bluuebunny/crossref_metadata_2025 huggingface dataset:
Cpg Oligodeoxynucleotide-Mediated Effects on B-Cell Chronic Lymphocytic Leukemia In Vitro Are Influenced by Cytogenetic Status and the Presence of Serum.
Authors: Jahrsdorfer Bernd , Blackwell Sue E. , Wooldridge James E. , Taylor Christiana M. , Weiner George J.
Published: April 2020

Immunostimulatory CpG-containing oligodeoxynucleotides (CpG ODN) are TLR9 agonists that mediate a number of immunologic effects in normal and malignant B cells including upregulation of immunogenic molecules. They are therefore felt to be attractive as potential components of immunotherapy for B cell chronic lymphocytic leukemia (B-CLL). The cytogenetic status of B-CLL is predictive of clinical prognosis, but little is known about how CpG ODN-mediated effects on B-CLL cells correlate with cytogenetic status. The present study was designed to explore the impact cytogenetic status has on in vitro B-CLL cell survival and immunogenicity. Since most in vitro studies to date have been performed under serum-low conditions, we also evaluated how the presence of autologous serum or plasma impacts on CpG ODN-mediated effects on B-CLL cells. B-CLL cytogenetic status by interphase FISH, as well as immunophenotype and cell survival in the absence or presence of CpG ODN was determined in 23 samples. CpG ODN decreased in vitro survival of B-CLL cells with good prognosis cytogenetics, but had little effect on cells with poor prognosis cytogenetics. In contrast, CpG ODN upregulated costimulatory and antigen-presenting molecules and enhanced allogeneic T cell response in samples with good and poor prognosis cytogenetics. The optimal concentration for CpG ODN-mediated effects in the presence of 100% autologous serum or plasma was higher (10–20 micrograms/ml ODN) than for serum-low conditions (2.5–5 micrograms/ml ODN). The observed inhibition of CpG ODN-mediated effects by serum directly correlated with a lower uptake of ODN into B-CLL cells in the presence of serum. In conclusion, CpG ODN induced changes in B-CLL consistent with enhanced immunogenicity in all samples studied, but induced apoptosis most effectively in the subset of B-CLL cells with good prognosis cytogenetics. Approximately 4-fold more CpG ODN was needed to induce these changes when serum was present as compared to low serum conditions due to decreased ODN uptake by the cells in the presence of serum. These studies suggest CpG ODN may be useful as immunotherapeutic agents for B-CLL irrespective of cytogenetic status because of their potential effects on immunogenicity. Higher systemic doses of CpG ODN than previously thought might be necessary to induce these changes because of the effect of serum proteins on ODN uptake.
Optophoresis Accurately Predicts Response to Chemotherapy in CLL.
Authors: McNeeley Patricia , Saven Alan , Kariv Ilona , Nieva Jorge , Marchand Philippe , Diver Jonathan , Riccitelli Samuel , Nerenberg Michael
Published: April 2020

In vitro drug resistance assays have shown efficacy in predicting response to chemotherapeutic drugs in a number of solid tumors. In chronic lymphocytic leukemia (CLL) many assays are limited by the difficulty involved in culturing these cells ex vivo. We have developed a novel, optical-based methodology that is sensitive to broad cellular physical characteristics, such as morphology, size, refractive index, density and surface properties. This measurement, known as Optophoresis, quantifies cell motion induced by exposure to a moving optical gradient, generated from a near-infrared laser beam (Forster AH, et al. Anal Biochem 2004, 327(1):14–22 and Wang MM, et al. Appl Opt 2003, 42(28):5765–73). In Optophoresis small numbers of cells are analyzed intact, in their native state. No labels are required for quantification of functional responses, but cell subpopulations may be identified using fluorescent tags. We have used this assay to predict response to chemotherapeutic agents in patients with CLL.

Methods: We performed Optophoresis with six drugs (fludarabine, chlorambucil, vincristine, cyclophosphamide, cladribine and prednisolone) on 74 CLL patient samples. 68 of these were classic B-CLL; there was one hairy cell variant, two were T-CLL, two evolved to PLL and one evolved to lymphoma. 21 of the samples were from patients for whom clinical data on response to chemotherapy was available for 33 drug treatments. Response to therapy was defined as a decrease in RAI stage.

Results: 21 patients aged 58 to 93 years; 71% Rai stage III or IV were included in this analysis. Six patients were evaluated prospectively, with the assay performed before chemotherapy administration, and 15 patients were evaluated retrospectively with the assay performed after a course of treatment. Optophoresis accurately predicted response to chemotherapy in 88% of the 33 treatments evaluated. In the four instances for which the Optophoresis results did not match the retrospective clinical outcome; the patient was historically sensitive to treatment and Optophoresis results indicated current resistance to the drug. Subsequent resistance to drug therapy is known to occur in a significant percentage of treated CLL patients.

Conclusion: Optophoresis of CLL cells accurately predicts response to chemotherapy in CLL. Further studies using results of Optophoresis to guide CLL treatment are warranted.
Combined Disruption of Both the MEK/ERK and the IL-6R/STAT3 Pathways Is Required To Induce Apoptosis of Multiple Myeloma Cells in the Presence of Bone Marrow Stromal Cells.
Authors: Chatterjee Manik , Stuehmer Thorsten , Herrmann Pia , Bommert Kurt , Dorken Bernd , Bargou Ralf C.
Published: April 2020

