bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccine-Induced Anti-Neuroblastoma Immunity Early after HSCT Arises in the Presence of Foxp3+CD25+CD4+ T Cells.
Authors: Jing Weiqing , Orentas Rimas J. , Johnson Bryon D.
Published: October 2019

Effective anti-neuroblastoma immunity can be induced in mice as early as 1 week after syngeneic HSCT by vaccination with neuroblastoma cells transfected to express CD80, CD86, CD54, and CD137L. However, effective vaccination at this early time point also requires adoptive transfer of T cells (2×107 splenocytes). Previous results in non-transplanted mice showed that T-reg depletion/blockade with anti-CD25 mAb (clone PC61) increased tumor vaccine efficacy. To determine if PC61 treatment might be effective early after HSCT, we first determined if CD4+CD25+Foxp3+ T-reg cells were present, derived either from host (i.e., they survived lethal TBI) or from the transferred spleen cells. Even though transferred splenocytes accelerated T cell reconstitution, HSCT recipient mice remained severely lymphopenic until 3 weeks after transplant. Furthermore, the percentage of Foxp3+ cells in the splenic CD4 fraction was significantly increased in transplanted mice, being as much as 3 times higher than that in non-transplanted mice (35% vs. 12%). During this time, the majority of CD25+Foxp3+CD4+ cells were derived from the adoptively-transferred T cells. Despite the presence of CD25+Foxp3+CD4+ T cells, protective anti-tumor immunity could still be efficiently induced. To examine whether depletion/inhibition of CD25+ T-reg cells could further enhance vaccine-induced immunity in transplanted mice, two strategies were tested: (a) physical depletion of CD25+ regulatory T cells from the adoptively-transferred T cells, and (b) anti-CD25 mAb treatment of the transplanted mice 4 days after HSCT to target both transferred and residual endogenous CD25+ cells. Immunomagnetic depletion eliminated more then 95% of the CD25+ T cells in the transferred population, but only 70% of Foxp3+CD4+ T cells. Adoptive transfer of CD25-depleted splenocytes at the time of HSCT resulted in modest increased tumor vaccine efficacy as compared to mice given non-depleted splenocytes. Two weeks after HSCT, both groups of mice contained similar percentages of Foxp3+CD4+ T cells in the blood and lymph nodes, but a lower frequency of Foxp3+CD4+ T cells was detected in the spleens of mice given CD25-depleted splenocytes (35% vs. 43%, p<0.01). Our second strategy, treatment of HSCT recipients with anti-CD25 mAb prior to tumor vaccination, negatively influenced the vaccine-induced anti-tumor response and resulted in decreased survival. This suggested that the antibody treatment inhibited effector cell function in the early post-transplant setting. IFN-γ ELISpot analysis demonstrated that vaccinated transplant recipients given anti-CD25 mAb had significantly decreased numbers of tumor-specific CD4 T cells in spleen. Collectively, these results suggest that anti-neuroblastoma immunity could be induced in the present of CD25+Foxp3+CD4+ T-reg cells early after HSCT; strategies that targeted at CD25 did not significantly increase the efficacy of the tumor vaccine.
Chronic Hemolytic Anemia in Two Patients Double Heterozygotes for Pyruvate Kinase and Another RBC Glycolitic Pathway Deficiency.
Authors: Pissard Serge , Saddedine Nafa , Vasson Aurelie , Ballayguier Marie-Odette , Galacteros Frederic , Wajcman Henri
Published: October 2019

Homozygous or compound heterozygous for Pyruvate Kinase (PK) deficiency are classical etiology for chronic non spherocytic hemolytic anemias while heterozygous carriers are free from disease. We report here 2 patients heterozygous for PK deficiency which displayed an unexpected marked chronic anemia. Enzymatic and molecular studies were performed to unravel the mechanism causing this phenotype. The first patient, a 60-year-old woman from Mali presented with Hb 10g/dL, MCV 98fL, and was free from any Hb abnormality. The second one was the first child of a healthy French Caucasian couple, and suffered since birth from a marked hemolytic anemia (Hb 7g/dL). We found that the first patient carried together an Arg569Met/Leu PK-R mutation (Pissard et al. Brit J Haematol, 2006, 133, 683-9) and the rare G6PD Santa Maria mutation (nt c.542 A>T, Asp181Val). The second patient had a PK splice site mutation (IVS4 + 10 G>T) and a new hexokinase mutation [c.1793_c.1836 +7(del 50)] which starts in exon 12 and ends in intron 12. It results in a protein troncated inside the glucose binding site. In this case, family study showed that the PK deficiency was inherited from the father and the HK deficiency from the mother. Enzymatic data are shown in the table. None of these enzymatic defects could alone, in the heterozygous state, be responsible for an hemolytic anemia. To explain why, in these two cases the combination of two defects resulted in a hemolytic disease, we hypothesized that the increase of the intra-erythrocytic 2,3-DPG level resulting from the PK-R deficiency might cause these disorders. It is well known that increase of the 2,3-DPG level dramatically change several properties of the RBCs such as a decrease in oxygen affinity and an inhibition of the G6PD activity (Tomoda A. Brit J Haematol, 1983, 54, 475 – 84). It has been shown that, when associated to a sickle cell trait, PK-R deficiency by increasing 2,3-DPG leads to sickle cell anemia (Cohen.Solal M. et al, Brit J Haematol, 1998, 103, 950-6). We propose that, in these two patients, the global mechanism leading to the disease results from the increased 2,3-DPG which cause a failure in the anti oxidant pathway. In the first case G6PD inhibition occurs along with a mutated enzyme and in the second one inhibition take place in a under supplied pentose phosphate pathway due to the hexokinase deficiency. Together with the diminished ATP supply of the cell, this decreased anti-oxidant activity might cause the hemolysis. Thus in any anemic heterozygous PK deficient patient, another RBC abnormality needs to be searched for.

enzymatic data patients Pk activity (5.9–8.1) g6pd activity (5.3–7.9) hexokinase activity (0.74–1.14) 2.3 DPG (11.7–15.3) nd : not determined 1 3.2 UI/g Hb 4.5 UI/g Hb nd 21.2 μM /g Hb 2 6.8 UI /g Hb 9.2 UI /g Hb 0.3 UI / g Hb 36.8 μM /g Hb
CD4+Treg in Immune Pathophysiology of Aplastic Anemia.
Authors: He Guangsheng , Zhou Ling , Wu De Pei , Sun Aining , Miao Miao
Published: October 2019

Objective: To explore the possible immune pathophysiology of CD4+T regulatory (CD4+Treg) in acquired aplastic anemia (AA).

