bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of Cytotoxic T Cell Responses Directed to HLA Class I Restricted Epitopes from the Aspergillus f16 Allergen.
Authors: Ramadan Gamal , Davies Barbara , Kurup Viswanath P. , Keever-Taylor Carolyn A.
Published: October 2019

Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients such as hematopoietic progenitor cell transplant patients. Studies both in patients with allergic bronchopulmonary aspergillosis and murine models demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. The role of CD8+ T cell response to A. fumigatus is less clear. Our efforts to develop effective immunotherapeutic approaches against A. fumigatus included preparation of 104 overlapping pentadecapeptides spanning the 427 aa coding region of the aspergillus allergen, Asp f16 previously shown to induce T cell responses. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Monocytes from healthy donors were treated with GM-CSF and IL-4 for 2-3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-alpha) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5/5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. After 4 weeks the line from donor #2 was strongly cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract-pulsed DC and peptide pool pulsed HLA Class I matched BLCL. Supernatant from this line killed fresh aspergillus conidia. Six of 21 smaller pools of 4-11 peptides showed reactivity. Specificity could be narrowed by screening peptides shared by the pools to 3 candidate peptides. Pool-pulsed BLCL matched for only 1 or 2 HLA alleles were used to demonstrate CTL restriction by HLA-B-3501. A database search of peptides likely to be restricted to B3501 identified the likely sequences as YFKYTAAAL, LPLCSAQTW, and GTRFPQTPM. Each induced similar reactivity when pulsed onto B-3501+ targets. CD8+ T cells steadily increased from 5.2% at week 3 to 19.0% after the 7th priming. CTL activity and IFNγ production were exclusively mediated by CD8+ T cells and CD107a was expressed by 42% of the CD8+ T cells in response to pool-pulsed BLCL indicating degranulation. CTL cross-reacted with pool pulsed B3503+ BLCL but not B3502+, or B3508+ BLCL. B3503+ BLCL presented YFKYTAAAL and to a lesser extent GTRFPQTPM but not peptide LPLCSAQTW. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD8+, HLA-Class I restricted Aspergillus-specific T cell response directed to multiple peptides contained within the pool. Further characterization of this system is in progress to identify additional immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.
Generation of Th1 T Cell Responses Directed to a HLA Class II Restricted Epitope from the Aspergillus f16 Allergen.
Authors: Ramadan Gamal , Davies Barbara , Kurup Viswanath P. , Keever-Taylor Carolyn A.
Published: October 2019

Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients, including hematopoietic progenitor cell transplant patients. Studies in patients with allergic bronchopulmonary aspergillosis as well as murine models have demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. A. fumigatus allergens have been identified based on reactivity to IgG and IgE antibodies in serum from patients with allergic bronchopulmonary aspergillosis. One allergen, Asp f16 was shown to induce a protective Th1 T cell response to infection with aspergillus conidia in murine studies. We prepared a series of 104 overlapping pentadecapeptides spanning the entire 427 aa coding region of Asp f16. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Autologous monocytes from healthy donors were treated with GM-CSF and IL-4 for 2–3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-α) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5 of 5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. As early as 3 weeks the T cell line from donor #1 had a high frequency of IFNγ-producing CD4+ T cells (24.5±5.4%) in response to the peptide pool, was cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract pulsed DC, and peptide pool pulsed autologous BLCL. Supernatant from this culture killed fresh aspergillus conidia. Screening of 21 smaller pools containing 4–11 peptides showed reactivity to three smaller pools and could be narrowed by screening individual peptides shared by the pools to a single candidate sequence of TWSIDGAVVRT that elicited IFNγ production and lytic activity equal to the entire pool. Peptide pulsed BLCL matched with the donor for only 1 or 2 HLA alleles were used to demonstrate that CTL activity and IFNγ production was restricted by HLA-DRB1- 0301. 23% of the CD4+ cells in the culture expressed CD107a in response to peptide pool indicating degranulation. PBMC from DRB1-0301+ individuals proliferated strongly in response to peptide TWSIDGAVVRT. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD4+, HLA-DR-restricted aspergillus-specific Th1 type T cell response directed to a single peptide contained within the pool. Further characterization of this system is in progress to identify other immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.
Acute Chest Syndrome Is Strongly Associated Parvo Virus B19 Seroconversion in Patients with Hemoglobin SC Disease.
Authors: Coates Thomas D. , Dampier Carlton , Kalinyak Karen , Lail Alice , Mentzer William , Piomelli Sergio , Radel Eva , Space Sharon , Howard Thomas
Published: October 2019

