bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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&lt;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&lt;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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&lt;0.05), and with dose-dependant(r=0.752, P&lt;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&lt;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

Abstract
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

Abstract
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&lt;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 (&gt;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

Abstract
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 &gt;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.