bluuebunny/crossref_metadata_2025 huggingface dataset:
ZAP-70 Expressed in CLL Is an Hsp90 Client Protein and Its Level of Expression Can Be Downregulated by Specific Hsp90 Inhibitors.
Authors: Prada Carlos E. , Loria Olivier , Kamal Adeela , Burrows Francis J. , Kipps Thomas J. , Castro Januario E.
Published: October 2019

Hsp90 is a ubiquitous molecular chaperone protein that is involved in the folding, activation and assembly of many client proteins, including mediators of signal transduction, cell-cycle control and transcriptional regulation. In order to exert its function on client proteins, Hsp90 requires the formation of an active protein complex composed of cochaperone molecules and an active ATP binding site. Proteins identified as Hsp90 client proteins include transmembrane tyrosine kinases [HER-2/neu, epidermal growth factor receptor (EGFR), MET and insulin-like growth factor-1 receptor (IGF-1R)], metastable signaling proteins (Akt, Raf-1 and IKK), mutated signaling proteins (p53, Kit, Flt3 and v-src), chimeric signaling proteins (NPM-ALK, Bcr-Abl), steroid receptors (androgen, estrogen and progesterone receptors), cell-cycle regulators (cdk4, cdk6) and apoptosis related proteins. It has been postulated that malignant progression and cancer prognosis may be associated with the presence of activated Hsp90 which exists in heightened complexes with cochaperone proteins.

The zeta-associated protein of 70 kD (ZAP-70), is a protein kinase found in a subset of patients with chronic lymphocytic leukemia (CLL) and its expression correlates with disease progression, poor clinical outcome, decreased overall survival and early requirement for treatment. ZAP-70 promotes phosphorylation of downstream signaling molecules after engagement of the B cell receptor (BCR). Since ZAP-70 is a marker of disease progression in CLL we hypothesized that its abnormal over-expression and function in CLL B cells may depend on activated Hsp90. To test this, we performed immunoprecipitation, western blot and flow cytometry analyses on CLL samples from patients that differ in their level of ZAP-70 expression (ZAP-70 positive and negative based on a cutoff expression of 20% by flow cytometry). We observed that in ZAP-70 positive CLL samples (n=4) the majority of Hsp90 present in the cytoplasm was in a complexed-activated form, whereas in ZAP-70 negative samples (n=4) most Hsp90 was found to be non-complexed. In addition, we found that ZAP-70 co-immunoprecipitated with Hsp90, suggesting that it is an Hsp90 client protein. In addition, the level of ZAP-70 expression was decreased 30–40% in cells treated for 24 hours with Hsp90 specific inhibitors at nanomolar concentrations.

Together, our data show that CLL B cells that express ZAP-70 have larger amounts of activated Hsp90 than those with negligible expression of this tyrosine kinase protein. In addition, we found that ZAP-70 is an Hsp90 client protein and its level of expression could be downregulated by using specific Hsp90 inhibitors. These findings suggest an important role for Hsp90 as a prognostic marker in CLL and provide the rationale to investigate, in clinical trials, the activity of Hsp90 inhibitors and their effect on ZAP-70 expression.
Rituximab-Mediated Inhibition of the Constitutive NIK/IKK/IκB/NF-κB Signaling Pathway in Non-Hodgkin’s Lymphoma (NHL) B-Cell Lines: Role in Chemo-Sensitization.
Authors: Jazirehi Ali R. , Huerta-Yepez Sara , Cheng Genhong , Bonavida Benjamin
Published: October 2019

The chimeric anti-CD20 antibody rituximab (Rituxan, IDEC-C2B8) is widely used in the clinical treatment of patients with non-Hodgkin’s lymphoma (NHL). Rituximab sensitizes NHL B-cell lines to drug-induced apoptosis via selective down-regulation of Bcl-xL expression (Jazirehi et al., Mol. Cancer Therapeutics 2:1183, 2003). We hypothesized that rituximab-mediated down-regulation of Bcl-xL expression may be due, in part, to inhibition of constitutive NF- κB activity that regulates Bcl- xL expression. This hypothesis was tested following treatment with rituximab of CD20+ drug-resistant Ramos (Bcl-2−/ Bcl-xL+) and Daudi (Bcl-2+/ Bcl-xL+) cell lines. Rituximab decreased the phosphorylation of NIK, IKK and κB-Iα, diminished IKK kinase activity and decreased NF- κB DNA-binding activity. These events and down-regulation of Bcl-xL expression occurred with similar kinetics and were observed 3–6 h post rituximab treatment. The role of NF- κB in the regulation of Bcl-xL transcription in both Ramos and Daudi cells was demonstrated by using 1) promoter reporter assays in which deletion of the two tandem NF- κB binding sites in the upstream promoter region significantly reduced the luciferase activity 2) IκB super-repressor expressing cells and 3) by NF- κB specific inhibitors. The underlying mechanism of the inhibition of the NF-κB signaling pathway by rituximab was shown to be due, in part, to upregulation of Raf-1 kinase inhibitor protein (RKIP) expression, thus, interrupting the NF- κB signaling pathway through the physical association between NIK and RKIP, which was concomitant with Bcl-xL down-regulation. The direct role of Bcl-xL in drug-resistance was evaluated by using Bcl-xL over-expressing Ramos cells, which exhibited higher resistance to drugs that was partially reversed by rituximab. These findings reveal a novel mechanism of action of rituximab-mediated signaling by inducing RKIP expression that negatively regulates the constitutive NF- κB pathway resulting in Bcl-xL down-regulation and chemo-sensitization of the NHL B-cells. Furthermore, these findings identify several targets, namely RKIP, Bcl-xL and the components of the NF- κB signaling pathway, for therapeutic intervention in combination with cytotoxic agents to reverse adaptive and acquired resistance of B-NHL.
Preclinical Evaluation of Hematide™, a Novel Erythropoietic Receptor Agonist for the Treatment of Anemia Caused by Kidney Disease.
Authors: Woodburn Kathryn W. , Fan Qing , Leuther Kerstin K. , Holmes Christopher P. , Zhang Jim J. , Velkovska Svetlana , Chen Min-jia , Schatz Peter J.
Published: October 2019