Parvovirus B19 replicates principally in human erythrocyte precursors resulting in temporary cessation of erythropoiesis. In patients with sickle hemoglobinopathies, parvovirus B19 infection has been associated with acute chest syndrome and may lead to transient aplastic crisis. Evidence of prior infection with parvovirus B19 is found in 50 to 60% of adults. Infection appears to be followed by lifelong immunity. The objectives of the Study of Seroprevalence and Incidence of Parvovirus B19Infection in Children with Sickle Hemoglobinopathies are to determine the prevalence of parvovirus B19 antibodies in children less than 16 years old; determine the rate of parvovirus B19 seroconversion; and determine whether parvovirus B19 seroconversion is associated with the occurrence of aplastic crisis or acute chest syndrome. Between April 2000 and June 2002 a total of 1039 children from eight Comprehensive Sickle Cell Centers (CSCC) were enrolled in the study and 898 had at least one follow up visit. Participants ranged in age from <1 month to 16 years at enrollment (mean age 7.2, + 4.5 years), 47% were female, 53% were male. 67% had homozygous Hb SS disease, 24% had Hb SC disease, 6% had Hb SB+ disease, 3% had Hb SB0 disease, and <1% had other S sickle cell disease. The seroprevalence of parvovirus B19 in the cohort was 35%. Prevalence varied significantly by age, ranging from a low of 8% for children 1–2 years of age to 69% for children 13+ years of age (p<0.0001). The greatest increase in seroprevalence occurred between 6 and 7 years of age. Adjusted for age, there were no significant differences in seroprevalence by gender, clinical center, geographic area of residence (northeast, southeast, midwest, and west) sickle hemoglobinopathy, number of siblings, hemoglobin level, reticulocyte %, or among recipients of treatment with transfusion or hydroxyurea. Parvovirus B19 seroconversion was strongly associated with an increased risk of aplastic crisis in both SS (odds ratio of 29, 95% CI (11.3, 74.4)) and SC patients (odds ratio of 110, 95% CI (9.6, 1270.4)), even when controlling for age. Seroconversion was associated with an increased risk of acute chest syndrome in SC patients, (odds ratio of 6.4, 95% CI (1.9, 21.9)) but did not reach statistical significance in SS patients.

These data show a clear causal relation between parvovirus B19 seroconversions and acute chest syndrome in subjects with hemoglobin SC disease and confirm the previously known association aplastic crisis.
The Role of BMP-4 in the Stromal-Maintenance of Haematopoietic Stem Cells.
Authors: Hutton Jonathon F. , Khor Fiona , Rozenkov Vlad , Lewis Ian D. , D’Andrea Richard J.
Published: October 2019

Haematopoietic stem cells (HSCs) expand in number during fetal liver hematopoiesis via a process that is not understood. Establishment of conditions supporting HSC maintenance and expansion ex vivo is critical for wider application of cord blood derived HSC. We have shown that bone morphogenic protein (BMP) 4 secreted by mouse foetal liver cells contributes to expansion of cord blood-derived HSCs during in vitro co-culture. Significant levels of BMP-4 mRNA and secreted protein were produced by the supportive murine foetal liver stromal cell line AFT024. Supplementing 14 day co-cultures of AFT024 and human cord blood CD34+ cells with the BMP-4 antagonist Noggin, or a neutralising BMP-4 antibody decreased the proportion of cells maintaining a CD34+CD38−CD33− primitive phenotype (by 27.6% and 37.2% respectively), decreased CFU-GM expansion (by 20.6% and 22.2% respectively), and caused a large reduction in net expansion of long-term culture-initiating cells (LTC-IC) (by 31.7% and 61.5% respectively). The ability of BMP4 to support the multipotency and self-renewal of hematopoietic stem cells is consistent with a role recently shown on embryonic stem cells and suggests that it may act generally as a stem cell maintenance factor. Elucidating further the role of HSC growth factors such as BMP-4 in ex vivo culture may lead to development of defined systems for the routine clinical expansion of HSC.
Negative Impact of Pre-Transplant Anemia on Long-Term Outcome after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Lazongas Chris , Wong Cindy J. , Sutton David M. , Lipton Jeffrey H. , Xenocostas Anargyros , Messner Hans A.
Published: October 2019

Background: Anemia is commonly present in patients with malignancies and is associated with reduced survival times. Recently, we described that low (<110 g/L) pre-transplant hemoglobin levels (PT-Hb) are associated with decreased early survival after allogeneic hematopoietic stem cell transplantation (alloHSCT)(Xenocostas et al. Transfusion2003;43:373–382). We are now presenting data on long-term survival and causes of death in BMT recipients with and without anemia prior to alloHSCT.

Study Design and Methods: A retrospective analysis of 511 patients consecutively transplanted between January 1995 and March 2000 was performed to evaluate survival, cause of death, and PT-Hb. The end date for follow-up was June 2002. The median follow-up time was 993 days. Causes of death were categorized either as relapse, treatment-related mortality (TRM), or other. PT-Hb levels were determined within 2 weeks prior to transplantation, after commencing conditioning chemotherapy. Comparisons between groups were done using chi-squared tests.