Methods: According to the markers on membrane, cytokines, and effective mechanisms of CD4+Treg, the levels of CD4+CD25+Treg,CD4+CTLA-4+Treg,CD4+ICOS+Treg,CD4+PD-1+Treg,CD3+CD8−IL-10+Treg,CD3+CD8−TGF-β1+Treg,CD3+CD8−IL-Treg in the bone marrow of 23 cases with AA at active phase, 10cases with AA at recovery phase, 15 normal controls, were measured, and the relationship between CD4+Treg and priming immune factor-CD28 or effective immune factor-IFN-γ were also evaluated respectively.

Results: [circ1]In the group of AA at active phase, at recovery phase, and normal controls, CD4+CD25+Treg: (3.36±2.05)%, (3.49±1.71)%, (2.40±0.77)%, CD4+CTLA-4+ Treg: (1.43±0.67)%, (3.46±2.26)%, (2.45±1.30)%, CD4+ICOS+Treg: (2.04±1.79)%, (2.95±2.23)%, (2.37±1.68)%, CD4+PD-1+Treg: (1.40±1.32)%, (2.34±0.87)%, (2.07±1.64)%, CD3+CD8−IL-10+Treg: (2.14±1.19)%, (2.37±1.20)%, (2.04±1.25)%, CD3+CD8−TGF-β1+Treg: (1.83±0.99)%, (1.52±0.73)%, (2.15±1.17)%, CD3+CD8−IL-4+Treg: (2.46±1.65)%, (2.53±1.61)%, (1.99±1.27)%; costimulatory of CD3+CD4+CD28+ was: (39.84±10.89)%, (22.72±9.08)%, 31.40±10.83)%, the Th1cells CD3+CD8−IFN-γ+ were: (11.13±4.96)%, (5.39±4.29)%, (4.21±2.11)%. [circ2] Contrast to normal controls, while CD4+CTLA-4+Treg of AA at active phase deceased markedly (p=<0.01), levels of other CD4+Treg which marked by membrane moleculars or cytokines: CD4+CD25+Treg, CD4+PD-1+Treg, CD4+ICOS+Treg, CD3+CD8−IL-10+Treg, CD3+CD8−TGF-β1+Treg, CD3+CD8−IL-4+Treg did not change significantly (p>0.05); [circ3] Contrast to normal controls, the ratio of membrane costimulatory of CD28/ICOS CD28/CTLA-4, CD28/PD-1 were all increased significantly (p<0.05) in AA at active phase; the ratio of cytokines in cell plasma of IFN-γ+IL-4, IFN-γ/TGF-β, and IFN-γ/IL-10, were also increased significantly (p<0.05).

Conclusion: [circ1] In AA, the positive regulatory costimulatory increased while the negative regulatory costimulatory did not change or decreased, which shifted the immune balance to intensification. [circ2] Not only at priming stage but also at effective stage, the immune strengthened factors were higher than attenuated factors in AA.
The Role of Nitric Oxide in γ-Globin Induction by Hydroxyurea.
Authors: Lou Tzu-Fang , Williams Ashley , Li Wei , Pace Betty S.
Published: October 2019

Hydroxyurea (HU) has been shown to induce fetal hemoglobin (HbF) synthesis through activation of the soluble guanosine cyclase/cyclic guanosine monophosphate signaling pathway. The release of NO from HU by heme iron is thought to be involved in this mechanism of HbF induction. Studies completed in sickle cell patients confirmed increased serum NO levels after oral HU therapy but NO generation in red blood cells and the effect on γ-gene transcription have not been extensively investigated. Therefore, we performed studies to quantify NO generated by HU in K562 cells and normal erythroid progenitors as a mechanism for γ-globin activation. NO levels were measured after drug treatments using the Nitric Oxide Assay Kit (Calbiochem) and γ-globin mRNA was measured using quantitative PCR. HU (100μM) increased NO 1.4 to 1.8-fold at 24–72 hrs in K562 cells compared to a 2.0 to 2.5-fold increase in NO produced by the known NO donor, deta-nonoate (DE; 400μM). NO levels were also measured in erythroid progenitors grown in liquid cultures; a 1.6-fold increase in NO was produced by 30μM HU after 48 hrs with comparable increases produced by 200μM DE. To understand the effects of HU on normal NO synthesis from L-arginine through the action of NO synthase (NOS), we performed studies with the NOS inhibitor, NG-Monomethyl-L-arginine (L-NMMA). Interestingly, HU increased NO levels 2.5-fold at 24 hrs when combined L-NMMA compared a 1.4-fold increase produced by HU alone; this pattern persisted up to 72 hrs. Parallel with these findings γ-globin activation by HU was augmented approximately 25% by L-NMMA; DE combined with L-NMMA did not produce the same effect. These data suggest a novel mechanism for NOS regulation by HU compared to DE. Subsequent studies were completed to determine if HbF synthesis could be augmented by combining NO donors since they have different mechanisms of action. HbF levels in K562 cells were measured by ELISA (Bethyl Laboratories) and normalized by total hemoglobin and protein. Treatment with HU or DE increased HbF 3.6-fold and 4.6-fold respectively; when HU was combined with DE an additive 7.6-fold increase in HbF was produced. These data confirm that HU treatment lead to NO generation in K562 cells and normal erythroid progenitors which plays a role in its mechanism of γ-globin activation. HU combined with DE had an additive effect on HbF synthesis. These findings are relevant to current research efforts to develop novel HbF inducers for therapy in sickle cell patients.
A Randomized Controlled Trial of Patient Controlled Analgesia Versus Continuous Infusion of Morphine during Vaso-Occlusive Crisis in Sickle Cell Disease.
Authors: van Eduard J. Beers , van Charlotte F.J. Tuijn , Nieuwkerk Pythia T. , Friederich Philip W. , Levi Marcel M. , Franken Jan H. , Biemond Bart J.
Published: October 2019