Hematide™ is a PEGylated synthetic dimeric peptide. Initial peptide sequences were identified by screening a human erythropoietin receptor (HuEPOr) with Recombinant Peptide Diversity libraries possessing > 1010 different chemical structures. Following extensive chemical analoging and introduction of novel architectural features, a compound containing a dimeric peptide having no detectable sequence identity to natural human EPO was created and this compound is substantially equipotent to natural EPO. The portion of the molecule containing the peptide was linked to polyethylene glycol to yield Hematide, a potent stimulator of erythropoiesis with sustained plasma persistence. Antibodies generated experimentally to Hematide peptide do not cross-react with recombinant human EPO and therefore this peptide-based HuEPOr agonist is unlikely to cause aplastic anemia and loss of potency for either recombinant erythropoietin receptor agonists or for the recipient patient’s endogenous EPO. Pharmacokinetic studies in rats, dogs and monkeys have demonstrated the extended plasma half-life of Hematide. In the rat, elimination half-life is between 21.5 to 30.7 hours, steady state volume of distribution (Vss) is between 42.7 to 65.6 mL/kg and clearance is slow at 1.3–2.3 mL/h/kg. In the dog, elimination half-life was 73.7 hours while clearance and Vss were 0.69 mL/h/kg and 69.6 mL/kg, respectively. Prolonged half-lives and slow clearance times are also evident in the monkey. At a dose of 1.35 mg/kg, a half-life of 58.4 hours was obtained with a clearance rate of 0.96 mL/h/kg. Erythropoietic activity of Hematide has been demonstrated in the normocythemic mouse, rat, dog, and monkey, and in the nephrectomized rat. Pharmacology studies following single dose administration in mice, rats (normocythemic and nephrectomized) and monkeys indicate that the rise in hemoglobin is dose-dependent. Erythropoietic activity was demonstrated in Sprague-Dawley rats when Hematide was given every week for 6 weeks at doses up to 1.35 mg/kg. There was a pronounced erythropoietic response and no anti-Hematide antibodies were detected in any of the dose groups. In conclusion, Hematide is a potent erythropoietin receptor agonist with prolonged half-life and slow clearance times. It is anticipated that similar prolonged clearance will be observed in the clinic, potentially enabling dosing intervals of 3 to 4 weeks that may translate into improved patient convenience and compliance.
Phosphatidylinositol 3-Kinase p85 Subunit-Dependent Interaction with BCR/ABL: Molecular Mechanism and Biological Consequences.
Authors: Ren Shuyue , Bolton Elisabeth , Skorski Tomasz
Published: October 2019

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.
Immune Tolerance with a High Purity Plasma Derived Factor VIII Containing Von Willebrand Factor in Haemophilia A Patients with High Responding Inhibitors.
Authors: Polack Benoit , Beurrier Philippe , Rothschild Chantal , Orsini Frédérique , Faradji Albert , Liennhard Anne , Fressinaud Edith , Goudemand Jenny
Published: October 2019

We report on 8 patients with severe haemophilia A and high responding anti-factor VIII (FVIII) inhibiting antibody in whom immune tolerance (IIT) was induced with a high purity plasma derived FVIII containing 14 to 20 IU/ml of von Willebrand factor (VWF) as a stabiliser. Their median ages at the diagnosis of the inhibitor was 10.6 months and ranged from 5 months to 9 years. At the onset of IIT, the median age was 1 year and 9 months and ranged from 9 months to 24.5 years. The median cumulative exposure days (CED) was 18.5 days and varied from 8 to 81 days when inhibitor was detected. The maximum level of inhibitor reached from 10 to 500 Bethesda Units (BU) with a median of 22.5 BU. Before inhibitor diagnosis 6 patients had received plasma derived high purity FVIII and 2 had received recombinant FVIII. The treatment schedule started with 50 IU/kg to 200 IU/kg FVIII every day. The time to reach an inhibitor level <1 BU was available for 5 patients; its median was 142 days and varied from 75 to 216 days. The treatment was considered successful when either the recovery or the half life of FVIII was normalized. The outcome was successful in 7 out of the 8 patients (87.5 %) who are now receiving FVIII concentrates either on demand or prophylactically. The last patient was considered as a partial success since the titre of his inhibitor was 0.8 BU and he could be treated by FVIII concentrates when needed. No definite IIT failure was observed in this cohort of patients. Since all the patients are by now back to FVIII treatment we can consider having a 100% success in IIT in this small cohort of patients. Therefore, despite the size of our cohort of patients, our study shows that high purity FVIII stabilized by VWF is an effective drug for the eradication of anti-FVIII inhibitors through induction of immune tolerance.