Results: The 180-day survival of patients with low PT-Hb levels (<110 g/L) was significantly worse than that of patients with PT-Hb levels ≥110 g/L (57.1% versus 83.1%, p<0.0001). The survival difference remained significant at 5 years (36.2% versus 59.4%, p<0.0001). The difference in 180-day survival was contributed to by an increase in TRM (36.1% versus 14.9%, p<0.0001) as well as a higher relapse rate (4.4% versus 0.3%, p=0.019 by Fisher’s exact test). For patients surviving more than 180 days, there was no difference in TRM (16.7% versus 16.7%, p=0.987) or relapse rate (12.0% versus 7.3%, p=0.150). No difference in the rate of other causes of death was found between the groups at either the 180-day or 5-year time points.

Conclusions: Pre-transplant anemia is an independent risk factor for increased mortality following alloHSCT. Relapse and treatment-related deaths are both more likely to occur early in the post-transplant course of patients experiencing pre-transplant anemia. Differences in long-term survival are predominantly related to treatment-related deaths rather than relapse.
Should Stem Cell Transplantation (SCT) Be Recommended for Any Child with AML in 1st CR?
Authors: Gibson Brenda , Hann Ian , Webb David , De Graaf Siebold , Stevens (deceased) Richard , Hills Robert , Wheatley Keith
Published: October 2019

Between 1988 and 2002, 868 children (0–15 years) were entered into MRC AML 10 (1988–95, n=341) and AML 12 (1995–2002, n=527) trials. Children were allocated to one of three MRC risk groups: good risk - patients with t(8,21),inv(16),t(15,17) irrespective of bone marrow status after course 1 or the presence of other genetic abnormalities; standard risk - patients with neither favourable nor adverse cytogenetics and not more than 15% blasts in the bone marrow after course 1; poor risk - patients with more than 15% blasts in the bone marrow after course 1 or with adverse abnormalities of -5,-7, del(5q), abn(3q), complex (>/-5 abnormalities) and without favourable genetic abnormalities. Outcome from CR - death in CR (DCR), relapse risk (RR), disease-free survival (DFS) and survival from CR (OSCR) - was analysed by MRC risk group.

RISK GROUP AML 10 AML 12 Good Standard Poor Good Standard Poor DCR (8yr %) 9 13 11 7 5 10 RR (8yr %) 35 40 66 21 37 53 DFS (8yr %) 59 52 31 74 60 42 OSCR (4yr %) 81 60 39 88 75 49 OSCR (8yr %) 78 57 37 84 72 49

In AML 10 all patients were eligible for SCT with a histocompatible sibling donor, but unrelated donor transplantation was not part of the protocol. Because of their favourable outcome in AML 10, good risk children were not eligible for SCT in AML 12 and part way through the trial a similar approach was adopted for standard risk patients, whilst SCT continued to be recommended for poor risk patients with their inferior outcome. Both sibling and unrelated donor transplantation were permitted. In AML 10 and AML 12, 38 of 139 (27%) poor risk children underwent SCT - 17 sibling allografts, 11 unrelated donor allografts and 10 autografts. The procedural mortality was: 6%, 55% and 0% respectively. Mantel-Byar analysis (to account for time to SCT) comparing transplanted with non-transplanted poor risk children showed no evidence of reduction in relapse risk (HR 1.02, 95% CI 0.58–1.79, p=0.9), disease-free survival (HR 1.47, 95% CI 0.87–2.50, p=0.16) or survival benefit (HR 1.64, CI 0.94–2.85, p=0.08 against SCT), both overall or for any type of SCT. The survival at 8 years from SCT was 41% for sibling allografts, 18% for unrelated donor allografts and 60% for autografts.

OUTCOME BY TREATMENT - AML 10 & AML 12 Poor Risk (all) Poor Risk (censored at SCT) DCR (8yr %) 11 5 RR (8yr %) 58 55 DFS (8yr %) 37 43 OSCR (4yr %) 45 50 OSCR (8yr %) 44 50

Outcome is relative and, whilst poor risk children still do worse than good and standard risk patients, the outcome for poor risk children has improved. A survival at 8 years from CR of about 50% for poor risk children in AML12 (and no deaths beyond 4 years suggesting that most of those who survive are cured), raises questions as to whether any children with AML should be transplanted in 1st CR given the mortality and morbidity of the procedure. The high mortality associated with unrelated donor transplantation requires further investigation.
Comparison of Closure Times and Bleeding Times in the Evaluation of Patients Referred for Mild Bleeding Disorder Evaluations.
Authors: Zeigler Zella Rose , Hassett Andrea L. Cortese
Published: October 2019