Intravenous morphine is the treatment of choice for severe pain during vaso-occlusive crisis in sickle cell disease (SCD). However, side-effects of morphine may hamper effective treatment and high plasma levels of morphine are associated with severe complications such as acute chest syndrome. Furthermore, adequate dosing remains a problem since no objective measurement of pain severity exists and analgesia should be titrated upon the patient’s reported pain. Patient-controlled analgesia (PCA) may therefore be an interesting alternative since patients can titrate the level of analgesia themselves. Although PCA is currently used for the treatment of vaso-occlusive crises in SCD, no randomized controlled trials have been performed in admitted patients with a vaso-occlusive crisis so far. In the present study, we conducted a randomized controlled trial to compare the administration of morphine with PCA versus CI in sickle cell patients with vaso-occlusive crises. Patients were randomized between PCA and CI of morphine within 24 hours after hospital admission. Endpoints of the study were: the mean and cumulative morphine dose, pain intensity, incidence of side-effects (nausea, constipation, pruritus and drowsiness) and quality of life (QoL). Pain intensity was measured daily using a ten-point-scale verbal pain score. Reduction of pain intensity was measured by subtracting a pain score on a ten point visual analogue scale (VAS) before randomization from the same measurement two days after randomization. Side-effects were measured four times per day on a 11-point numerical rating scale. QoL was measured using the 36-item Short Form Healthy Survey (SF36). Twenty five consecutive episodes of vaso-occlusive crisis in 19 patients with SCD were included in the study. No difference in the mean daily pain scores were found between the two groups (4.9 versus 5.3). However, patients in the PCA-group demonstrated to have a strongly reduced mean and cumulative morphine consumption as compared to the patients in the CI-group (0.5 mg/h versus 2.4 mg/h (P<0.001) and 33 mg versus 260 mg (P=0.018) respectively). The lower mean and cumulative morphine consumption in the PCA-group led to significant less nausea and constipation during treatment as compared to the CI-group (area under the curve respectively 11 versus 18 (P= 0.045) and 30 versus 45 (P= 0.021)). Furthermore, a non-significant reduction in the duration of hospital admission of 3 days was observed in the PCA-group. Patient controlled analgesia results in adequate pain relief at a much lower morphine consumption and should considered to be first choice in morphine administration to sickle cell patients admitted with vaso-occlusive crisis.

Figure. Mean morphine dose per patient Figure. Mean morphine dose per patient
Time Series Analysis of the Pain Diary Data Obtained during the Multicenter Study for Hydroxyurea (MSH) Clinical Trial.
Authors: McCarthy William F. , Bauserman Robert L. , Barton Bruce A. , Guo Nan , Ballas Samir K. , Smith Wally
Published: October 2019

The Multicenter Study of Hydroxyurea in Sickle Cell Anemia (MSH) was a randomized double-blind placebo-controlled trial to test whether hydroxyurea could reduce the rate of painful crises in adult patients who had at least three painful crises per year. Daily pain diaries were collected biweekly for 299 MSH patients. Patients rated daily pain on a linear scale 0 through 9. The daily pain data was collected to provide useful information regarding the efficacy of hydroxyurea in reducing the rate of painful crises. The objective of this analysis was to determine if there was an underlying time series pattern associated with the MSH pain diary data, especially one indicative of a calendar or seasonal association. An Unobserved Component Model (UCM) was used to determine, based on empirical data, if there was a cycle and/or trend associated with the time series. The times series started on December 1991 and ended on December 1994. The series was equally spaced and univariate. The equal spacing was a monthly interval. The average pain score for each two- week diary period was used to create each unit of analysis. This analysis considered three groupings of patients: all patients, hydroxyurea patients and placebo patients. The results of the modeling indicated that there was indeed both a cycle and a trend associated with the MSH pain diary data. This is the case for each group of patients considered. All three groups had a cycle pattern that seemed to show a seasonal behavior of average pain scores (the changing of the season seemed to show an approximate transition from trough (Spring) to peak (Fall/Winter) and peak to trough). There was a downward trend in average pain scores for both the all patientsgroup and the hydroxyurea patientsgroup. However, there was an upward trend in average pain score for the placebo patientsgroup. This may reflect the result shown in the MSH Clinical Trial, that HU usage reduced the average pain scores reported by patients.
AHSP Promoter Homopolymer in Beta-Thalassemia Syndromes.
Authors: Galanello Renzo , Satta Stefania , Cipollina Maria Dolores , Perra Chiara , Perseu Luciana
Published: October 2019

Alpha hemoglobin stabilizing protein (AHSP) is an erythroid protein that binds specifically to the alpha globin chains to form a stable complex, preventing their precipitation before the assembly of the hemoglobin tetramere. Studies have shown that

AHSP is required for normal erythropoiesis, knock-out mice has a phenotype similar to beta thalassemia, loss of AHSP worsens the severity of the disease in thalassemic mice.