Patients data

Patient Age at FVIII 1st infussion Age at inhibitor diagnosis CED at inhibitor diagnosis Age at IIT Inhibitor max.titre (BU) Time (d) <1 BU IIT results N.A.; not available 1 9m 1y 9 1y 3m 52 93 Success 2 2d 4y 3m 81 4y 3m 21 N.A. Success 3 8y 10m 9y 33 24y 6m 30 N.A. Success 4 8m 10m 28 1y 8m 24 75 Success 5 8m 8.5m 8 9m 14.5 - Partial success 6 1y 1m 1y 10m 21 3y 1m 10 142 Success 7 6m 9m 10 11m 20 216 Success 8 4m 5m 16 1y 9m 500 176 Success
HIV Envelope Protein gp120 Stimulates Expression of Specific Chemokines in Lymphatic Endothelial Cells.
Authors: Zhang Xuefeng , Wang Jian Feng , Groopman Jerome E.
Published: October 2019

Lymphoid organs are the major anatomical home of HIV, where the virus replicates during both the acute and chronic phases of infections. In this regard, there are significantly more infected cells in lymph nodes (LNs) than in circulating blood, and these infected cells are a major reservoir of infectious HIV. Certain chemokines like CCL19 (MIP-3β) and CCL21 (SLC) play key roles in immune cell trafficking to LNs. They induce specific homing of naïve T cells and dendritic cells into the T cell zone of secondary lymphoid organs. There, the T cells become activated by the dendritic cells. A network of channels composed of lymphatic endothelium exists in LNs that provides a route for this dendritic cell and T cell movement. To date, how this lymphatic endothelium may contribute to the pathogenesis of HIV infection has not been studied. This prompted us to investigate whether HIV may alter immune cell trafficking via interaction with this lymphatic network. Lymphatic endothelial cells (LEC) were separated from primary dermal microvascular endothelial cells. The phenotype of LEC was confirmed by immunostaining with specific lymphatic markers including VEGFR-3, LYVE-1, and podoplanin. Since HIV envelope proteins are presented to endothelial cells in the microenvironment, we studied the effects of X4 gp120 on LEC. Using a pathway specific cDNA array, we detected enhanced expression of a restricted repertoire of chemokines in LEC upon HIV-1 gp120 stimulation. Gp120 upregulated expression of the chemokine genes GRO-α, GRO-γ, MIP-3β, and SDF-1α and β in LEC. These chemokines can act to enhance T cell and dendritic cell homing to LNs. Furthermore, we also detected GRO-α, SDF-1, and SLC proteins in culture supernatants of the gp120-treated LEC. We did not observe upregulation of the chemokines RANTES and MCP-1 upon gp120 stimulation. Since dendritic cells mediate the HIV infectivity of CD4+ T cells by presenting HIV particles, our study suggests that HIV-1 gp120-induced production of a restricted repertoire of chemokines in LEC may accelerate the trafficking of infected dendritic cells to LNs and foster HIV infection in this reservoir.
Abbreviated Treatment with Short-Course Dose-Adjusted EPOCH and Rituximab (DA-EPOCH-R) Is Highly Effective in AIDS-Related Lymphoma (ARL).
Authors: Dunleavy Kieron , Little Richard , Gea-Banacloche Juan , Grant Nicole , Steinberg Seth , Yarchoan Robert , Pittaluga Stefania , Jaffe Elaine , Janik John , Wilson Wyndham H.
Published: October 2019

In ARL, rituximab may slightly improve CHOP response but is associated with greater toxicity (Proc Am Soc Hem102:1488, 2003). We hypothesized that the addition of rituximab to DA-EPOCH may increase fractional tumor cell kill and allow fewer treatment cycles and lower immune suppression. Patients received DA-EPOCH-R (E=etoposide 50 mg/m2/d, O=vincristine 0.4 mg/m2/d and H=doxorubicin 10 mg/m2/d all CIV d 1–4 (96 hours); C=cyclophosphamide 750 mg/m2 IV d5; P=prednisone 60 mg/m2 PO qd d1–5 and R=rituximab 375 mg/m2 IV d1 and 5) with G-CSF. Prophylactic IT MTX x 6 was administered. HAART was discontinued before and recommenced after DA-EPOCH-R. Unlike our previous study of DA-EPOCH in ARL (BLOOD101:4653, 2003) where the dose of C was lower and based on CD4 cell count, all patients on this study received full dose C on cycle 1 with subsequent reduction if the ANC nadir was < 500/mm3 for ≥ 2 days. Patients received 1 cycle beyond CR, based on CT and PET, for a minimum of 3 cycles. Characteristics of 21 patients include median (range) age 39 (9–61) years; IPI 3 (0–4); ECOG PS 2 (1–4); CD4 212 (0–674) cells/mm3 and HIV viral load 53100 (0– 286472) RNA copies/mL. Additionally, male sex 17 (81%); LDH> nl 15 (71); stage IV 15 (71%) and histology with diffuse large B-cell 9 (90%) and Burkitt’s lymphoma 2 (10%). The 18 patients who completed treatment (2 TE; 1 NE) received a median (range) of 3 (3–5) cycles. Responses are CR/CRu 15 (83%); PR 1 (6%) and NR 2 (11%). At 19 mos median follow-up, overall PFS and OS are both 77%, and both 90% in patients with CD4 > 100 cells/mm3. Treatment outcome of DA-EPOCH-R is similar to DA-EPOCH (CR 74% and PFS 73% at 53 months) but with significantly shorter treatment (median cycles 3 vs. 6). Toxicity on 57 cycles include ANC < 500/mm3 on 27 (47%); platelets < 50,000/mm3 on 15 (26%) and; fever/neutropenia on 20 (35%) cycles. DA-EPOCH-R produced a median (range) CD4 cell decrement of 64 (−541 to + 239) cells/mm3 compared to 189 (−973 to +19) with DA-EPOCH. Hematological toxicity is higher with DA-EPOCH-R compared to DA-EPOCH with ANC < 500/mm3 47% vs 30% and fever/neutropenia 35% vs. 13%, respectively, likely due to higher C dose intensity and/or rituximab. Other toxicities are similar. Abbreviated DA-EPOCH-R is equivalent to DA-EPOCH x 6 and appears to produce less CD4 cell loss. Accrual continues.