The bleeding time has largely been replaced by closure times using a PFA-100 device for the evaluation of patients with suspected platelet function defects or von Willebrand’s disease. Initial studies showed concordance with both tests being abnormal in well defined thrombocytopathies (thrombasthenia, Bernard-Soulier syndrome, storage pool deficiency, and aspirin-like defects) and in von Willebrand’s disease. However recent studies have suggested that the closure time may be particularly insensitive in the more common platelet secretion disorders. This is a retrospective medical record review study to compare the results of closure times and bleeding times performed the same day in a consecutive series of patients evaluated for mild bleeding disorders. Incusion criteria were all patients referred to the PI at the Hemostasis and Thrombosis outpatient clinic at the Institute for Transfusion Medicine for such an evaluation, who had both closure and bleeding times performed on the same day. Exclusions were patients who did not have these tests performed on the same day. Platelet function testing included platelet aggregation in platelet rich plasma (epinephrine-11 micromolar, ADP 2.5, 5, 10 and 20 micromolar, collagen 2 mg/ml, high and low dose Ristocetin), adenine nucleotides.If these were normal, whole blood aggregation and ATP secretion were also assessed. Von Willebrand parameters (on at least three different dates) and von Willebrand multimers were measured. Thirty-two patients were evaluated and of these, two had normal tests. The remaining thirty patients (8M:20F) had a median age of 51 (range = 18 to 80). Seventeen pts (57%) were referred for abormal surgical bleeding, four (13%) were evaluated for bleeding (CNS in two, menorrhagia in two), one pt for excessive bruising and the remainder because of abnormal tests.Their diagnoses were platelet secretion defects (n=20), storage pool deficiency (n=5), aspirin-like defects (n=4) and von Willebrand’s disease type 1 (n=1). Bleeding times and closure times were both abnormal in only 3/30 pts (10%).CT coll/epi and CT coll/ADP results were abnormal in 5/30 (17%) and in 3/30 (10%) respectively. By contast, bleeding times were abnormal in 19/30 (63%). Only two/30 pts (7%) had isolated prolongation of the closure times. Of note, 9/30 patients (30%) had normal values in all three screening assay parameters. Using ROC analysis, the area under the curve was not significant for closure times with p values for CT:coll/epi of 0.387 and for CT:coll/ADP of 0.362. However, the p value for bleeding times was significant at <0.001. Using a cutoff bleeding time of greater or equal to 7.5 minutes, the sensitivity was 67% and specificity was 100% for this group of patients. We conclude that the closure time is not a good screening assay for patients with the more common thrombocytopathies. Unfortunately bleeding times also remain insensitive for screening for these disorders.
Phase II Study of CC-5013 (Revlimid) and Rituximab in Waldenström’s Macroglobulinemia: Preliminary Safety and Efficacy Results.
Authors: Treon Steven P. , Patterson Christopher J. , Hunter Zachary R. , Branagan Andrew R.
Published: October 2019

INTRODUCTION: In previous studies, we observed that CC-5013 enhanced rituximab mediated ADCC killing of lymphoplasmacytic cells (BJH2005; 128:192). As such, we conducted a phase II study of CC-5013 with rituximab in WM patients naïve to either agent. Intended therapy was as follows: Weeks 1-48 CC-5013 (25 mg po qD for 3 weeks, then 1 week off); Weeks 2–5, 13–16 Rituximab (375 mg/m2/week).

METHODS: Twelve patients were enrolled, 10 of whom were previously untreated with a median age of 65 (range 53–76 yrs), along with baseline BM involvement of 50 (range 5–90%), serum IgM of 4175 (range 1180–7130 mg/dL), hematocrit of 31.9% (range 24–36.6%), and B2M of 3.5 (range 1.8–6.0 mg/L).

RESULTS: Four patients were taken off study due to intolerance to therapy (3 to CC-5013, 1 to CC-5013 and Rituximab) and were non-evaluable. Unexpected acute decreases in hematocrit occurred in 10/12 (85%) patients within the first 2 weeks of therapy, resulting in hospitalization for 4 patients. The median decrease in hematocrit, which coincided with CC-5013 administration was 4.2% (range 1.7-7.5%), and was not associated with blood loss, hemolysis, or generalized myelosuppression. Other grade 3/4 toxicities attributed to CC-5013 therapy were expected and included neutropenia (n=4); tinnitus (n=2); thrombocytopenia (1); myositis (n=1); and rash (n=1). Five patients had a spike in serum IgM levels following rituximab, accompanied with epistaxis (n=1), headaches and blurry vision (n=2) requiring plasmapheresis. Toxicities led to the discontinuation and dose reduction of CC-5013 for 8 and 4 patients, respectively, and discontinuation of rituximab in 1 patient due to recurring allergic reactions. Discontinuation of CC-5013 within the first 28 days of therapy occurred in 6 out of 8 patients. Responses among the 8 evaluable patients: PR (n=3); MR (n=4); SD (n=1); ORR 88% among evaluable patients. In two patients with bulky adenopathy and splenomegaly, a significant reduction in the node/spleen size was noted by week 12. Both of these pts have subsequently achieved a PR. With a median follow-up of 7+ months, no patient who received at least 2 months of CC-5013 have progressed.