The IL-6R/STAT3 pathway has been reported to critically contribute to the pathogenesis of multiple myeloma (MM) and to protect MM cells from apoptosis. However, recently we could demonstrate that MM cells become independent of the IL-6R/STAT3 pathway if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6-independent pathways that exert a pro-survival effect. It was therfore the aim of this study to analyze the underlying mechanism of this phenomenon. Pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and MAPK in parts via IL-6R-independent mechanisms. Abolition of MEK1, 2 activity with PD98059, or of ERK1,2 through siRNA constructs, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. Thus, disruption of the MEK/ERK pathway restores IL-6/STAT3 dependence of MM cells in the presence of BMSCs indicating that BMSC-mediated induction of the MEK/MAPK pathway is the mechanism by which BMSCs render MM cells IL-6/STAT3 idependent. Consequently, in the presence of cells from the BM microenvironment the combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This effect was observed with MM cell lines and with primary MM cells and might have direct implications for the development of future therapeutic strategies for MM.
Prognostic Significance of BCL-2 Expression in Patients with Multiple Myeloma.
Authors: Hashmi Mehmood , Kojouri Kiarash , Yang Jian , Patel Sunil , Khan Mohammad , Sylvester-Kohrt Nancy , Kamble Rammurti , Selby George B. , Ozer Howard , Kharfan-Dabaja Mohamed A.
Published: April 2020

Background: multiple myeloma (MM) remains an incurable disease that accounts for approximately 10% of all hematologic cancers. Patients (pts) with MM treated with conventional chemotherapy have a median overall survival of 36–42 months. Various adverse prognostic factors have been described in MM, including elevated B2 microglobulin, hypoalbuminemia, cytogenetic abnormalities of chromosome 13 (13q14 del, monosomy 13), and elevated CRP among others. BCL-2 mediates anti-apoptotic pathways and its expression has been implicated with lack of response to certain chemotherapeutic agents and unfavorable prognosis in different human malignancies.

Objective: to evaluate the prognostic significance of BCL-2 expression in patients with MM.

Methods: adult pts who were diagnosed with MM at our institution since 1/1/90 were retrospectively analyzed. Expression of BCL-2 was determined by immunohistochemistry (IHC) staining of deparafinized bone marrow specimens. Median survival was calculated for BCL-2 (+) and BCL-2 (−) groups. Survival curves were estimated according to the Kaplan-Meier method, and were compared with the use of the log-rank test. Median survivals were also compared by using Wilcoxon-Mann-Whitney test. Two-tailed alpha level of 0.05 was considered statistically significant. SAS® software (Cary, NC) was used for statistical analysis.

Results: sixty-six patients were initially identified. Forty-four pts were excluded because bone marrow samples were not available for IHC staining. Twenty-two pts (11 males, 11 females), with a median age at the time of diagnosis of 64 years (range: 46 to 83 years) were included for this analysis. Fourteen pts (64%) had positive BCL-2 bone marrow specimens, including 4 mild, 3 moderate, and 7 strong staining, while for 8 pts (36%), bone marrow specimens did not stain for BCL-2. Twelve pts (86%) from BCL-2 (+) group and 4 pts (50%) from BCL-2 (−) group died during the period of follow-up; 8 pts are still alive at the time of this analysis, 23 to 71 months (median: 39 months) after diagnosis. The median survival among BCL-2 (+) pts was 35 months (95% CI: 24 – 69 months) and among BCL-2 (−) pts was 47 months (95% CI: 13 months - N/A). Overall survival did not differ between the two groups (p-values for log-rank and Wilcoxon-Mann-Whitney tests, 0.8256 and 0.7072, respectively).

Conclusion: although our study did not show correlation between BCL-2 expression and overall survival, median survival among BCL-2 (−) pts with MM was 34% longer than BCL-2 (+) pts. The authors recognize the limitations associated with this analysis including the retrospective nature of the study, small sample size, and exclusion of a large number of patients. This study highlights the need for prospective evaluation of the prognostic significance of BCL-2 expression among pts with MM.
Characteristics of Immunoglobulin Deficiency in Patients with Untreated Multiple Myeloma Stage II and III.
Authors: Hamel Markus , Poenisch Wolfram , Hasenclever Dirk , Gelbrich Goetz , Loeffler Markus , Ali Haifa K. Al , Lehmann Stefanie , Niederwieser Dietger , Merkle Karlheinz
Published: April 2020

Introduction: It is well known that multiple myeloma (MM) is often associated with immunoglobulin (Ig) deficiency and increased risk of infection. We evaluated the Ig levels in 150 patients with MM stage II and III as part of a phase III randomised study comparing the effectiveness of melphalane and prednisolone versus bendamustine and prednisolone.

Patients and methods: 150 patients at a median age of 68 years, with untreated MM stage II, n = 19, and stage III, n = 131, were evaluated. MM type was IgG, IgA, light chain, and biclonal in 106 (70,7 %), 32 (21,3 %), 11 (7,5 %), 1 (0,7 %) patients respectively. Serum Ig levels were measured in all patients prior to chemotherapy. Normal values for IgG were 7,02 – 14,6 g/l, for IgA 0,24 – 3,81 g/l, and for IgM 0,54 – 2,27 g/l. Only Ig classes other than the myeloma protein class were evaluated.

Results: Patients with IgG MM had a median serum IgA of 0,4 g/l (range 0 – 7,9), and a median IgM level of 0,2 g/l (0 – 5,6). In 106 patients with IgG MM, IgA serum levels were below, within normal, and above normal values in 36 (34 %), 69 (65,1 %), and 1 (0,9 %) patients, respectively. On the other hand, IgM levels in these patients were below, within normal, and above normal values in 94 (88,7 %), 9 (8,5 %), and 3 (2,8 %) patients, respectively. Median IgG for IgA MM patients was 3,7 g/l (range 0,1 – 11,8), and median IgM in these 32 patients was 0,2 g/l (range 0 – 1,3). IgG levels were below and within normal in 28 (87,5 %), 4 (12,5 %) patients, respectively. Similarly, IgM level were below, and within normal range in 30 (93,8 %), and 2 (6,3 %) patients with IgA MM. Lastly, median IgG in patients with light chain myeloma was 6,3 g/l (range 2,1 – 9,7), median IgA, and median IgM were 0,4 g/l (range 0,1 – 1,7), and 0,3 g/l (range 0,1 – 0,8), respectively. IgG, IgA, and IgM were below normal values in 8 (72,7 %), 3 (27,3 %), and 9 (81,8 %), respectively.