Figure Figure
Adolescents with Hodgkin Lymphoma Treated on Adult and Pediatric Protocols Have Distinct Therapeutic Outcome.
Authors: Mottl Alexis A. , Fisher Susan G. , Fisher Richard I. , Constine Louis S. , Friedberg Jonathan W.
Published: October 2019

Hodgkin Lymphoma (HL) is a B cell malignancy, highly curable in the modern era (1989–2004). It is the most common cancer in the 15–19 age range. In the United States, adolescents with de novo HL may be treated on either pediatric or adult protocols. To identify outcomes of adolescents with HL, we conducted a comprehensive literature review and statistical analysis of all treatment trials (N > 50 pts) published in the past 15 years. Trials were selected for their relevance to treatment and disease outcome in adolescents with the following search criteria: Hodgkins disease, therapy, treatment, adolescents (13–18 years), and adults (19–44 years). The search retrieved 120 randomized control trials and 57 Phase II trials. 117 trials were eliminated due to inappropriate histology, non-clinical endpoints, topic reviews or otherwise not meeting established criteria. 79 studies were eligible for analysis. 15 pediatric treatment protocols (13 multi-center, 7 randomized) met established criteria. 7 identified the number of adolescents on trial (N=958/2675; 36%). Only 3 of these studies evaluated adolescent outcome; one trial demonstrated an inferior event-free survival for the adolescent subgroup vs. all children (72.0% vs. 89.0%; p-value 0.006, Chi Square Test). 57 adult treatment studies (39 multi-center, 44 randomized) were reviewed. 7 identified the number of adolescents on trial (N=422/2522; 17%). Only one provided information on adolescent outcome, which experienced a superior overall survival compared with the entire population (86.0% vs. 73.1%; p-value 0.030, Chi Square Test). None of the trials with both pediatric and adult subjects (7 multi-center, 4 randomized) provided information on the adolescent subgroup. In comparing pediatric and adult trials, we found remarkable differences in therapeutic approaches (all pediatric studies included XRT as a treatment component compared to 58% of adult studies) and in trial design (77% of adult studies were randomized compared to 47% of pediatric studies; p-value 0.0234, Fishers Exact Test). The adolescent population with HL is significantly understudied despite reasonable clinical trial participation and relatively high prevalence. In fact, only 4 published trials provided outcome information on adolescents with HL; in 2, outcome was statistically different from the rest of the study population, suggesting this subgroup may benefit from a specialized treatment approach. It is critical for future treatment trials to analyze results of the adolescent population to determine whether unique biological features, therapeutic practices, or host factors contribute to prognosis and outcome.
Assessment of the Economic Value of the Intercept Blood System for Platelets in Belgium.
Authors: Moeremans Karen , Warie Hans , Annemans Lieven , Digneffe Toon , Toté Jacques , Corash Laurence
Published: October 2019

Objectives: Despite transfusion safety measures, a residual risk of infection remains. New emerging pathogens continue to threaten blood safety, requiring novel approaches to prevent transmission as from the time of emergence. INTERCEPT Blood System for platelets (IBSP) inactivates different types of pathogens including viruses, bacteria and parasites, aiming at eliminating the residual risk of screened infections, the risk of other existing pathogens and the risk of transmitting emerging pathogens. The objective was to evaluate the potential incremental cost-effectiveness ratio (ICER) associated with implementing IBSP in Belgium.

Methods: A decision model compares a “world with IBSP” to a “world without IBSP”. It calculates lifetime costs and “quality adjusted life years” (QALYs) following platelet transfusion in patients with different oncological and surgical indications, taking into account disease specific life expectancy and the potential impact of transfusion transmitted infections. Transfusion safety data were obtained from Belgian Red Cross, costs from Belgian health insurance. The health economic consequences of HIV, HCV and HBV transmission were modelled on the basis of clinical and economic literature. In addition a HCV-like emerging pathogen was simulated at different risk levels based on current and historical hepatitis, HIV and West Nile Virus transmission data. Different scenarios for additional benefits (elimination of current safety measures) were considered.

Results: A wide range of ICERs was observed, highly sensitive to the risk of emerging pathogen transmission, underlying disease and age. In the most conservative scenario with emerging risk of 1/100,000 and no additional benefits, the ICER ranged from 3,355,308€/QALY to 165,051€/QALY. The mean threshold of emerging infection risk for dominance of IBS ranged from 1/1,074transfusions to 1/2,277transfusions depending on additional benefits.