CONCLUSION: The response data achieved with CC-5013 and rituximab in this study is encouraging, though the dose and schedule of CC-5013 administration, and mechanism for the unexpected acute drops in hematocrit observed in WM patients warrants further investigation. Such studies are in progress in our laboratory.
IL-7 Effects on Thymocyte Progenitors.
Authors: Kassar Nahed El , Choudhury Baishakhi , Flomerfelt Francis , Lucas Philip J. , Kapoor Veena , Telford William , Bare Catherine V. , Mahmud Semhar , Bhandoola Avinash , Gress Ronald E.
Published: October 2019

IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.
Phase I/II, Nine Month Study Results of Enzyme Replacement Therapy with Gene Activated Human Glucocerebrosidase (GA-GCB) in Patients with Type I Gaucher Disease.
Authors: Zimran Ari , Fratazzi Candida , Mensah Robert , Elstein Deborah
Published: October 2019

AIMS: Evaluation of safety and clinical activity of Gene-Activated ® human glucocerebrosidase (GA-GCB) as a new intravenous enzyme replacement therapy (ERT) for patients with type I Gaucher disease participating in a 9 month, open-label Phase I/II clinical study.

BACKGROUND: GA-GCB is human glucocerebrosidase (GCB) produced in a well-characterized, continuous human cell line using proprietary gene activation technology. GA-GCB has an identical amino acid sequence to the naturally occurring human enzyme and contains terminal mannose residues that target the enzyme to macrophages, the primary target cells in Gaucher disease. Nine month study results of GA-GCB treatment will be presented.

METHODS: Twelve adult patients (7 females /5 males) with type I Gaucher disease with clinically significant anemia, thrombocytopenia, hepatomegaly and/or splenomegaly, were enrolled in a 9 month Phase I/II clinical trial. Patients received GA-GCB every other week for a total of 40 weeks (20 infusions). A staggered dose escalation of GA-GCB was performed in the first 3 patients: these patients received 15 U/kg at the first infusion, 30 U/kg at the second infusion, and then 60 U/kg IV every other week for a total of 20 IV infusions. The remaining 9 patients received 60 U/kg beginning with their first dose. Anti-GA-GCB antibodies were tested in all patients during the 9 months of treatment. All patients were routinely evaluated for hemoglobin, platelets, liver and spleen volumes, infusion reactions and adverse events. In addition, serum samples were analyzed for disease biomarkers: chitotriosidase and CCL18.

RESULTS: Eleven of the 12 patients who were enrolled in the trial have been treated for nine months. One patient withdrew consent after three injections for reasons not related to treatment. Dose escalation from 15 U/kg to 60 U/kg was well tolerated in the first three patients. The remaining patients received 60 U/kg every other week throughout the course of the study. Anti-GA-GCB antibody test results were negative for all patients. Mean hemoglobin and platelet values, below the normal range at baseline, improved after 9 months of GA-GCB treatment to levels consistent with the therapeutic goals for ERT in Gaucher disease (Seminars in Hematology 2004). Analysis of the liver and spleen volumes decreased following 9 months of treatment. In addition, serum chitotriosidase and CCL18 levels decreased over the 9 months of GA-GCB treatment.

CONCLUSION: ERT with GA-GCB was well tolerated and demonstrated clinical activity in well-established clinical markers in patients with type 1 Gaucher disease when administered IV every other week at 60 U/kg over 9 months.

Results suggest that GA-GCB holds promise as a new therapeutic option for ERT in Gaucher disease.
Helicobater Pylori Infection and Clinical Response to Eradication Therapy in Patients with Immune Thrombocytopenic Purpura (ITP).
Authors: Fabris Fabrizio , Allemand Emanuele , Scandellari Raffaella , Vettore Silvia , Randi Maria Luigia , Luzzatto Guido
Published: October 2019

We performed a prospective study in order to investigate the controversial association between HP infection and immune thrombocytopenic purpura (ITP). We studied the prevalence of HP infection and the efficacy of its eradication in 57 consecutive adult patients admitted to our ward for ITP. Thirty-three patients were females and 24 were males (median age 58 years; mean platelet count 68±42 x109/L). HP infection was demonstrated by the stool antigen test in 28 patients (49.1%). Such prevalence increased with the age. No statistical difference by gender, platelet count, disease duration, presence of specific platelet autoantibodies (MACE) and therapy regimens was observed between HP-positive and negative patients. On the other HP positive patients were significantly older than HP negative ones (62 versus 54 years, p<0,03) and presented more gastroenteric symptoms (32% versus 7%, p<0,05). Thirteen patients with chronic ITP were given HP eradication since their platelet count was above 20x109/L and they do not need for starting or modifying therapy of ITP for at least 6 months. HP eradication was performed by standard triple therapy based on omeprazole plus clarithromycin and amoxicillin for 1 week. The bacteria was eradicated in all cases. Twelve HP negative ITP patients were followed as controls. The mean platelet count was significantly increased in ITP patients after 3 and 6 months from HP eradication. However, considering as end-point, a platelet count increase of more than 50% over the basal value, the platelet response to the HP eradication appears to be better in eradicated patients only after 3 months of follow-up (83% of positive response versus 57%). We also studied in HP-positive patients the presence of antibodies against CagA citotoxic protein by ELISA method. Seventeen of the patients have anti-CagA antibodies (56%) but the positivity did not correlate with platelet response to the eradication. In conclusion the prevalence of HP infection in our patients with ITP was comparable to that observed in the adult population from the same geographic area. Infection is not associated with clinical or laboratory distinctive features with the exception of tan older age and gastroenteric symptoms. The eradication of the HP infection seems to improve mean platelet count in ITP patients but larger prospective studies are required to validate this approach.