Therefore, altered AHSP expression or function could account for some of the variability in beta-thalassemia phenotype. At position -160bp upstream of exon 1 of the AHSP gene, two main homopolymers (T18 and T15) have been reported that may influence the AHSP expression and hence the hematological phenotype. In particular the frequence of the shorter T15 homopolymer, which is associated with a lower AHSP expression, was much higher than expected (on the basis of the allele frequency in the healthy population) in a group of 9 patients with thalassemia intermedia, due the compound heterozygosity for beta-thalassemia and the triple alpha globin gene (Mei et al, 2006). We have studied the frequency of T15 and T18 homolpolymers in a group of 20 normal subjects and in patients with different thalassemia syndromes, i.e. beta zero 39 homozygotes with thalassemia major (14 patients) and intermedia (19), compound heterozygotes for beta-thalassemia and triple alpha globin gene (13) and simple carriers for beta thalassemia with thalassemia intermedia phenotype (5). We found the following genotype distribution reported in figure1. There is no difference in the frequency of the genotypes among the groups, except in the severe beta-thalassemia carriers, where the short T15 homopolymer is more common. These findings are consistent with the reduced AHSP expression previously reported in the reticulocytes and in the erythroid cultures of these unusually severe beta thalassemia carriers. (Galanello et al, abs 1881. ASH 2003). Therefore, only in the peculiar group of beta-thalassemia carriers with the clinical features of thalassemia intermedia, the AHSP genotype appears to be a relevant contributory factor in the hematological phenotype.

Figure Figure
The Involvement of Regulatory T Cells in Immunopathophysiology of Autoimmune Neutropenia in Infancy.
Authors: Nakamura Kazuhiro , Miki Mizuka , Kawaguchi Hiroshi , Sato Takashi , Kurita Emi , Onodera Rie , Hiraoka Asako , Kobayashi Masao
Published: October 2019

CD4+25+ regulatory T cells (Treg) have been recognized to play a significant role in the immunopathogenesis of autoimmune disease, tumors, and organ transplantation. Several human autoimmune diseases in adulthood have been reported to associate with alternations in Treg. To date little is known about the standard value of Treg in infancy and childhood and its involvement in pediatric autoimmune disease. Autoimmune neutropenia (AIN) in infancy is a common disorder characterized by chronic neutropenia, the detection of antineutrophil antibodies in sera, and spontaneous resolution of neutropenia within several months to a few years. In this study, to elucidate the involvement of Treg in AIN known as a representative autoimmune disease in infancy and early childhood, we examined the frequencies of Treg in various normal age groups and AIN patient. Blood samples from normal neonates (n=22), infants (1 month through 3 years in age, n=21), adults (n=12) and AIN patients (1 month through 3 year in age, n=22) were obtained after informed consent. The frequency of Treg was assessed by the expression of CD4, CD25, and FOXP3 using flow cytometry. As shown in Table, the frequency of CD4+25high Treg known as pure Treg fraction was highest in neonates and decreased by aging. Notably CD4+25high Treg in AIN patients was fewer than age-matched control infants (P<0.01). Furthermore, the ratio of intracytosolic FOXP3 expression of each Treg fraction in AIN patients was fewer than age-matched control infants (P<0.05). The spontaneous disappearance of antineutrophil antibody and the restoration of the frequency of CD4+25high cells that preceded the normalization of the neutrophil count were found in two patients with AIN. These results suggest that Treg could contribute to the immunopathophysiology of AIN in infancy.

Percentage of CD25+, CD25high, and CD25low in CD4+ cells Neonates Infants Adults Patients with AIN Date represent mean±SD. CD25+ 11.9±2.6 10.1±2.0 18.5±3.9 9.5±1.9 CD25high 8.3±2.7 5.6±1.9 4.2±1.1 4.0±1.6 CD25low 3.6±1.2 4.5±1.9 14.3±3.6 5.5±1.5
The Outcome of 129 Korean Children with Hemophagocytic Lymphohistiocytosis.
Authors: Youn Hoi Soo , Song Joon Sup , Im Ho Joon , Seo Jong Jin , Moon Hyung Nam , Park Byoung Gyu , Kim Thad T. , Kang Hyoung Jin , Shin Hee Young , Ahn Hyo Seop , Yoo Keon Hee , Sung Ki Woong , Koo Hong Hoe , Jeong Dae Chul , Cho Bin , Kim Hack Ki , Lee Mee Jeong , Park Eun Sil , Kim Hwang Min , Lim Young Tak , Kim Heung Sik , Lyu Chuhl Joo , Kim Sun Young , Park Jun Eun , Lee Young Ho , You Chur Woo , Kim Ji Yoon , Kang Im Ju , Lee Kun Soo , Kook Hoon , Hwang Tai Ju
Published: October 2019