Conclusion: Considering the high value society places on prevention of accidental injury, and the ICER of recent implementations in transfusion medicine (NAT testing: up to €2.3 million/lifeyear), IBSP can be considered cost-effective and even a dominant strategy taking into account the potential risk of emergence of a new pathogen in the future.
Tumor Cell Apoptosis Induced after Treatment with Adenovirally Expressed TRAIL in Vivo.
Authors: Wang Jishi , Fang Qin , Wu Yingya , Hu Xiaoyan , Abraham N.G.
Published: October 2019

Tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) is a naturally occurring death inducing ligand that induces apoptosis preferentially in malignant cells as normal cells are protected by expression of decoy receptors. Activation of TRAIL-R1 and R2 leads to the recruitment of procaspase-8 to the receptor, through an adaptor protein FADD, leading to formation of the death inducing complex (DISC), which leads to activation of caspase 8 followed by caspase 3 activation resulting in apoptosis. Our previous in vitro studies indicated that TRAIL induces apoptosis in tumor cells but not in normal cells. In order to improve the delivery of TRAIL to tumor cells, we constructed a recombinant adenovirus pAd5CMV-NpA- TRAIL vector, the TRAIL transgene was placed under the control of the CMV promoter. Ad-CMV-EGFP was constructed and used as control. They were amplified in HEK 293 cells and the titer of both was 1–1.5x1010pfu/ml.

C57 mice were injected with the adenoviral vectors (200ul) in the tail vein. We examined the expression of EGFP in mice tissues from animals administered adenoviral pAd5CMV-NpA-EGFP vector, and the presence of the EGFP gene was examined in the heart, lung, liver, and kidney of the animals. The results showed that EGFP was detectable in these tissues. Positive signals were obtained 1 and 2 weeks after injection of adenoviral- EGFP construct, the highest tissue levels of EGFP were obtained 1 week after EGFP gene transfer. Western blots revealed strong EGFP expression in the tissues 5 days after Ad-CMVEGFP injection.

In a model for metastatic cancer, tail vein infusion of pAd5CMV-NpA- TRAIL (200ul) resulted in elimination of preestablished liver metastases. Intravenous injection of this vector caused increased TRAIL expressions in tissues in tumor-bearing melanoma C57 mice, which we attributed to factors released from apoptotic tumor cells. Liver histology analyzed at day 7–14 after virus injection did not show signs of hepatocellular damage. This new oncolytic vector represents a potentially efficient means for gene therapy of metastatic cancer. We found that TRAIL can systemically inhibit tumor growth and reduce the size of tumors. In addtion, we also investigated whether pretreatment of TRAIL can prevent the tumor formation in mice treated with B16 melanoma. Normal C57 mice that received 5 days of treatment of TRAIL prior to implanting tumors resulted in the inhibitoty effect on tumor growth when compared to control mice.
Blockade of Inhibitor Formation by B-Cell Delivered Tolerance to Immunodominant A2 and C2 Domains.
Authors: Lei Tei Chi , Scott David W.
Published: October 2019

A major impediment in the treatment of hemophilia is the formation of inhibitory antibodies, which occurs in approximately 25–30% of Hemophilia A patients treated with therapeutic Factor VIII (fVIII). We have focused on the development of a gene therapy protocol for tolerance induction, with an emphasis on the elimination of inhibitor production. Our lab has demonstrated that LPS-stimulated B-cell blasts, transfected with a retrovirus encoding an IgG-peptide fusion protein, such as fVIII domains, are tolerogenic in both normal and primed recipients. Last year, we reported (http://www.abstracts-on-line.com/abstracts/hemphiladelphia03; Scott and Lei 2003) that specific tolerance to the immunodominant epitopes in the C2 domain of fVIII (a major target of inhibitors) could be induced by our protocol. However, the immune response to full length fVIII was only modestly affected. Most inhibitory antibodies are reactive with conformational epitopes on the exposed surfaces of the A2, as well as the C2, domain of fVIII. Therefore, in this study, we inserted residues S2173-Y2332 of the C2 domain and S373-R740 of the A2 domain onto the IgG heavy chain backbone, respectively, to induce tolerance in hemophilic mice. Specific tolerance to each domain was induced by this protocol. Importantly, a combination of A2-IgG and C2-IgG expressing B cells induced tolerance to the full length fVIII molecule, a result which supports the dominance of these domains in the immune response to fVIII. Tolerance was manifested in terms of ELISA, T-cell proliferation and especially Bethesda Unit titers (95% reduction). Similar results were obtained even when treatment was initiated after priming injections of fVIII. In conclusion, this protocol offers great promise for prevention and potential reversal of this serious complication of fVIII replacement therapy. (Supported by HL61883 and a Laboratory Grant from the National Hemophilia Foundation).
Red Blood Cell Hemolysis and Suppression of Erythropoiesis Strongly Upregulate mRNA for Iron Binding Lipocalin (NGAL/24p3) in the Liver.
Authors: Necas Emanuel , Vokurka Martin , Krijt Jan
Published: October 2019