Platelet response to the HP eradication in HP positive ITP patients and in HP negative controls Platelets (x 109/L, mean ± SD) basal time 1 month 3 months 6 months 12 months * p<0.05 vs basal platelet count ITP Patients 13 HP+ eradicated 74±43 141±142 129±73* 104±54* 160±117 12 HP- controls (not eradicated) 62±35 76±33 73±40 78±51 76±48
FcRγ-Dependent Facilitating Cells Are Direct Inducers of Regulatory T Cells.
Authors: Taylor Kendra N. , Patil Vivek R. Shinde , Chittenden Meredith , Colson Yolonda L.
Published: October 2019

Facilitating cells (FC) are CD8+/αβγδTCR− bone marrow-derived cells that promote allogeneic stem cell (SC) engraftment without graft vs. host disease (GVHD) and induce donor-specific transplantation tolerance. However, the mechanism of FC-mediated allogeneic SC engraftment is not known. We have previously demonstrated that allogeneic SC engraftment promoted by FC result in no clinical evidence of GVHD and is associated with increased number of CD4+25+ regulatory T cells post transplant compared to recipients of bone marrow T cells that develop severe GVHD. This is consistent with the hypothesis that FC facilitate allogeneic SC engraftment and induce tolerance through the induction of a regulatory T cell network. Here we report that CpG activation of toll-like receptor 9 (TLR9) on FC induce CD4+25− naïve T cell differentiation into CD4+25+ regulatory T cells. CpG stimulated and unstimulated CD8+αβγδTCR− FC isolated from bone marrow of C57/BL6 mice by flow cytometric cell sorting FC were co-cultured with splenic CD4+25− T cells for 5 days. CpG stimulated FC co-culture gave rise to CD4+25+ T cells as determined by mRNA expression of the CD4+25+ regulatory T cell marker, FoxP3. In contrast, unstimulated FC in co-culture did not induce regulatory T cells. Because FcRγ is the dominant ITAM receptor (immunoreceptor tyrosine-based activating motif), on FC and given that in vivo studies have demonstrated a requirement for FcRγ expression on FC, we hypothesized that FcRγ gene expression is increased within activated FC. FcRγ gene expression in CpG stimulated and unstimulated FC were compared by real-time PCR analysis. CpG-mediated TLR signaling within FC result in increased gene expression of FcRγ. Taken together, these studies demonstrate that CpG stimulated FC induce the generation of CD4+25+ regulatory T cells in vitro and CpG activation results in increased gene expression of FcRγ, suggesting a requirement for FcRγ signaling in FC-mediated induction of regulatory T cells. These findings provide the first mechanistic evidence that FC are direct inducers of regulatory T cells. Further characterization of cooperative TLR and FcRγ signaling pathways in FC will be critical to defining the mechanism of FC-mediated SC engraftment and the identification of potential therapeutic targets for the clinical induction of tolerance in the future.
Establishment and Characterization of a Human Acute Myelocytic Leukemic Cell Line, SH-2, Carrying t(16;17)(q24;q12).
Authors: Qiu Hui Y. , Xue Yong Q. , Pan Jin L. , Wu Ya F. , Zhang Jun , Wang Yong , Shen Juan , Dai Ai P.
Published: October 2019

We present a novel human myeloid leukemic cell line, designated as SH-2 which was established from the bone marrow of a patient with acute myelocytic leukemia (AML-M2a) carrying t(16;17)(q24;q12) translocation. The cell line has proliferated continuously in vitro for more than 12 months. Its morphology showed typical features of acute myelocytic leukemia (AML). The cell line’s immunoprofile was accordant with AML (positivity for CD13, CD33, CD38, CD117, CD16, CD56 and MPO). Karyotypic analysis revealed the translocation t(16;17)(q24;q12), monosomy 17 and trisomy 19. The apoptosis related genes such as bcl-2, Fas and GST-πtranscription were detected by RT-PCR. Meanwhile, MDR1, MRP and LRP transcription were not detected by RT-PCR. The deletion of p53 gene and the translocation between chromosomes 16 and 17 were confirmed by FISH method. The SH-2 cells grew colonies in in vitro methylcellulose cultures. Tumor masses were found in 1/2 mice injected by the tail vein with the SH-2 cell line after two months. Infection of the EBV and the mycoplasma were also excluded. Cell line authentication by STR showed that the primary leukemia cell of the patient and the SH-2 cell line originated from same individual. SH-2 cells were proliferated by the addition of cytokines such as IL-3, GM-CSF and SCF. two point mutations in exon 5 of the p53 gene were detected in the SH-2 cells by PCR analysis and direct sequencing showing the conversion of T to G in both codon 349 and 417. The establishment of an myelocytic leukemia cell line with t(16;17)(q24;q12) could be valuable for the study of leukemogenesis and for the research of cloning the new gene involved in the t(16;17)(q24;q12) translocation.
Relative Effectiveness of Phenylhydrazine Treatment and Hemorrhage in the Production of an Erythropoietic Factor
Authors: JACOBSEN ELLY M. , DAVIS A. K. , ALPEN EDWARD L.
Published: October 2019