Chemotherapy and immunotherapy based treatments improved survival of patients with hemophagocytic lymphohistiocytosis(HLH), but the outcome is still unsatisfactory. We analyzed the putative prognostic factors in a nationwide cohort of patients with HLH. Retrospective data recruitment for the patients diagnosed as HLH during the past 10-year period from 1996 to 2005 was carried out by the Histiocytosis Working Party of the Korean Society of Hematology. The HLH diagnostic criteria of the Histiocyte Society were strictly applied to confirm the eligibility of patients for this study. We analyzed the outcome of pediatric patients with HLH according to the age at diagnosis, sex, central nervous system(CNS) involvement, disease condition(familial or secondary), treatment modalities and disease state after 2 months of initial treatment. One hundred twenty nine patients from 19 centers fulfilled the diagnostic criteria(n=112) and/or had affected siblings together with some of the criteria(n=17). The male to female ratio was 0.95:1. The probability of 3 year overall survival(OS) in HLH patients was 41% with a median follow-up of 51 months. The 3 year OS in patients under 12 months of age at presentation(n=23) was 21.7%, and 44.3% in those over 12 months of age(n=106)(p=0.001). The 3 year OS in patients with CNS involvement(n=16) was 29.1%, and 44.4% in patients without CNS involvement(n=112)(p=0.01). The 3 year OS in patients with active state after 2 months of initial treatment(n=63) was 14.1% compared to 77.2% in those with inactive state(n=61)(p=0.0001). The 3 year OS in patients who received hematopoietic stem cell transplantation(HSCT)(n=17) was 82.3%, and 35.2% in patients treated with chemoimmunotherapy only(n=112)(p=0.03). Among the HSCT patients, complete remission was obtained in 14 patients except 3 other patients who died of infection and graft failure at early post-transplant period. The reasons for HSCT were active disease after chemoimmunotherapy(n=8), relapsed disease(n=5), and familial HLH(n=4). Other prognostic factors were not significantly correlated with outcome in our survey. The age and CNS involvement at diagnosis, disease state after 2 months of initial treatment were important prognostic factors which affected the outcome of HLH significantly in this cohort. This survey also demonstrated excellent outcome of familial or relapsed, persistent secondary HLH after HSCT compared to chemoimmunotherapy only.
Prion Clearance Capacity Is Improved by Modification of a Filtration Step in the Gamunex® (IGIV) Manufacturing Process.
Authors: Mink Michael , Hartwell Randal , Bauman Patricia , Burdick Michael , Kislan Michele , Nelson Mark , Oquendo Renea’ , Pifat Dominique , Cai Kang
Published: October 2019

Prions are misfolded pathogenic proteins associated with the transmission of transmissible spongiform encephalopathies (TSEs). Although no TSE transmission through human plasma products has been documented to date, the risk of transmission exists. To provide assurance for product safety with respect to the risk of TSE transmission, this study evaluates the prion clearance potential of a modified intravenous immunoglobulin (Gamunex®) manufacturing process (Figure 1).

Figure 1. Gamunex® Manufacturing Process Figure 1. Gamunex® Manufacturing Process

In the process prior to modification, an intermediate material was subjected to cloth filtration. In the modified process, the cloth filtration is replaced with a depth filtration to improve pathogen removal capacity. Both processes were studied using validated scaled-down models, along with a spiked prion agent. The modified process reduced the prion level to below the limit of detection by Western blot assay. The animal bioassay for infectivity confirmed that the modified process has a marked improvement in prion clearance. This modified process has been implemented in actual Gamunex® manufacturing process. Gamunex® is the only liquid IGIV with FDA approved prion clearance data.
Prognostic Factors for HIV-Associated Lymphoma - Experience in a Tertiary Referral Center in Hong Kong.
Authors: Lau SzeMan June , Lee ManPo , Chan CheukHung , Li ChungKi Patrick
Published: October 2019

Objective: To review the clinical and pathological profile of HIV-associated lymphomas in the local population in order to identify prognostic factors that may define a subgroup of patients who may have better outcome.

Methods: The clinical records of a cohort of 874 cases of HIV infection first seen in our institute from March 1994 to March 2006 were reviewed.

Results: Twenty patients with HIV-associated lymphomas were identified. All were males. Median age was 42 (18 – 72) years. Three had primary central nervous system lymphoma (PCNSL) and 17 had systemic non-Hodgkin’s lymphoma (NHL). All PCNSLs were of B-cell origin. For systemic NHLs, 15 patients had aggressive B-cell lymphoma (diffuse large B cell lymphoma: 12, burkitt-like lymphoma: 2, mantle cell lymphoma: 1) and 2 patients had plasmablastic lymphoma. For the 3 patients with PCNSL, 1 patient deteriorated rapidly and died 12 days after diagnosis, 1 patient survived 20 months after whole-brain radiation therapy (WBRT) and died of CMV pneumonia later and 1 patient is still surviving at 88 months (mons) after WBRT while maintaining on highly active anti-retroviral therapy (HAART). For patients with systemic NHL (n = 17), 2 patients had spontaneous disease regression upon treatment with HAART, 6 received curative intent chemotherapy, 3 received palliative treatment with chemotherapy or radiotherapy, 6 patients could not receive any treatment because of poor performance status and rapid deterioration after diagnosis. The following baseline variables were included as potential predictors of survival among all patients with systemic NHL: prior AIDS diagnosis; CD4 cell count; use of HAART and treatment intent. Number of adverse factors according to International Prognostic Index (IPI) for aggressive NHL (stage III or IV disease, age > 60 years, elevated lactate dehydrogenase, ECOG performance status ≥ 2, ≥ 2 extranodal sites) was calculated. Use of HAART was a favorable prognostic factor for overall survival (p = 0.008). Median overall survival for patients receiving HAART (n = 11) was 15 mons versus 1.6 mons for those without (n = 6). Patients with ≤ 2 adverse factors according to IPI were associated with longer overall survival (p = 0.0002). Patients with IPI > 2 (n = 11) had median survival of 2 mons whereas median survival for patients with IPI ≤ 2 (n = 6) had not been reached (range of follow up duration: 7 to 120 mons).

Conclusion: In our cohort, for patients with HIV-associated systemic NHL, use of HAART and IPI score ≤ 2 were associated with better survival.

Figure Figure Figure Figure
Hedgehog Pathway: A New Target for B-Cell Lymphomas an Multiple Myeloma.
Authors: Dierks Christine , Grbic Jovana , Zirlik Katja , Mertelsman Roland H. , Warmuth Markus
Published: October 2019

The hedgehog pathway (Hh) is one of several key developmental pathways whose deregulation is known to induce tumor formations in mice and humans including medulloblastoma or basal cell carcinoma. Certain human solid tumors (prostate cancer, pancreatic cancer) have been identified which require sustained Hh-Gli signaling for proliferation.