A member of the lipocalin family of proteins, NGAL/24p3, was demonstrated to bind iron and was suggested to participate in a non-transferrin dependent iron transport mechanism capable to deliver iron to the cytoplasm (Kaplan 2002, Yang et al. 2003). Therefore, we have studied mRNA for NGAL/24p3 levels in the liver tissue during its loading with iron released from senescent and damaged red blood cells. Red blood cell hemolysis was induced in mice by administration of phenylhydrazine (PHZ). Suppression of iron reutilization for erythropoiesis was achieved by a total body sublethal irradiation (5 Gy). Samples of liver tissue were collected 16 hrs or 48 hrs after PHZ and 40 hrs after irradiation. Combined treatment consisted from irradiation followed by PHZ administration 40 hrs later. The irradiation suppressed 24 hrs incorporation of 59Fe into blood from 46,5 % in controls to 1.2 % in irradiated mice, indicating a significant suppression of erythropoiesis. PHZ administration alone decreased hematocrit from 44.7 % to 38.7 %, reflecting degree of the red blood cell hemolysis. The combined treatment by irradiation and PHZ resulted in the elevation of the liver iron content from 43.8 to 106.7 micrograms/g wet tissue 16 hours after PHZ, indicating a significant loading of the liver tissue with iron. All these treatments increased mRNA for NGAL/24p3 levels as determined by real-time PCR, significantly. After the combined treatment the increase reached almost three orders of magnitude. We further compared the response of NGAL/24p3 mRNA to the response of hepcidin and transferrin-1 receptor (TfR-1) mRNAs, both known to be sensitive to the liver iron content. Hepcidin mRNA increased significantly after the treatment with irradiation, PHZ, or combination of irradiation and PHZ but the increase was less pronounced compared to that of NGAL/24p3 mRNA. TfR-1 mRNA significantly decreased 48 hours after the combined treatment only. As an indicator of the acute phase response, the mRNA for interleukin-6 was determined and it did not change after the treatments used. The results demonstrate that mRNA for the putative iron transport molecule NGAL/24p3 was strongly upregulated by experimental maneuvers that lead to accumulation of iron in the liver tissue.
Initial Screening Transferrin Saturations, Serum Ferritin Concentrations, and HFE Genotypes in Whites and Blacks in the Hemochromatosis and Iron Overload Screening (HEIRS) Study.
Authors: Barton James C. , Acton Ronald T. , Dawkins Fitzroy W. , Adams Paul C. , Lovato Laura , Leiendecker-Foster Cathy , McLaren Christine E. , Reboussin David M. , Speechley Mark R. , Gordeuk Victor R. , McLaren Gordon D. , Sholinsky Phyliss , Harris Emily L.
Published: October 2019

We compared initial screening data of 44,149 whites (17,043 men; 27,106 women) and 26,947 blacks (9,788 men; 17,159 women) who reported no previous diagnosis of hemochromatosis or iron overload from a primary care-based sample of ~100,000 adults ≥25 years recruited from 5 Field Centers. Each underwent transferrin saturation (TfSat) and serum ferritin (SF) measurements without regard to fasting, and HFE C282Y and H63D genotyping. We observed these mean TfSat and SF values and percentages of participants with elevated biochemical measurements (TfSat >50% and SF >300 ng/mL, men; TfSat >45% and SF >200 ng/mL, women):

Participants Mean % TfSat (95% CI) Elevated TfSat, % Participants Mean SF, ng/mL (95% CI) Elevated SF, % Participants White men 32.4 (32.3, 32.6) 7.05 150.9 (149.1, 152.8) 19.49 Black men 29.3 (29.1, 29.5) 4.03 178.4 (175.5, 181.3) 25.95 p Value <0.0001 <0.0001 <0.0001 <0.0001 White women 27.5 (27.3, 27.6) 6.62 63.0 (62.3, 63.7) 8.74 Black women 23.3 (23.1, 23.4) 3.04 68.3 (67.2, 69.4) 15.43 p Value <0.0001 <0.0001 <0.0001 <0.0001

In participants grouped by decade, mean TfSat was greater in white men than in black men, and in white women than in black women (all p<0.0001). Mean SF was greater in black men than in white men in all decades except 25–34 years, and in black women than in white women in decades ≥45 years. SF increased with age to ~45 years in white men and ~50 years in black men, and then decreased. In white women, SF rose with age until ~62 years and less rapidly to age ≥80. In black women, SF rose with age until ~62 years and then decreased. C282Y allele frequency was 0.0700 (95% CI: 0.0683, 0.0717) in whites and 0.0119 (0.0110, 0.0129) in blacks. H63D allele frequency was 0.1532 (0.1509, 0.1556) in whites and 0.0299 (0.0284, 0.0313) in blacks. Frequencies of genotypes with C282Y or H63D were greater in whites than in blacks (all p<0.0001). The prevalence of elevated TfSat and SF plus genotype C282Y/C282Y was 0.003 in whites and 0.00004 in blacks (p<0.0001). The prevalence of elevated TfSat and SF plus genotype C282Y/C282Y, C282Y/H63D, or H63D/H63D was 0.006 in whites and 0.0003 in blacks (p<0.0001). We conclude that: 1) the prevalence of elevated TfSat is higher in whites and the prevalence of elevated SF is higher in blacks; 2) SF levels increase with age; 3) HFE C282Y and H63D alleles occur more frequently in whites than in blacks; and 4) the prevalence of elevated TfSat and SF plus genotypes with two common HFE mutations is greater in whites than in blacks.
Minimal Residual Disease(MRD) in Childhood Acute Lymphoblastic Leukemia(ALL) in Relapse. A Children’s Oncology Group (COG) Study.
Authors: Borowitz Michael J. , Devidas Meenakshi , Hunger Stephen P. , Carroll William L. , Raetz Elizabeth
Published: October 2019