Active plasma extract has been prepared from rabbits made anemic by phenylhydrazine treatment, but in every case where rabbits were made equally anemic by bleeding the only peripheral evidence of erythropoietic activity found after injection of plasma filtrate was the inducement of a moderate reticulocytosis in a heterologous recipient.

In rabbits bled on successive days until their hematocrits were equal to paired animals made anemic by phenylhydrazine injection, there is, nevertheless, a lower oxygen carrying capacity in the phenylhydrazine injected animals. The latter have methemoglobin concenstrations amounting to approximately 24 per cent of the total blood pigment while in the bled rabbits the methemoglobin level is practically zero.

Plasma extracts from phenylhydrazine treated rabbits with apparently normal or very slightly damaged livers produce factor comparable in potency to that obtained from bled animals.

The most highly active preparations are obtained in phenylhydrazine treated rabbits which have severely damaged livers.

The proposed hypothesis is that the factor is produced in quantity only when the effective hematocrit is below 12 to 15 per cent and when the liver is not capable of rapid active destruction of the factor or is not producing an otherwise normally occurring inhibitor.

The factor obtained from phenylhydrazine treated rabbits with liver damage is markedly active as shown by significant increase in blood volume and red cell mass, an elevated red blood cell count and hematocrit, and an intense reticulocytosis.
Anti-A Agglutinins in Pooled Plasma as a Cause of Hemolytic Anemia
Authors: RUTZKY JULIUS , COHEN FLOSSIE , ZUELZER WOLF W.
Published: October 2019

Five sets of observations are presented showing the development of hemolytic anemia in group A patients as the result of prolonged administration of relatively large amounts of pooled plasma. During the hemolytic episodes, spherocytosis, increase in osmotic fragility, rising bilirubin levels and sometimes clinical jaundice and splenomegaly were observed. Antibody attached to the recipients’ erythrocytes was demonstrated by means of the direct Coombs test, and in several instances free antibody of anti-A specificity was found in the recipient’s plasma during the hemolytic episode.

Random testing of commercial pooled plasma revealed a number of instances of astonishingly high anti-A titers, indicating that neither dilution in the pool nor neutralization by natural A substance in the component A plasmas can be relied upon.

The pools implicated in the production of hemolytic reactions had anti-A1 titers of 1:64 in saline or higher, and with the usual technics showed no clear-cut "immune" characteristics except in one instance. The administration of substantial amounts of plasma pools of low titer (1:16) was not followed by adverse reactions in several instances. On the other hand, the administration of originally high titered plasmas, neutralized in vitro to a saline titer of 1:16 resulted in blood destruction, indicating that under conditions of prolonged massive administration, specific soluble substance, though effective in vitro, is inadequate in vivo. Whether the adverse effects of the moderately high and high titered plasma pools were the result of purely quantitative factors connected with the introduction of large amounts of natural anti-A agglutinins or whether special immune isoantibodies not detected by the methods used were responsible could not be determined by the data at hand.

Prolonged administration of untitered pooled plasma to recipients of blood groups A and probably B is potentially dangerous.
Studies on the Specificity of Autoantibodies in Acquired Hemolytic Anemia
Authors: CROWLEY LEONARD V. , BOURONCLE BERTHA A.
Published: October 2019

A significant proportion of patients with autoimmune hemolytic anemia form autoantibodies of blood group specificity, rather than nonspecific autoantibodies.

Ashby survival studies suggest that in such cases properly selected blood, lacking the antigens against which autoantibody has been formed, can be transfused successfully.

These concepts are important not only for a better understanding of the nature of autoantibodies, but also can be applied to the practical management of patients with autoimmune hemolytic anemia.
Dose Attenuation of R-CHOP for the Treatment of Elderly Patients with Diffuse Large B Cell Lymphoma.
Authors: Choi Young Jin , Lee Kyung Woo , Shin Ho-Jin , Chung Jooseop , Cho Goon-Jae
Published: October 2019

Backgrounds: More than half of the patients with newly diagnosed aggressive lymphomas are older than 60 years. The addition of rituximab to the conventional CHOP regimen, administered every 21 days, has conclusively demonstrated to lead to a significant improvement of the outcome in elderly patients with DLBCL and surivival benefit. The aim of this study was to evaluate the efficacy and toxicity of dose attenuated CHOP with rituximab 3 weekly in untreated elderly DLBCL.