Here, we show for the first time a crucial role of Hedgehog signalling in hematopoietic malignancies. In our experiments, we demonstrate that the Hh pathway is essential for survival of more than 70% of primary lymphomas extracted from transgenic Eμ-Myc mice or Cdkn2a−/− mice (INK4Arf−/−). 34 Myc-lymphomas (10 B-cell lymphomas, 12 plasmablastomas, 10 plasmocytomas, 2 mixed lymphomas) and 10 Cdkn2a−/− lymphomas were isolated from bone marrow, spleen or lymph nodes from diseased mice and expanded on bone marrow stroma cells from Cdkn2a−/− mice. Inhibition of Hh signaling by Cyclopamine (2–5 μM), an alkaloid which inhibits smoothened activation, induced apoptosis in 80% of B-cell lymphomas, 82% of plasmablastomas and 60% of multiple myelomas without affecting stroma proliferation. Protein or transcript levels of Hh pathway downstream targets, such as Gli1, Ptch or Bcl2 but not BMI1 were downregulated after 24h treatment with Cyclopamine. Overexpression of Bcl2 or Gli3 could inhibit apoptosis induction by Cyclopamine.

High levels of Ihh protein could be detected in our stroma cell line as well as in bone marrow stroma from various mouse strains. Stimulation of lymphoma cells with recombinant Shh or Ihh protein could overcome apoptosis induction induced by removal of the stromal layer, indicating Ihh as a mediator between stroma and lymphoma cells.

Injection of luciferazed lymphoma cells into C57Bl6 mice induced lymphoma formation within 2–6 weeks, as shown by Xenogen luciferase imaging. Subcutaneous treatment of lymphoma injected mice with Cyclopamine could inhibit lymphoma formation and even reduce lymphoma mass in mice with already fully developed lymphomas, indicating stroma-lymphoma interaction through hedgehog signaling as an important survival mechanism for lymphoma cells also in vivo.

Our data demonstrates that proliferation and survival of a majority of B-cell lymphomas and plasmacytomas is dependent on intact Hh signaling in vitro and in vivo, suggesting disruption of this pathway as a novel approach in lymphoma therapy.
Dendritic Cell-Induced T Cell Non-Responsiveness Is Mediated by FOXP3+ and TGF-beta+IL-10+ CD4+ T Cells.
Authors: Berneman Zwi N. , Cools Nathalie , Van Tendeloo Viggo F.I. , Lenjou Marc , Nijs Griet , Van Bockstaele Dirk R. , Ponsaerts Peter
Published: October 2019

Dendritic cells (DC), the professional antigen presenting cells of the immune system, exert important functions both in induction of T cell immunity as well as of tolerance. Previously, it was accepted that the main function of immature DC (iDC) in their in vivo steady state condition is to maintain peripheral tolerance to self-antigens and that these iDC mature upon encounter of so-called danger signals and subsequently promote T cell immunity. However, a growing body of experimental evidence now indicates that traditional DC maturation can no longer be used to distinguish between tolerogenic and immunogenic properties of DC. In this study, we compared the in vitro stimulatory capacity of immature DC (iDC), cytokine cocktail-matured DC (CC-mDC) and poly I:C-matured DC (pIC-mDC) in the absence and presence of antigen. All investigated DC types could induce at least 2 subsets of regulatory T cells. We observed a significant increase in both the number of functionally suppressive transforming growth factor (TGF)-beta+ interleukin (IL)-10+ T cells as well as of CD4+CD25+FOXP3+ T cells within DC/T cell co-cultures as compared to T cell cultures without DC. The induction of these regulatory T cells correlates with in vitro T cell non-responsiveness after co-culture with iDC and CC-mDC, while stimulation with pIC-mDC resulted in reproducible cytomegalovirus pp65 or influenza M1 matrix peptide-specific T cell activation as compared to control cultures in the absence of DC. In addition, the T cell non-responsiveness after stimulation with iDC was shown to be mediated by TGF-beta and IL-10. Moreover, the suppressive capacity of CD4+ T cells activated by iDC and CC-mDC was shown to be transferable when these CD4+ T cells were added to an established T cell response. In contrast, addition of CD4+ T cells stimulated by pIC-mDC made responder T cells refractory to their suppressive activity. In conclusion, we hypothesize that DC have a complementary role in inducing both regulatory T cells and effector T cells, where the final result of antigen-specific T cell activation will depend on the activation state of the DC. This emphasizes the need for proper DC activation when T cell immunity is the desired effect, especially when used in clinical trials.
Identification of Human Cd8+ and Cd4+ Lymphocytes with Unique Drug Resistance Activity by Imaging the Drug Transport Kinetics at the Single Cell Resolution.
Authors: Shirihai Orian , Biran Israel , Bertelsen Kirk M. , Greenblatt David J. , Deutsch Motti , Zurgil Naomi
Published: October 2019

Drug resistance activity of transporters such as P-glycoprotein within lymphocytes may have significant consequences to the pharmacologic treatment of HIV, immune suppression, autoimmune diseases, and leukemia, as many drugs are substrates of the MDR transporters. Variability in MDR activity may be a significant barrier in the evaluation of the predicted effect of a drug and on the therapeutic dosage necessary to overcome MDR activity. Current methodologies to measure MDR, e.g. FACS, do not provide kinetic measurements of dye efflux at the resolution of the individual cell thereby missing the existence of functionally different subpopulations. To overcome these limitations, we developed a new imaging device that is comprised of thousands of transparent pico-wells (20 micron in diameter) organized in a honeycomb structure that enables real-time visualization and sequential quantification of drug transport rate from thousands of individual living lymphocytes, thereby generating MDR kinetic data. Fluorescence microscopy was used to measure DiOC2 dye (a common fluorescent probe for P-glycoprotein transport) retention from individual human CD4+ or CD8+ lymphocytes over time. Kinetic analysis of dye export from CD4+ and CD8+ cells revealed the occurrence of subpopulations of cells with unique sensitivity to MDR inhibitors. When the MDR inhibitor Verapamil was used, CD8+ lymphocytes demonstrated a greater magnitude of inhibition compared to CD4+ lymphocytes. Remarkably, CD4+ lymphocytes contained a sizable subpopulation which was verapamil insensitive, suggesting the existence of a non-P-glycoprotein drug export activity in these cells. These findings indicate that functional MDR assays, which are based on transport kinetics, can efficiently dissect the heterogeneous lymphocyte cell populations and identify unique subsets of cells with increased activity.