In spite of the excellent outcome of children with ALL, those who relapse fare poorly. To date, time to relapse is the most powerful predictor of outcome. Most studies indicate that children with early marrow relapse (ER), occurring within 36 months of diagnosis, have a long-term event-free survival (EFS) of 10–20%, while those with a late marrow relapse (LR) have an EFS of 40–45%. MRD, detected either by flow cytometry or polymerase chain reaction is an important prognostic factor in patients with newly diagnosed ALL, and recent studies indicate that it is prognostic of outcome following relapse. However studies in relapse are more limited and generally only one time point at end induction has been tested. COG AALL01P2 is a pilot study designed to administer three sequential blocks of intensive therapy to children and adolescents with first marrow relapse of ALL with the goal of improving remission rates and achieving a lower level of disease burden prior to post induction therapy such as bone marrow transplantation. We measured MRD in 52 patients on COG AALL01P2 by flow cytometry using the 4-color combinations CD20-FITC/CD10-PE-CD45-PerCP/CD19-APC; CD34/9/45/19; and CD38/58/45/19. We detected MRD in 35 patients (67%) after the first block, a much higher figure than the 28% seen at end induction in more than 2000 patients with newly diagnosed ALL, consistent with the poor prognosis of relapse patients. Of 32 ER patients, 26(81%) were MRD+ compared to only 45% (9/20) of LR patients (p<.01). Serial MRD samples measured after each block of therapy were collected in 32 patients, and several patterns of kinetic changes in MRD levels were noted. Twenty-two patients were classified as responders: 7 patients had no detectable disease at any time point(R1); 9 patients were initially MRD positive, but became negative(R2); and 6 additional patients showed at least a 1 log decrement in MRD(R3). No patient negative at any time point became positive at a later time point. Six patients had >1% MRD at all time points tested, while 4 patients with a level <1% on at least one timepoint had an increase of at least 1 log in MRD burden between 2 timepoints. All 10 of these latter patients were ER while all 7 R1 patients were LR. While it is too early to correlate MRD with EFS, our results show that the incidence of MRD is different in ER and LR patients in a pattern that correlates well with expected outcome. Serial measurements of MRD can be used to identify different patterns of treatment response and may be useful to dissect out effective components of therapy. Moreover, it may be possible to assess efficacy of novel agents added to this backbone of therapy by measuring changes in MRD response.
Unmutated Immunoglobulin Heavy Chain Variable Region Genes and Disease Progression Post Splenectomy Are Poor Prognostic Factors in Splenic Marginal Zone Lymphoma.
Authors: Oscier David Graham , Mould Sarah J. , Gardiner Anne C. , Glide Sharron , Parker Anton E. , Davis Zadie A. , Orchard Jenny A.
Published: October 2019

Splenic marginal zone lymphoma (SMZL) is generally an indolent disorder which has a median survival of 10 – 13 years, but a minority of patients pursue a more aggressive clinical course. A number of adverse prognostic factors including the level of haemoglobin, lymphocyte count and platelet count, B2 microglobulin, presence of a paraprotein, IgVH gene mutational status and p53 loss or mutation have been identified, but these have not been consistent among recent series. We have studied 89 patients with SMZL, diagnosed on the basis of lymphocyte morphology, immunophenotype and marrow and splenic histology, when available, and have evaluated the impact of presenting Hb, lymphocyte count, spleen size, presence of a paraprotein, cytogenetic abnormalities and IgVH gene mutational status on both the need for treatment and on overall survival. In 43 patients the diagnosis was made following a routine blood count performed for an incidental reason. The M:F ratio was 1:1.78, median age at presentation was 67.5 years (range; 49 – 91) and mean follow-up was 78.1 months. 63/89 (71%)patients presented with palpable splenomegaly, and a further 5 developed splenomegaly during the course of their disease. 42/89 (47%) patients required treatment of whom 29 (32%) underwent splenectomy. 36 patients have not required treatment, 25 have received an alkylating agent, 12 have received fludarabine and 4 were treated with rituximab.15/29 (17%) received chemotherapy for progressive disease post splenectomy. In six patients there was transformation to a high grade lymphoma. 14 patients (16%) died, 11 of whom had a disease-related death. 24/83 patients (29%) had a paraprotein, 67/87 (77%) had an abnormal karyotype of whom 39 (45%) had an abnormality of 7q. Using interphase FISH, 7/78((9%) showed trisomy 3 and 7/74(9%) had p53 loss. Of the patients with high grade transformation, 3/6 had p53 loss. 47/67 patients (70%) had mutated IgVH genes, and 15 cases utilised the VH1-02 gene segment. Only spleen size >10cm (p=0.0002) and Hb <100 gm/l (p=0.008) correlated with the need for treatment. Only unmutated IgVH genes (p=0.032) and the need for further therapy following initial treatment with splenectomy (p=0.034) correlated with overall survival.The median survival of unmutated cases was 113 months and was not reached for mutated cases.The median survival of patients for whom splenectomy has been their only treatment has not been reached, but was 110 months for patients requiring additional treatment post splenectomy. There is a need for larger, multi-centre studies to identify poor risk patients with SMZL.
Pre-Transplantation FDG-PET Is the Best Predictor for Outcome in Relapsed Lymphoma Patients Treated with Autologous Stem Cell Transplantation.
Authors: Schot Bart , Sluiter Wim , van Imhoff Gustaaf , Pruim Jan , de Brouwer Miryam , Verhoogt Carolien , Vaalburg Wim , Vellenga Edo
Published: October 2019

Intensive chemotherapy followed by autologous stem cell transplantation (ASCT) is the prefered treatment for young patients with chemosensitive relapsed aggressive lymphoma (NHL) and Hodgkin’s disease (HD). FDG-PET appears to be an excellent tool to evaluate chemosensitivity in lymphoma. In the present study we analyzed the predictive value for outcome of pre-transplantation PET as compared to initial prognostic factors at relapse.