Conclusion: Most patients with untreated MM have markedly reduced serum Ig levels. The correlation of reduced Ig level to the incidence of infections is being evaluated. The effect of chemotherapy on Ig levels is currently being assessed in our phase III study.
Recombinant Human Erythropoietin (rHuEpo) as an Anti-Myeloma Immunomodulatory Agent.
Authors: Prutci-Sagiv Sari , Katz Odelia , Yeyni Lidor , Skaat Alon , Barzilay Eran , Neumann Drorit , Mittelman Moshe
Published: April 2020

Recombinant human erythropoietin (rHuEpo), the main growth regulator of red blood cells, has been used in clinical practice for the treatment of cancer-related anemia. Recent studies suggest that rHuEpo has additional biological effects; including our finding that rHuEpo significantly prolonged the expected survival of six end-stage multiple myeloma (MM) patients whose expected survival was <6 months (Mittelman et al., 2004, EJH 72, 155–65). The course of disease in these six MM patients led us to hypothesize that Epo might have an anti-neoplastic or anti-myeloma effect. This latter observation was further supported by studies, demonstrating a T cell mediated anti-myeloma effect of rHuEpo in mice models (Mittelman et al., 2001, PNAS 98, 5181–6). The mechanisms underlying the effects of Epo on the immune system are not fully clear. Our data show the effects of rHuEpo on the T-cell as well as the B-cell lineage. We found that the ratio of CD4+ to CD8+ T cells, which is decreased in MM patients, increases to the normal range in MM patients who are receiving rHuEpo treatment. rHuEpo-treated MM patients show a significant improvement in the level of mitogen-mediated activation of mononuclear cells, as measured by the early activator marker CD69. Furthermore, mononuclear proliferation in rHuEpo-treated MM patients was increased compared with MM patients who were not on rHuEpo treatment. Elucidating the anti-neoplastic effects of Epo treatment and obtaining a better insight into the immunomodulatory features of these effects will open new avenues in the treatment of hematological malignancies such as MM and related disorders. Moreover, the better understanding of the immunomodulatory properties of Epo may also be applied in other diseases manifesting immune abnormalities and immunocompromised subjects.
Effects of A771726, the Active Metabolite of Leflunomide,on Multiple Myeloma Cells Proliferation in Vitro.
Authors: Yang Shaosong , Venkataraman Girish , Krett Nancy L. , Rosen Steve T.
Published: April 2020

Multiple Myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated plasma cell in the bone morrow, which accounts for 10% of all hematological cancers. Despite major advance of last decade, MM remains an incurable disease in need of new therapeutic approaches. Leflunomide was originally developed as an immunosuppressive drug capable of alleviating the severity of autoimmune disease and preventing allograft and xenograft rejection and it has been FDA approved to use in the treatment of rheumatoid arthritis (RA) since 1998. It rapidly converted in gastrointestinal tract and plasma to open ring metabolite A771726. In current study, we examine the effects of A771726 to the growth of MM cell lines in vivo and show that A771726 inhibits the proliferation of four different well-characterized MM cell lines (MM.1, U266, RPMI8226 and MDR10V) in a dose- and time- dependent manner, regardless their sensitivity to the conventional chemotherapies. Cell cycle analyses further demonstrate that G1-S phase transition is blocked by A771726 in those cells. Moreover, we find that, at higher concentration, A771726 is able to induce apoptosis in at least two of the cell lines tested as measured by an increase of the cells with subG1 DNA content, an increasing in caspase activity and cleavage of caspase substrate. Previous studies suggested that A771726 exerts its anti-proliferate function mostly by inhibiting de novo pyrimidine synthesis in lymphocyte and supplementation of the cultures with exogenous uridine could prevent A771726-induced cell cycle block by restoring the intracellular UTP and CTP lever. However, we find that additions of exogenous uridine can prevent neither A771726-induced cell cycle block nor apoptosis in the MM cell lines tested, suggesting a novel mechanism. It shall be noted that pharmacologically, the highest concentration of A771726 (200uM) used in our study is readily achievable in the plasma through current drug regiment of Leflunomide in RA patients. Since its approval, Leflunomide has been proved to be an effective antirheumatic drug with mild side effects. Given the results from current study, we are tempting to suggest that Leflunomide might also provide a safe alternative in the treatment of MM. The vast knowledge gained from clinical use of Leflunomide in last six year will greatly help us greatly in testing this hypothesis in a clinical setting in a near future.
Severe Osteomyelitis of the Jaws in Long Term Survivorsmultiple Myeloma Patients Treated with Biphosphonates.A New Clinical Entity.
Authors: Lugassy Gilles , Nemets Anatoly
Published: April 2020

Multiple Myeloma is a frequent and usually fatal lymphoproliferative disorder with a median survival of around 30 months.Supportive therapy includes monthly intravenous injections of biphosphonates as prevention, or therapy,of lytic bone disease.