Figure Figure
Cyclosporine Toxicity and Vascular Endothelial Growth Factor (VEGF).
Authors: Kubisz Peter , Grandtnerova Barbara , Laca Ludovit , Stasko Jan
Published: October 2019

Vascular endothelial growth factor (VEGF) is a potent angiogenic and endothelial cell-specific mitogen. Expression of VEGF mRNA is increased under hypoxia/ischemia conditions. Its role in the setting of clinical kidney transplantation (Tx) is currently under investigation. Majority of works are dealing with gene polymorphisms and/or renal tissue expression. The aim of the study was to evaluate serum VEGF concentrations after kidney Tx and its relationship to graft outcome. Thirty-five adult patients (17 male, 18 female) treated with cyclosporine A or tacrolimus, at least 1 month after Tx and 22 healthy control (17 male, 5 female). Patients with acute rejection or infection were excluded. Serum VEGF (S-VEGF) concentrations were measured by Quantikine Immunoassay, RD Systems. S-VEGF concentrations were significantly higher after Tx than in healthy control (556 ± 463 pg/ml, vs 145 ± 74 pg/ml, p< 0.0001). No correlation was found with age, gender, time after Tx or type of calcineurin inhibitor. However, a significant correlation was found between basaline S-VEGF and S-creatinine (p< 0.05), S-VEGF and S-creatinine six months after Tx (p< 0.01) and between S-VEGF and cyclosporine toxicity defined as a gingival hyperplasia or biopsy proven nephrotoxicity (202 ± 121 pg/ml in stable patients vs 741 ± 436 pg/ml in cyclosporine toxicity group, p< 0.001). During six months of follow up, the kidney graft function was stable in 100% of patients with S-VEGF concentration ≤ 145 pg/ml but only in 50% of patients with S-VEGF concentration > 145 pg/ml (p < 0,05). We conclude that the S-VEGF concentration seems to be a marker of cyclosporine toxicity and prospective graft function deterioration. S-VEGF could be helpful to select patients who would have benefit from an early switch of immunosuppression.
The MYST Domain Histone Acetyltransferase TIP60 Interacts with and Is as a Coactivator of the Myeloid Transcription Factor C/EBPα.
Authors: Bararia Deepak , Trivedi Arun K. , Zada Abdul Peer , Behre Hermann M. , Behre Gerhard , Bohlander Stefan K.
Published: October 2019

Recently, we and others have shown that protein-protein interactions play an important role in the pathogenesis of leukemia, and that the transcription factor C/EBPα is a key player in granulopoiesis and leukemogenesis. In the present study, we sought to identify C/EBPα interacting proteins. A glutathione-S-transferase-C/EBPα fusion protein was used to pull down interacting proteins from U937 nuclear extracts. These proteins were analyzed by 2-D gel electrophoresis, 1-D nano LC and identified by MALDI or MALDI-TOF/TOF. Several novel C/EBPα interactors were identified including known proteins like Rb, hnRNP and E2F4. Two novel interactions, one of cell cycle regulator protein MCM5 and the other with the MYST domain histone acetyl transferase TIP60 with C/EBPα were further confirmed by using pull-down and co-immunoprecipitation experiments. TP60 was able to markedly enhance C/EBPα mediated transcription in reporter gene assays, suggesting that TIP60 is a co-activator of C/EBPα. This co-activator function of TIP60 was dependent on an intact histone acetyl transferase domain and on the C/EBPα DNA binding domain. TIP60 was found to be associated with the human C/EBPα promoter in-vivo in a chromatin immunoprecipitation assay with a concomitant increase in histone H3 and H4 acetylation. Furthermore, we observed a lower expression of TIP60 mRNA in U937 CD11b− compared to retinoic acid induced U937 CD11b+ cells suggesting that higher TIP60 expression is associated with myeloid differentiation. There was also a marked correlation between the expression levels of TIP60 and C/EBPa in normal bone marrow, chronic myeloid leukemia and acute myeloid leukemia samples with t(8;21), inv(16) and t(15;17). This finding further confirms the functional synergism between C/EBPα and TIP60 and suggests that TIP60 might be an important player in leukemogenesis.
Inhibition of the TGFβ Pathway in Primary Effusion Lymphoma.
Authors: Di Bartolo Daniel , Cesarman Ethel
Published: October 2019

Kaposi’s sarcoma-associated herpesvirus (KSHV) interferes with a number of cellular pathways involved in apoptosis and cell cycle regulation. Since TGFβ is a potent inducer of cell cycle arrest and apoptosis in hematopoietic lineages, we were interested in examining whether TGFβ signaling is blocked in primary effusion lymphoma (PEL) and if so whether KSHV plays a role in this repression. Treatment with TGFβ1 had no effect on either cell cycle or apoptosis in several PEL cell lines. The defect in this pathway was found to be a lack of the TGFβ type II receptor (TβRII), expression. Upon transfection of TβRII, PEL cell lines become responsive to TGFβ1 treatment. We are currently examining how TβRII transcription is regulated in PEL. The lack of TβRII expression in some tumors has been attributed to altered chromatin structure due to hypoacetylation. Treatment with both a demethylating agent and a histone deacetylase inhibitor resulted in expression of this receptor in PEL. Several KSHV latent gene products are known to interact with or recruit p300, a histone acetyltransferase, for other cellular processes. It is possible that the interaction of one or more of these viral gene products with p300 may serve to prevent it from acetylating histones associated with the TβRII gene and thereby repress its transcription. We have also found by ELISA that PEL cells secrete a higher level of TGFβ1 than other TGF β responsive B cell lines. Increased secretion of TGFβ1 in conjunction with downregulation of TβRII may be a mechanism by which KSHV creates an immunosuppressed environment while allowing infected cells to grow free from the constraints of this pathway.
MKC-1 Is a Novel Agent That Induces Cell Cycle Arrest and Disrupts Multiple Survival Pathways in Hematologic Cancers.
Authors: Denny Trisha A. , Chen Xiaoru , Waters Cassandra L. , Burke Patricia A. , Fletcher Graham C. , Xu John , Sidor Carolyn F. , Minden Mark D. , LaVallee Theresa M. , Bray Mark R.
Published: October 2019