Methods: Clinical and PET data from all consecutive patients with relapsed lymphoma (aggressive NHL and HD) were assessed. All patients were treated with DHAP-VIM-DHAP, followed by ASCT if responsive (at least PR based on CT after DHAP-VIM). Whole-body FDG-PET was performed after DHAP-VIM. Visual analysis was obtained in each scan and standardized uptake value (SUV) of the 3 most intense lesions in attenuation-corrected scans. Minimal follow-up after ASCT was 6 months. Predictive factors used were secondary age-adjusted IPI (sAAIPI) for NHL and secondary IPS (sIPS) for HD, duration of first remission and LDH at relapse. Univariate and multivariate Cox regression models and ROC analysis were used to assess predictive factors for relapse/progressive disease (PD) and Fisher’s exact test to assess relative risk.

Results: Between 1999 and 2003, 93 relapsed lymphoma patients were treated. 68/93 (73%) patients had responsive disease and ultimately 63/93 (68%) were transplanted. Post transplantation 36 (25 NHL and 11 HD) were in continued complete remission after a median follow up of 20 months.

PET was performed in 84 (63 NHL and 21 HD). All scans were used for visual assessment. SUV could be assessed in 70% of the scans. PET was positive in 76% and negative in 24% of the patients. Relapse/PD was observed in 44/64 (69%) PET-positive and only in 6/20 (30%) PET-negative patients (RR 5.1 (1.7–15.3), p=0.002).

SUV (p=0.001), sAAIPI/sIPS (p=0.023) and LDH (p=0.028) were predictive for outcome. In a multivariate Cox regression model adding separate parameters to SUV, no additional significant predictor was found. There was no difference between NHL and HD. ROC analysis showed a maximal predictive value at a SUV of 1.8 with a sensitivity and specificity of 0.66. A SUV ≥ 6.0 after DHAP-VIM was highly predictive for relapse/PD (spec. 0.95, sens. 0.27).

Conclusion: Pre-transplantation PET is the best predictor for outcome in patients with relapsed aggressive lymphoma as compared to other prognostic factors. Persisting lymphoma lesions on PET with a SUV ≥ 6.0 after reinduction chemotherapy predict progressive disease. Pre-transplantation PET may be helpful in the selection of patients for ASCT.
Fas or Fas Ligand Expression Can Determine the Clinical Outcome of Germinal Center Type in Diffuse Large B Cell Lymphoma.
Authors: Kojima Yasushi , Tsurumi Hisashi , Goto Naoe , Sawada Michio , Yamada Toshiki , Hara Takeshi , Takahashi Takeshi , Yamada Tetsuya , Saio Masanao , Tomita Eiichi , Takami Tsuyoshi , Moriwaki Hisataka
Published: October 2019

In recent years, diffuse large B cell lymphoma (DLBCL) has been classified by a cDNA microarray, an oligonucreotide microarray, or a tissue microarray. From the prognostic point of view, DLBCL consists of germinal center B-cell-like (GC) type, activated B-cell-like (ABC) type and type 3. ABC type and type 3 can be collectively categorized as non-GC (NGC) type. GC type has favorable prognosis compared with NGC type. Escape from apoptosis is considered to be an important mechanism for the progression of lymphoma. Fas is the major protein which leads to apoptosis by binding with Fas-ligand (FasL). We evaluated the prognostic significance of such markers as CD10, Bcl-6, MUM1, Fas and FasL, as well as GC or NGC type with paraffin embedded sections from 69 DLBCL patients immunohistochemicaly. The patients were 40 men and 29 women with median age of 67 years old (range, 20–82 years old). The median follow-up of surviving patients was 43 months. The 3-year OS for the entire group was 56%. There was no significant difference in sex, age, clinical stage, lactate dehydrogenase, or International Prognostic Index between GC and NGC type. Expression of CD10 was seen in 29% (20 of 69 of the patients), Bcl-6 in 27% (19 of 69), MUM1 in 27% (19 of 69), Fas in 51% (36 of 69), and FasL in 50% (35 of 69). We divided 69 DLBCLs to 26 GC type (CD10 positive or Bcl-6 positive and MUM1 negative) and 43 NGC type (the other). Positive CD10 was the best marker to indicate favorable overall survival (p=0.0156). GC type had tendency to have better overall survival than NGC type, though it was not significant (p=0.0723). Although Fas or FasL expression was not significant for overall survival in all DLBCL, it predicted significantly a longer over all survival (Fas; p=0.0021, FasL; p=0.0165) in GC type. These results may suggest that the presence of a subtype of DLBCL in which Fas/FasL system works effectively. This group is approximately 20% of DLBCL and mainly belongs to the GC type. Fas expression in GC type of DLBCL may predict the prognosis and be useful to choose the appropriate therapy. Furthermore, CD10 was more significant than GC type and, thus, we may need to build the strict consensus for GC type.