We report three cases of Multiple Myeloma patients who developed severe osteomyelitis of the jaws while receiving prolonged biphosphonate therapy.All three patients are very long survivors of Myeloma,being alive 6 to 12 years after diagnosis.They have been treated with biphosphonates for more than 4 years, receiving large doses of Pamidronate(3420 to 4680 mg) and lesser doses of Zoledronate(16 to 72 mg).Jaw infection was unprovoked in wo parients and was cused by dental extraction in the third.Diagnosis of osteomyelitis was confirmed by histology in all three patients, while in two we could demonstrate the presence of Actinomyces.Therapy with hyperbaric oxygen of osteomyelitis refractory to prolonged antibiotherapy, was curative in one patient. To the best of our knowledge, these are the first cases of osteomyelitis of the jaw reported in the literature in Myeloma patients treated with chronic administration of intravenous biphosphonates.Recent reports have alerted physicians of a ’’ growing epidemics’’ of Pamidronate and Zoledronate induced avascular necrosis of the jaw, most of them provoked by teeth removal.Both of these biphosphonates have strong antiosteoclastic activity, and Zoledronate is antiangeogenic, all properties which contribute to the development of bone necrosis.Since the jaws are the only bones in the skeleton exposed to the external environment, the association of bone necrosis with secondary chronic infections may lead to osteomyelitis.Even though it is not possible to prove beyond doubt an etiological link between biphosphonate therapy and the occurrence of osteomyelitis of the jaws, the association of Myeloma with severe immunosupression, the very prolonged biphosphonate therapy in our three patients, and the localization to the jaw, are in favor of a clear relationship between therapy with Zolandronate, Pamidronate and osteomyelitis of the jaws. With the expected life prolongation of Myeloma patients due to recent therapeutic breakthroughs and the increasing use of antiangeogenic agents, it is crucial to be aware of the possibility of a rise in the number of osteomyelitis of the jaws in Myeloma patients.
Serum Free Light Chain (FLC) Measurement in Multiple Myeloma (MM) Patients (pts) Correlate with Known Monoclonal Paraprotein, Disease Stage and Therapy Response.
Authors: Engelhardt Monika , Räpple Daniel , Weis Andreas , Bisse Emanuel , Ihorst Gabriele
Published: April 2020

So far, data based on small patient (pt) population suggest that the measurement of serum FLC from MM pts undergoing high-dose chemotherapy (HDCT) with stem cell transplantation (SCT) may be a sensitive marker for monitoring therapy success and for early detection of relapse. For further evaluation of the impact of FLCs on the assessment of treatment efficacy of standard- (ST) and HDCT with SCT, we performed a prospective analysis on serial serum specimens from 86 MM and 9 control pts. Measurement of FLC concentration was performed with the commercially available Freelite™ kit (Binding Site). For statistical analysis, pts’ clinical history, age at diagnosis, sex, current state of disease, karyotype and serum parameters, such as ß2-microglobulin, calcium levels and serum creatinine were evaluated. In the control group (NHL=6, AML=1, non-hematological disease=2), median concentrations of kappa(k)- and lambda(l)- FLC were 9.8 mg/l and 12.8 mg/l, respectively, corresponding to reference intervals for healthy individuals with normal kappa(k)/lambda(l)-ratios. In MM, 40 (46.5%) pts displayed kappa(k)-FLC levels above the upper range of 19mg/l, 26 (30%) had lambda(l)-FLC levels above the upper range of 26 mg/l and 9 pts (10.4%) had both elevated kappa(k)- and lambda(l)-FLC serum levels. An abnormal kappa(k)/lambda(l)-ratio was observed in 45 (52,3%) MM pts. Pts with a known kappa(k)-paraprotein (n=58) had a median FLC kappa(k)-concentration of 38 mg/l, but lambda(l)-FLC within the normal range. For pts with a known lambda(l)-paraprotein (n=27), reciprocal findings (76.4 mg/l for lambda(l)- vs kappa(k)-FLC in the normal range) were observed. Pts with responsive disease (CR, PR and SD) had both kappa(k)- and lambda(l)- FLC levels within the normal range, whereas newly diagnosed pts (ED) and those with PD had kappa(k)- FLC levels approx. 3-times the normal range, with lambda(l)- FLC levels at the upper limit of normal. Pts receiving ST as compared with HDCT had higher FLC levels. This is also observed in pts with amyloidosis, renal impairment or PD. Our results suggest that serum FLC assay allows monitoring of the therapy response and early detection of relapse. Determination of FLCs is also important, when evaluating new therapeutic substances, and for detection of prognostic patterns for better risk-based stratification of treatment.
Osteoblastic Activation and Response to Velcade® in Patients with Relapsed/Refractory Multiple Myeloma (MM).
Authors: Zangari Maurizio , Lee Choon-Kee , Barlogie Bart , Najarian Kevin , Kang Seung-Hee , Yaccoby Shmuel , Esseltine Dixie , Stover Julie M. , Tricot Guido J.
Published: April 2020

The molecular targets of anti-tumor activity elicited by VELCADE may vary among tumor types. The extent to which each target is critical to the inhibition of tumor growth may also differ. In MM patients, we have previously reported a statistical correlation between the elevation of total alkaline phosphatase (ALP) levels (in the absence of liver function abnormalities) and response to combination treatment with VELCADE, Thalidomide, and Dexamethasone (Zangari, et al, Blood, 2003:102(11):687a). Changes in ALP levels have recently been analyzed in patients treated with single agent VELCADE on the Millennium-sponsored trial 025 evaluating sequential multi-chemistry profiles obtained from a total of 182 patients with relapsed/refractory MM. Median age was 58 years and 61% were male. Patients were divided into two groups based on their myeloma response. Group A included patients with no response and/or progressive disease following VELCADE therapy. Group B included patients who achieved ≥ 25% paraprotein reduction. Elevation of ALP, in the absence of liver function abnormalities, was observed exclusively in responding patients (Group B). Using the Wilcoxon Rank-Sum test, the difference in elevation of ALP after completion of two cycles of VELCADE between the two groups was statistically significant (P=0.02) [Figure1]. In an attempt to determine the source of the ALP increases, prospective serial measurements of ALP, bone-specific alkaline phosphatase (BALP) and intact parathyroid hormone (PTH) were obtained in three MM patients treated with single agent VELCADE at our institution. The rise in ALP activity was associated with rapid paraprotein response and to a parallel increase in BALP and PTH levels, indicating increased osteoblastic activity [Figure2]. Our experience represents the first clinical evidence in MM patients that VELCADE promotes osteoblastic activity, which typically correlates with anti-myeloma activity.
Cytokines Behaviour in Multiple Myeloma during Zoledronic Acid Treatment.
Authors: Avvisati Giuseppe , Petrucci Maria T. , Annibali Ombretta , Bartolozzi Francesco , Giannetti Barbara , Levi Anna , Battistoni Fabrizio , Gavasci Michele , Dicuonzo Giordano , Vincenzi Bruno , Santini Daniele , Foa Robin , Mandelli Franco
Published: April 2020