MKC-1 is a novel, orally active cell cycle inhibitor with in vitro and in vivo activity against a wide range of human solid tumor cell lines, including multi-drug resistant cell lines. MKC-1 has been tested in over 270 patients to date and is currently in Phase II clinical trials. The strong pre-clinical activity of MKC-1 towards solid tumor lines and signs of efficacy in the initial clinical evaluation with lack of neuropathy and cardiotoxicity suggests that MKC-1 may also be of clinical benefit in the treatment of hematopoietic cancers. The antiproliferative activity of MKC-1 was examined against a panel of hematopoietic cell lines including HL-60, U937, MV4;11, THP-1, Jurkat, and OCI-AML 1–5. MKC-1 showed potent and dose-dependent activity towards these cell lines, with IC50 values in the range of 20 – 400 nM. MKC-1 also inhibited in vitro growth of primary cells derived from AML and CML patients. Additionally, MKC-1 showed enhanced activity with Ara-C in combination studies in vitro when added either simultaneously or sequentially using the cell line OCI-AML 4. Binding studies have shown that MKC-1 binds to the colchicine binding site of tubulin and to members of the importin beta family of proteins. Consistent with these results, cell cycle arrest in the G2/M phase of the cell cycle followed by apoptosis was observed in cell lines and patient samples treated with MKC-1. Immunofluorescence analysis of cells treated with MKC-1 revealed that the drug induced a disruption of the microtubule network and the formation of aberrant mitotic spindles. Furthermore, MKC-1 was also shown to induce a dose-dependent reduction in the levels of both phospho-Akt and phospho-p70S6K kinases through Western blot analysis of treated THP-1 cells. In conclusion, our results indicate MKC-1 arrests the cell cycle and disrupts multiple survival pathways to induce apoptosis in hematopoietic cell lines and patient samples. These results suggest that MKC-1 may have clinical potential in the treatment of leukemia either alone or in combination with other agents. Phase I trials in hematological cancers are currently being explored.
Re-Evaluation of Prognostic Value of Seric LDH and β2-Microglobulin Levels among 248 CLL with a Median Follow-Up Time More Than 6 Years.
Authors: Desablens Bernard , Garidi Réda , Gruson Bérengère , Vaida Iona , Brevet Marie
Published: October 2019

Except in MD Anderson Center, seric β2-m is not recognized as a strong prognostic factor in CLL and seric LDH too, despite this factor is more and more important among lymphoma with a continuous prognostic value. So we studied 248 CLL aged less than 75 yrs, without elevated creatininemia or evident hemolysis. There were 201 pts with stage A, 29 B and 18 C and sex-ratio M/F was 1.48. Median survival time is 10 yrs and median “corrected” (i.e., without not CLL-related deaths including those due to a solid tumor because this complication is independent of initial prognostic factors of CLL) survival time is 10.9 yrs. LDH values range from 0.55 to 3.54 N, and mean and median values are 1.05±0.40 and 0.96 N. LDH level is only correlated with blood lymphocytosis and hemoglobin levels studied as continuous values (p=0.015 and 0.002). When looking at “corrected” survival, all cut-off values from 0.75 N, N, 1.25 N… to 2.5 N are statistically significant but the best one is 1.25 N (p=6 10−6). β2-m values range from 0.81 to 16.5 mg per l, and mean and median values are 2.67±1.75 and 2.15 mg. When studied as continuous values, β2-m is linked to age, number of lymphoid areas according to Binet’s system, and hemoglobin and platelets levels (p=0.0009, <10−4, <10−4 and 0.003) but age was ruled out among patients less than 70 yrs. For “corrected” survival, all cut-off values except 1.25 mg were significant and for example p value at 4 mg level as usually used by MD Anderson Center, is 5 10−5. However we find that the best cut-off value is 3 mg (p<10−6) and this value is statistically independent from LDH one in Cox model. We tried to combine these 2 cut-off values. Product of observed LDH/1.25 N × observed β2-m/3 mg were pertinent to isolate pts with a very poor prognosis (median “corrected” survival time about 6 yrs) but size of pejorative subgroups were small (≈25% of all pts) and other subgroups were not statistically significant. When adding ratios, results were similar. So we find that the best system is to count the number of pejoratives factors, i.e., LDH > 1.25 N and β2-m > 3 mg. Global median survival times of the 3 defined groups were respectively > 10.8 yrs (160 pts), 6.5 yrs (68 pts) and 4.2 yrs (20 pts) with a p value < 10−6. This system is also efficient among stage A pts (p=10−5). When we compare this prognostic system with Binet’s and Rai’s prognostic systems, Cox model rules out Binet’s one and keeps Rai’s system and ours (p=0.01 and 0.0005). Among patients with stage A, Cox model keeps only this LDH and β2-m system. We conclude on the strong prognostic values of seric LDH and β2-m initial levels in CLL and we claim that these 2 simple biological parameters have to been compared with “modern” biological prognostic factors of CLL.