cells that produced IFNγ were captured using the IFNγ-secretion assay and were enriched using the CliniMACS system. The phenotype of the cells, both pre and post enrichment was evaluated by flow cytometry using monoclonal antibodies against CD3, CD4 and IFNγ. Enriched IFNγ producing T cells were cultured with irradiated feeder cells in the presence of IL-2 for 18 days. Alloreactivity experiments were performed by mixing 105 enriched T cells at day 0 and day 18 of culture with 105 third party cells for seven days. Proliferation was assessed by the 3H-thymidine incorporation assay. Experiments to date have shown that pp65 is capable of stimulating both CD4+ and CD8+ T cells to secrete IFNγ. Flow cytometric analyses revealed that prior to enrichment, IFNγ+ cells represented 0.13% of the CD3+/CD4+ cell population and 2.1% of the CD3+/CD4− cell population. Whereas, following magnetic enrichment and an 18-day expansion phase, the proportion of IFNγ+ cells increased significantly (554-fold and 6-fold) to 72% and 13.3% in the CD3+/CD4+ and CD3+/CD4− subfractions, respectively. The alloreactivity of the expanded positive cells had decreased by an average 95.5% (SD=5.7, n=3) compared to prior to enrichment and cell expansion. The proliferation of the expanded cells was still high (Stimulation Index >3), but considerably reduced compared to the cells at day 0. The cytotoxicity of these cells will be assessed to determine their suitability for immunotherapeutic use post-SCT. Large scale CMV-specific T cell capture experiments, performed under GMP conditions, are ongoing.
Control of Cell Death Levels Using Th9402-Based PDT Treatment on Fresh PBMC, and Potential Application to Patients with cGvHD.
Authors: Levesque Annie , Savard Ann-Louise , Roy Denis-Claude , Foss Francine , Scotto Christian
Published: April 2020

Recently, extracorporeal photopheresis (ECP) has shown interesting clinical activity for the treatment of drug-refractory chronic graft-vs-host disease (cGvHD), inducing Th1/Th2 immunomodulation to restore immune tolerance. Several studies indicate that target cell apoptosis may have a role in the control of cGVHD, and increasing apoptotic levels may favor immune modulation. We have developed an approach to eliminate immunoreactive cells using the Theralux™ photodynamic cell therapy (PDT) system based on the use of the rhodamine-derivative TH9402 illuminated ex vivo with a visible light source (l=514nm). The capacity of TH9402 PDT to induce increasing levels of T cell apoptosis has not been investigated. The induction of apoptotic cell death was studied using peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers (HV) and cGvHD patients. We found a good correlation between PDT conditions and levels of cell death induced. In comparison to controls showing 8±4 percent (%) of apoptosis, as measured by TUNEL assay on cells harvested 3 days post-treatment, PBMC from HV subjected to PDT using 0.33, 0.66, or 1.32 micromolar of TH9402 showed 28±16%, 49±17%, and 78±11% of apoptosis, respectively. These studies have also shown that the intra- and inter-donor variability in TH9402 incorporation are very low (~5% and 10%, respectively). To ensure that these findings could also be applied to a clinical setting, PBMC from cGvHD patients were treated with 0.33 and 1.32 micromolar of TH9402 to trigger either low or high levels of apoptosis. PBMC from cGvHD patients showed a sensitivity similar to that of PBMC from HV, with 38±9% and 73±13% of apoptosis when treated with 0.33 and 1.32 micromolar TH9402, respectively. Data obtained from the analysis of various cell death parameters such as the loss of DΨm, AnV/PI staining, TUNEL assay and agarose DNA ladder on PBMC and human Jurkat T cells treated with escalating doses of TH9402 suggested strongly the higher sensitivity of proliferating cells to the treatment. Moreover, together with the use of the caspase-inhibitor ZVAD-fmk, these data suggested that upon irradiation, the photoactivation of TH9402 will trigger the formation of reactive oxygen species (ROS) and the release of proapoptotic factors from mitochondria triggering various cell death mechanisms, such as caspase-dependent apoptosis, caspase-independent apoptosis, or a mixture of apoptosis and necrosis. Finally, preliminary data showed that PDT-treated cells were able to induce in vitro the maturation of monocyte-derived dendritic cells. Based on these data, we are beginning a pilot clinical study evaluating two controlled PDT conditions inducing low and high levels of apoptosis in order to assess the efficacy and biological effect of TH9402-based ECP to treat cGVHD in humans.
Selection of CD34+ HSC from Pooled Cord Blood Samples Does Not Result in Co-Purification of Potential Alloreactive CD3+ T Cells.
Authors: Gilmore Gary L. , DePasquale Darlene K. , Lister John , Shadduck Richard K.
Published: April 2020

Most systems for studying the ex vivo expansion of human UCB-HSC require enrichment of the HSC population, typically by CD34 selection. Because the number of HSC present in a single UCB collection is limited, one approach to increase the total HSC is to pool multiple UCB samples. A potential complicating factor with this method is that alloreactive T lymphocytes present in whole UCB samples would be expected to bind to cells displaying allogeneic HLA, including HSC, and would therefore co-purify with CD34+ UCB-HSC during isolation. Such complexes would be excluded from ISHAGE analysis by the side scatter gate, which is set to exclude aggregates. Alloreactive T cells would be expected to contain cytotoxic T lymphocytes [CTL], which could potentially inhibit or completely abrogate HSC expansion.