The main pathological feature underlying the bone disease in multiple myeloma is an uncoupling of bone resorption from bone formation so that resorption predominates. In the bone marrow (BM), MM plasmacells are in close contact with stromal elements and induce the release of several cytokines which modify the BM microenviroment, upregulating RANKL expression and secretion by both stroma and osteoblasts. Destruction of bone matrix induced by MM and its cellular interactions is accompanied by further release of cytokines. Bisphosphonates are chemical compounds which selectively concentrate at the interface of the active osteoclasts and the bone resorption surface where they inhibit osteoclast activity.

Recently, in solid tumors, Santini et al have demostred that zoledronic acid could have an in vivo antiangiogenic property through a significant and long-lasting reduction in serum VEGF levels (Clin Cancer Res2003;9:2893–7). Taking into account that angiogenesis plays an important role in MM progression, we have investigated the behaviour of IL6, PDGF, IGF, TNFα and VEGF in a cohort of 28 (16 males and 13 females) consecutive MM pts with lytic bone lesions treated with 4 mg of zoledronic acid. Venous blood samples were drawn just before the beginning of drug infusion and at 1,2,7,21 (before the subsequent infusion) days after the zoledronic acid infusion. Serum samples were then tested for PDGF, VEGF, IL6, TNFα , and IGF with the R & D quantitative kits (R & D Systems, Minneapolis, USA), according to the manufacturer’s instructions. Basal cytokine levels were compared with the values observed at 1, 2, 7 and 21 days using the Wilcoxon’s test for nonparametric-dependent continuous variables. Differently from what observed by Santini et al in solid tumors, serum VEGF median levels did not decrease;on the contrary, it significantly increased at days 7 (P=0.0047). Moreover, IL 6 and TNFα levels significantly increased after 1 (P=0.0000 and 0.0001 respectively) and 2, ( P=0.0000 and 0.0021 respectively) days. Furthermore, PDGF significantly decreased (P=0.005) after 2 days following zoledronic acid infusion. Among these pts, 8 (28.5%) were receiving also Interferon; no statistically significant differences in the levels of VEGF, IL6, TNFa and IGF between the two groups were observed. Only the PDGF levels have shown a statistically significant decrease in the interferon group at 0, 1, 2, 7, 21 days (P<0.05). In conclusion, the cytokines’ behaviour after the administration of zoledronic acid in MM is different from that observed in solid tumors; in particular, we did not observe a decrease in VEGF levels. This behaviour may partially explain why zoledronic acid has no effect on MM progression as originally postulated.
Anti-NK Cell Treatment Induces Stable Mixed Chimerism in MHC-Mismatched, T-Cell Depleted, Nonmyeloablative Bone Marrow Transplantation.
Authors: Cho Seok Goo , Shuto Yukinobu , Soda Yasushi , Nakazaki Yukoh , Izawa Kyoko , Uchimaru Kaoru , Takahashi Satoshi , Tani Kenzaburo , Tojo Arinobu , Asano Shigetaka
Published: April 2020

Objective: To clarify Natural killer (NK) cell-mediated resistance under cytoreductive conditioning and T-cell depleted bone marrow transplantation (TCD-BMT), we investigated the effects of host NK cell depletion on engraftment and induction of stable mixed chimerism.

Methods: BALB/c mice (H-2kd) were injected intraperitoneally with anti-asialoGM1 antibody (anti-NK Ab) on day -1. On day 0, they received TBI at a dose of 500 cGy, followed by intravenous infusion of 2 x 107 T-cell-depleted (TCD) bone marrow (BM) cells from C57BL/6 mice (H-2kb). Early engraftment and chimerism were determined by the relative ratio of peripheral blood (PB) lymphocytes expressing either H-2kd or H-2kb on day +21. Long-term engraftment and chimerism were evaluated on PB and spleen by multi-color flow cytometry.

Results: Although no recipients treated with TBI alone showed engraftment, all the recipients conditioned with anti-NK Ab and TBI showed successful engraftment as well as a donor-dominant pattern of mixed chimerism in both PB and spleen. Spleen cells from recipients with mixed chimerism showed specific tolerance to both host and donor strains, but not to a third-party (C3H/He). None of the reconstituted mice showed signs of graft versus host disease, and all survived up to day +330.

Conclusion: These observations indicate that host NK cell depletion may be used to reduce the intensity of conditioning regimens for engraftment of TCD grafts, and can contribute to establishment of stable mixed chimerism in MHC-mismatched NMT.
Oligotide, a Defibrotide Derivative, Protects Human Microvascular Endothelial Cells Against Fludarabine-Induced Activation, Damage and Allogenicity.
Authors: Eissner Guenther , Iacobelli Massimo , Blueml Susanna , Andreesen Reinhard , Holler Ernst
Published: April 2020

Fludarabine is a non-myeloablative immunosuppressant increasingly used as a component of alternative reduced intensity conditioning regimens prior to allogeneic stem cell transplantation (SCT). However, we have previously shown that 2-Fluoroadenine 9-beta-D-arabinofuranoside (F-Ara) as the active metabolized form of fludarabine induces damage, activation, and allogenicity in human microvascular endothelial cells (HMEC). We had also identified the pharmaceutic compound Defibrotide (DF), originally used in the treatment of veno-occlusive disease and thrombotic microangiopathy, as being protective against F-Ara induced dysfunction of HMEC, importantly, without affecting the anti-leukemic effect of F-Ara. In the present report we show that a recently developed derivative of DF, Oligotide, similarly downregulates F-Ara induced activation and damage of HMEC as well as their antigenicity for allogeneic CD8+ T cells. In addition, Oligotide could also block F-Ara mediated transendothelial migration of peripheral blood cells across the HMEC barrier. Taken together, these observations argue for a potential clinical use of both, DF and Oligotide in pre transplant conditioning.
Antigen Specificity and Immunoregulation in Syngeneic Graft-Versus-Host Disease.
Authors: Hess Allan D. , Thoburn Christopher J. , Bright Emilie C. , Miura Yuji
Published: April 2020

Syngeneic graft-vs-host disease (SGVHD) is a T cell dependent autoaggression syndrome induced by administering Cyclosporine following syngeneic bone marrow transplantation. The SGVHD autoreactive T cells recognize the MHC class II-invariant chain peptide complex (MHC class II-CLIP) and can be separated into functional subsets based on their differential dependence on the N- and C-terminal peptide flanking domains of CLIP. The present studies were undertaken to determine whether the N- or C-terminal flanking domain dependent subsets of CLIP reactive T cells reside within the CD4+CD25+ regulatory compartment. Multi-color flow cytometry was used to identify and isolate CD4+ (FITC) CD25+ (PE) T cells. Antigen-specific T cells within this compartment were identified with a soluble MHC class II-Ig chimeric construct (bioinylated, Cychrome avidin counterstaining) loaded with variants of CLIP containing the MHC class II binding domain and having either the N- or C-terminal flanking regions (N-CLIP, CLIP-C). Approximately 8.5% of the cells within the normal CD4+ lymphocyte population were CD25+. Both N-CLIP (1.1%) and CLIP-C (4.8%) reactive T cells coould be detected in the CD4+CD25+ population. Assessment of CD28, CTLA4, B7.1 and B7.2 mRNA expression levels by quantitative PCR directly ex vivo, revealed remarkable differences between the N-CLIP and CLIP-C specific CD4+CD25+ T cells. Although CD28 mRNA levels were comparable for both subsets, B7.2 and CTLA4 mRNA transcript levels were significantly increased (>50 fold) in the CLIP-C+CD4+CD25+ T cells compared to the N-CLIP specific subset. On the other hand, levels of B7.1 mRNA were increased >10 fold in N-CLIP+CD4+CD25+ T cells. Additional studies assessing mRNA transcript levels for the regulatory transcription factor Foxp3, also revealed a disparity between the N-CLIP and C-CLIP specific subsets. mRNA transcript levels for Foxp3 were markedly increased (>35 fold) in the CLIP-C dependent subset compared to the levels detected in N-CLIP+CD4+CD25+ T cells. Low levels of cytokine (IL-2, IL-4, interferon-γ) mRNA transcripts were detected in both subsets. Interestingly, intradermal immunization of normal animals with the peptides presented on dendritic cells increased mRNA transcript levels for type 1 cytokines in the N-CLIP reactive subset and type 2 cytokines in the CLIP-C dependent subset. Taken together, the results indicate that the CLIP-C antigen specific CD4+CD25+ cells have a profile consistent with regulatory T cells whereas the profile of the N-CLIP+CD4+CD25+ lymphocytes is more characteristic of activated T helper cells. The ability to identify and isolate regulatory T cells ex vivo and to modify their activity by immunization provides opportunities to both enhance and monitor the re-establishment of self-tolerance.
Can Addition of GM-CSF in Adult Haplo-Identical Transplant Who Cannot Benefit from NK Alloreactivity Improve the Relapse Rate of Advanced Leukemia: A 3 Step Study.
Authors: Lewalle Philippe , Delforge Alain , Nowak Berengere , Misplon Virginie , Lagneaux Laurence , Theunissen Koen , Bennani Jalil , Bron Dominique , Martiat Philippe
Published: April 2020

Haplo-identical transplant is now established as a procedure of choice for patients who lack a compatible donor. It might even be the best choice for AML, provided there is a GvH NK alloreactivity. However, patients are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study trying improve transplant related mortality, relapse rate and immunity: (1): G-CSF + DLI, (2): GM-CSF + DLI, (3): patient and disease adapted strategy. Thirty-four consecutive leukemia patients, aged 18–55, were investigated (20 very poor risk, 11 poor risk and 3 better risk). GvH type NK alloreactivity was chosen when possible (19/34) and balanced across the 3 groups. In the first 9 patients, G-CSF was used pot-transplant and prophylactic DLI were given at month 1, 2 and 3. The use of G-CSF and 1 to 3 DLI (104 CD3/kg) was found safe. It resulted in faster CD4 recovery and a low rate of infections. However, it was insufficient to induce a protective GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI (104 CD3/kg) at day 30 unless aGVHD (3 pts). The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CSF + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD grade 2 or more: 9/12; price to pay: GVHD resulting in 5 deaths in total. Median time to relapse in the 21 first patients was 6 months (range 4 to 9). Step 3 (14 patients) consists of a patient adapted strategy: no more aspecific DLI (selected anti-CMV and aspergillus DLI planned in all patients); in myeloid disorders with NK alloreactivity: no GF. In the other cases, GM-CSF (at a reduced total dose of 500 mcg) is given from day 5 to day 9. The follow-up of these 14 patients, although promising (2 relapses), is currently short (median 5 months), compared to the median of relapse in the 2 first groups (6 months) but will be updated for the meeting (10 months and 17 patients). Overall, TRM at day 100 is 3/29, reflecting the good tolerance of the conditioning in a heavily pre-treated population (median age : 43). Overall NR-mortality at one year is 8/26, but was greater in the GM-CSF + DLI group, reflecting the impact of severe aGVHD. In the current group, its is 2/14 at 3 months. We conclude that the third strategy might improve the outcome and the relapse rate without exposing patients to unnecessary severe GVHD.
Mechanisms of Alpha Hemoglobin Stabilizing Protein (AHSP) Actions.
Authors: Zhou Suiping , Gell David , Kong Yi , Li Jianqing , Mackay Joel P. , Weiss Mitchell J. , Gow Andres J.
Published: April 2020