In order to determine whether CD3+ T lymphocytes co-purify with CD34+ UCB-HSC in pooled samples, UCB pools were prepared containing 2 to 4 UCB samples. CD34+ HSC were isolated by MACS and analyzed by flow cytometry and antibodies to human CD45, CD34 and CD3, with and without ISHAGE gates. Samples of CD34+ HSC purified from a single UCB collection were analyzed concurrently to give the background value for co-purification of syngeneic T cells and formation of T:HSC aggregates in the CD34-selected products. We found a limited number of CD3+ T cells present in CD34+ HSC isolated from single UCB collections [mean = 0.25%, range = 0 – 1.1%]. That value was slightly elevated when pools of UCB were used [mean = 0.44%; range = 0 – 1.9%]. There are few, if any, CD34+/CD3+ cells that can be detected by either the standard ISHAGE gating or by gating merely on CD45+ cells with no side scatter gating [0.02 – 0.04%]. This was true of CD34+ HSC isolated from either pooled UCB or single UCB collections.

Based on these results, we conclude that there is not significant co-purification of CD3+ T cells with CD34+ UCB-HSC, and that any such complexes that form are not found at any greater frequency in UCB pools than in single UCB collections.
Optimum Temperature for Maintaining the Viability of CD34+ Cells during Storage and Transport of Fresh Hemopoietic Progenitor Cells.
Authors: Antonenas Vicki , Garvin Frances , Webb Melina , Sartor Mary , Bradstock Kenneth F. , Gottlieb David J.
Published: April 2020

Background and Aim: The optimum storage and transport of freshly harvested hemopoetic progenitor cells (HPC) in the liquid state is not specified in the JACIE and FACT guidelines. Depending upon transplant centre, there is a range of reported ideal temperatures (1°C to 24°C) for HPC storage and transport but little data exists to justify the recommendations. Due to the limitations of Trypan Blue viability assays and CFU-GM colony assays, we used a no-lyse, CD34 assay (Sartor et al, Bone Marrow Transplantation 2005) to determine the optimum storage and transport temperature for maintaining viability of CD34+ stem cells in freshly harvested HPC.

Method: Samples were aseptically removed from 46 fresh HPC harvests (34 PBSC & 12 BM) and stored at refrigerated temperature (2°– 8°C), room temperature (18°– 24°C) and 37°C, for up to 72 hours. Samples were analysed for viable CD34+ cells/ml at 0, 24, 48 and 72 hours.

Results: The mean viable CD34+ yield prior to storage was 7.7x106/kg (range: 0.7 – 30.3). No viable CD34+ cells remained after storage at 37°C for 24 hours. The mean % loss of viable CD34+ cells at refrigerated and room temperatures can be summarized as follows:

Conclusion: These results demonstrate that the optimum temperature to maintain the viability of CD34+ stem cells for up to 72 hours during storage and transport of freshly harvested HPC is 2°– 8°C.

Mean % loss or gain of CD34+ cells on storage Time Refrigerated Temperature Room Temperature N Mean Range N Mean Range 24 hrs 39 −9.4 +15 to −42 23 −21.9 +17 to −59 48 hrs 31 −19.4 +7 to −69 20 −30.7 +3 to −68 72 hrs 29 −28.0 +4 to −53 18 −43.3 +11 to −70
Comparison of Umbilical Cord Collection Methods: Impact on Cellularity and Microbial Contamination Rates.
Authors: Foster Karen A. , Chasan-Taber Scott , Dracker Robert A. , Gunter Kurt C.
Published: April 2020

Introduction

Umbilical cord blood collections are typically performed using one of two methods: the syringe collection method and the gravity collection bag method. Collection volume, total nucleated cell (TNC) count, total CD34 positive cell count and percent with microbial contamination were analyzed to determine differences between collections performed using these two methods.

Methods

Units were processed using a hydroxyethyl starch red blood cell depletion method. TNC counts were measured using a Beckman Coulter HmX Analyzer. Total CD34 positive cell counts and viability were measured using the Beckman Coulter Epics XL-MCL flow cytometer. Microbial testing was performed using the VersaTrek system. Data related to collection volume and cellular content were analyzed by a Wilcoxin rank sum test of the paired differences [95% confidence interval (CI) for the difference between the control and experimental (syringe collections) results].

Results and Conclusions

The total cord blood volume collected via the syringe method was not significantly lower than that collected using the gravity collection bag method. However, the TNC count and total CD34 positive cell count from syringe collections were significantly inferior to the TNC count and CD34 positive cell count from gravity bag collections. The rate of microbial contamination of units collected in syringes was 3.3-fold higher than that of units collected using gravity collection bags. Cord blood banks should train collection sites on optimal collection methods and monitor the compliance of all collection sites with standardized collection procedures.

Summary of Results Gravity Bag Collections Syringe Collections Collection Volume N 27,121 55 Mean 66 mL 60 mL Standard Deviation +/− 30 mL +/− 24 mL Wilcoxin rank sum test (p-value) 0.0945 TNC Count (Post Processing) N 27,121 55 Mean 7.5 x 10^8 5.0 x 10^8 Standard Deviation +/− 4.6 x 10^8 +/− 3.5 x 10^8 Wilcoxin rank sum test (p-value) <0.0001 Total CD 34 Positive Cells (Post Processing) N 20,495 51 Mean 2.76 x 10^6 1.77 x 10^6 Standard Deviation +/− 2.87 x 10^6 +/− 1.32 x 10^6 Wilcoxin rank sum test (p-value) < 0.0001 Microbial Contamination Rate 7.1 % 23.6%