Alpha hemoglobin stabilizing protein (AHSP) is an abundant erythroid factor that specifically binds and stabilizes free α hemoglobin (Kihm et al., Nature 417:758, Gell et al., JBC, 277:40602). In mice, AHSP deficiency causes oxidative stress, mild anemia, and reticulocytosis. Furthermore, loss of AHSP exacerbates α globin precipitation and anemia in a murine model for β thalassemia. These data suggest that AHSP operates as a molecular chaperone to stabilize free α hemoglobin and inhibit its ability to catalyze redox chemistry. We investigated how AHSP associates with α hemoglobin and thereby reduces its oxidative capacity. By NMR and mutational analysis, we determined the structure of AHSP and defined its α globin-interacting surfaces. AHSP consists of three linked α helices. Key α globin-binding residues lie along helices 1 and 2 in a region that resembles the G and H helical fold of β globin. Hence, AHSP presents a surface resembling that of β hemoglobin and thus promotes the formation of a “pseudo α/β interface”. To investigate how formation of this complex affects α hemoglobin-mediated redox chemistry, we examined the ability of AHSP to inhibit peroxide-mediated oxidation of the redox-sensitive dye TMPD. Oxy-α hemoglobin alone catalyzed TMPD oxidation at a rate of 140 μMmin−1; preincubation with AHSP reduced this rate significantly, to 65 μMmin−1. Furthermore, AHSP reduced both heme loss from α hemoglobin and its ability to oxidize hemoglobin A. AHSP binding to oxy- α hemoglobin induced a spectral shift in the UV/visible range, suggesting that stabilization might be associated with structural alterations in the heme moiety. Specifically, the Soret peak was reduced and shifted leftward, while the 541 and 576 nm peaks of the visible region were reduced with concomitant rises at 500 nm and at the region above 580 nm. These alterations suggest that heme-bound iron is converted to a ferric (Fe3+) form that is liganded at all 6 coordinate positions, and therefore, can no longer catalyze redox chemistry. Consistent with this possibility, addition of KCN to the α hemoglobin-AHSP complex produced cyanomethemoglobin-AHSP, but at a slower rate than reaction of KCN with ferric-α hemoglobin alone. In addition, electron paramagnetic resonance and resonance raman spectroscopy showed that the heme iron exists in a low spin state within the α hemoglobin-AHSP complex. The oxidized complex was insensitive to pH alteration, indicating that iron was not bound by hydroxyl ion, which typically occurs at alkaline conditions. Together, our findings illustrate a potential mechanism whereby AHSP renders α hemoglobin chemically inert by inhibiting the reactivity of heme-bound iron. We propose that within the AHSP-α hemoglobin dimer, the oxidized heme is held within a bi-histidyl form, thus blocking its ability to act as a redox catalyst.
The Kinetics of Etoposide (VP-16) Peripheral Blood Progenitor Cell (PBPC) Mobilization.
Authors: Bolwell Brian J. , Kuczkowski Elizabeth , Rybicki Lisa , Pohlman Brad , Andresen Steven , Sobecks Ronald , Baker Janet , Cherni Kelly , Bernhard Laura , Vellani Alison , Berry Nicole , Sungren Shawnda , Foster Leslie , Kalaycio Matt
Published: April 2020

The kinetics of G-CSF PBPC mobilization are well described. CD34+cells in peripheral blood rise four days after G-CSF administration, and decline after the eighth day. VP-16 is increasingly used with G-CSF as a mobilizing regimen; however, the kinetics of mobilization are sparsely described. We retrospectively reviewed 275 patients (pts) with NHL or HD who received VP-16 plus G-CSF as a primary PBPC mobilizing regimen. 214 (78%) had NHL; 31% received prior radiation therapy; 63% were male; 85% had responsive disease. Pts received VP-16 (2 gm/m2) followed by daily G-CSF (10 mcg/kg). All pts experienced a significant WBC nadir. Pts began pheresis when their WBC recovered to 5,000. Pts were pheresed for at least 2 days or until 7.0 x 106 CD34+ cells/kg were collected; pheresis continued until a minimum of 2.0 x 106 CD34+ cells were collected. WBC nadir occurred day +6 after VP-16 and WBC recovery to 5,000 occurred day +13 (median) after VP-16. Pts were pheresed for a median of 3 days which yielded a median of 9.7 x 106 CD34+ cells/kg (range, 2.0–100.1 x 106). 72% of pts began collection on day +12, day +13, or day +14. The average CD34+ cell collection was maximal on the first 3 days of pheresis, but reasonable yields continued to be obtained for approximately 10 days of pheresis, as shown below:

Figure Figure

81% collected ≥ 5 x 106 CD34+ cells/kg, and 72% collected ≥ 7 x 106 CD34+ cells/kg. The platelet count on the first day of pheresis correlated with the ability to collect 5 and 7 x 106 CD34+ cells/kg (p<0.001). 50 of 275 (18%) pts did not begin pheresis until day +15 or longer after VP-16 administration. The variables that correlated with these problematic pts was the platelet count at day 1 of pheresis and refractory disease at the time of mobilization; of 24 pts with refractory disease, 11 (46%) had poor WBC recovery after VP-16 and initiated mobilization on a median of day +16 (p<0.001). We conclude that VP-16 + G-CSF is a very effective mobilizing regimen. The majority of pts mobilize large numbers of CD34+ cells requiring only 3 days of leukopheresis for excellent yields. Unlike the kinetics of G-CSF for PBPC mobilization in which yields of CD34+ cells are optimal for only 3–4 days, CD34+ cell collection continues for approximately 10 days from the initiation of pheresis. A minority of pts have poor WBC recovery after VP-16 administration, and these pts tend to be those with refractory disease.
Association of Circulating CD34+ Cell Count with Liver Conditions.
Authors: Masuya Masahiro , Katayama Naoyuki , Ohishi Kohshi , Shiraki Katsuya , Uemoto Shinji , Nobori Tsutomu , Shiku Hiroshi
Published: April 2020

Hematopoietic stem and progenitor cells, which express CD34 antigen, circulate at a very low frequency in peripheral blood in steady state. CD34+ cells are highly mobilized after myeloablative chemotherapy or the administration of various cytokines such as granulocyte colony-stimulating factor and thrombopoietin (TPO). As TPO is mainly produced by the liver, liver conditions may be associated with hematopoiesis, especially thrombopoiesis. In fact, thrombocytopenia is common in patients with chronic liver diseases. To clarify the association of CD34+ cell mobilization with liver conditions, we examined the number of circulating CD34+ cells in 88 patients with various stages of hepatitis C virus-associated chronic liver diseases; 5 healthy carriers, 43 patients with chronic hepatitis (CH), 19 patients with liver cirrhosis (LC), and 21 patients with LC and hepatocellular carcinoma (HCC). Moreover, to assess the basal number of circulating CD34+ cells, 16 healthy controls were included. The evaluation of CD34+ cells has been carried out by flow cytometry, by applying conventional protocols. The number of circulating CD34+ cells was significantly diminished with progression of liver disease: healthy controls, 3.78 ± 3.93 cells/μL; healthy carrier, 1.89 ± 0.94 cells/μL; CH, 1.49 ± 1.41 cells/μL; LC, 0.66 ± 0.83 cells/μL; and LC with HCC, 0.58 ± 0.19 cells/μL. Next, we analyzed platelet count and number of circulating CD34+ cells before and after living donor liver transplantation (LDLT) in 12 patients who underwent LDLT for LC and/or HCC. A 4-fold increase in platelet level (from 68 x 109/L to 234 x 109/L) and a 5-fold increase in number of circulating CD34+ cells (from 0.78 cells/μL to 3.52 cells/μL) were observed after LDLT. These results suggest that not only thrombopoiesis but also mobilization of CD34+ cells from bone marrow into PB depends on liver conditions.
Bayesian Multilevel Evaluation of Stem Cell Cryopreservation at the First National Umbilical Cord Blood Bank from the Public Health Sector in Mexico.
Authors: Calderon-Garciduenas Eva D. , Sanchez-Guerrero Sergio A. , Fernandez-Torres Javier , Millan-Rocha Miriam , Marin-Lopez Antonio , Valiente-Banuet Leopoldo , Perez-Huizar Monica
Published: April 2020

Introduction. The umbilical cord stem cell bank was created in Mexico City in june 2003 due to the need to have access to pluripotential stem cells to cover the hematological and immunological pediatric needs. We have so far 300 units of umbilical stem cells available to the medical community. The bank has been designed with a completely automatic process and the standards are based in NETCORD-FAHCT outlines.

Objective. In the present study we made a balance of the evaluation of umbilical cord stem cell process including the maternal setting, the standarization of the methodology to obtain the stem cells, compared to other institutions around the world.

Material and methods. Bayesian analysis allow us to evaluate our procedures at the different levels. Bayesian networks are directed acyclic graphs (DAGs) where the nodes are random variables and certain independence assumptions hold.

Results. In table 1 we show the results of the first 300 units process with the automatic process. The arcs in a bayesian network specify the independence assumptions that must hold between the random variables and the global dependence of the total factors. Figure 1.

Conclusion. Through the bayesian analysis, we found a direct influence of the collected volume, the time between the collection and the procedure, and the maternal unit, with respect to the number of recovered cells, specifically with CD34+ the viable ones as well as the totals.

Figure 1. Bayesian Analysis between neonatal and process factors. Weight baby (PRN); sex (SRN); cord blood unit collected volume (ml); initial white cell (GBTI); total final white cell(GBTF); % total CD34+ ( PCD34T); % viable CD34+ (PCD34V); total CD43+ (CD34T); viable CD34+ (CD34VA); ginecology unit (UH); time hour (TH). Figure 1. Bayesian Analysis between neonatal and process factors. Weight baby (PRN); sex (SRN); cord blood unit collected volume (ml); initial white cell (GBTI); total final white cell(GBTF); % total CD34+ ( PCD34T); % viable CD34+ (PCD34V); total CD43+ (CD34T); viable CD34+ (CD34VA); ginecology unit (UH); time hour (TH).

Results of the first 300 units process with these technology

Initial nuclear cells/ml Collectionvolume Initial total nuclear cells % CD 34total Viable% CD 34 Recovery% Average 11.4 x 106/ml 102.8 ml 12.2 x108 0.32 0.31 80 Acceptance criterion 7–20 x 106 > 75 ml >8 x 108 0.1– 0.3 0.1– 0.3 >60%