Methods: CHOP regimen consisted of cyclophosphamide(600mg/m2), doxorubicin(30mg/m2), vincristine (1mg/m2) on day 1, and prednisolone (20mg bid) given on day 1 to 5 every 3 weeks. Rituximab (375mg/m2) was administered the same day as CHOP.

Results: This study included 37 patients with DLBCL with a median age of 70 years (range:64–81). Seventy six percent had an international prognostic index score>1, 35% had stage IV disease. Toxicity was calculated over 192 cycles administered. Grade 4 neutropenia developed in 2.6% of cycles and no grade 4 non-hematologic toxicity didn’t developed except asthenia (1 person). The overall response rate was 83.3% (complete response rate: 72.2%). 2 year overall and event free survival rates were 79.8% and 67%, respectively.

Conclusions: Dose attenuated R-CHOP is effective and tolerable for elderly patients with DLBCL.
No Significant Expression of Wt1 Gene in Multiple Myeloma Patients at Diagnosis: Is Wt1 Gene Expression Useful Marker for Minimal Residual Disease in Multiple Myeloma?.
Authors: Ozkul Yusuf , Caglayan Ahmet O. , Kocyigit Ismail , Saatci Cetin , Akalin Hilal , Demir Muzaffer , Altuntas Fevzi , Cetin Mustafa , Eser Bulent , Kaynar Leylagul
Published: October 2019

Monitoring of multiple myeloma patients during and after treatment for the presence of remaining myeloma cells (minimal residual disease, MRD) has been shown to give major insight into the effectiveness of treatment. However, so far applicabile genetic markers amenable to sensitive detection by PCR for MRD in myeloma patients. Previouse studies showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease in leukemia and myelodysplastic syndrome. In this study was to measure, by real-time polymerase chain reaction, levels of WT1 expression, in order to find a possible association between this gene and the minimall residual disease. We investigated peripheral blood WT1 expression level in 41 newly diagnosed multiple myeloma cases using quantitative reverse transcriptase polymerase chain reaction. All patients carried normal WT1-mRNA levels. WT1 expression analyse may be not useful genetic marker for monitoring MRD in routine clinical use of multiple myeloma patients.
IL-6 and IGF-1 Act in Synergy with HGF in Myeloma Cells by Modulating the Ras-MAPK Pathway.
Authors: Hov Håkon , Tian Erming , Waage Anders , Børset Magne , Sundan Anders
Published: October 2019

Multiple myeloma is an incurable malignancy of plasma cells homing to the bone marrow. Myeloma cells are dependent on factors in their microenvironment for survival and expansion. HGF may be produced both by myeloma cells and the bone marrow microenvironment and serum levels of HGF are known to be a prognostic factor in multiple myeloma. Both IL-6 and IGF-1 are known growth factors for myeloma cells. In the human myeloma cell line (HMCL) INA-6, HGF alone had low effect, but together with IL-6 and IGF-1 it became a potent growth factor increasing thymidin incorporation two- to three-fold above the levels obtained with IL-6 or IGF-1 alone. Similar results were obtained for the myeloma cell line OH-2. The ANBL-6 cell line harbours an autocrine growth promoting HGF-loop. When inhibiting this autocrine HGF loop with a specific c-Met receptor tyrosine kinase inhibitor (PHA-665752), IL-6- and IGF-1-induced proliferation was reduced by 80% and 50% respectively. Thus, in the prescence of HGF, both IL-6 and IGF-1 are dependent on the HGF-receptor c-Met for full effect on cell proliferation. There seems to be two interconnected explanations for the synergy between HGF- and either IL-6- or IGF-1-signalling in myeloma cells. IL-6 and IGF-1 treatment increased the expression of c-Met in INA-6 cells Secondly, we found that HGF was unique among the three growth factors in activating both Ras and p44/42 MAPK in INA-6 cells. Also in ANBL-6 cells, IGF and IL-6 was dependent on functional c-Met signalling to fully activate this pathway. Thus, the reason for synergy between HGF and IL-6 or IGF-1 seemed to be that full activation of the Ras-Mapk pathway through Gab1 and SHP-2 by these cytokines was dependent on operating c-Met signalling. Taken together, HGF and c-Met signalling would be attractive targets for therapy of multiple myeloma.
Recombinant Human Erythropoietin May Negatively Influence Survival in Newly Diagnosed Patients with Multiple Myeloma: A Single Center Experience in 246 Patients.
Authors: Katodritou Eirini , Verrou Evgenia , Banti Anastasia , Gastari Vassiliki , Mihou Dimitra , Hadjiaggelidou Christina , Markala Dimitra , Lazaridou Anna , Terpos Evangelos , Zervas Konstantinos
Published: October 2019

Anemia is a common clinical problem in patients with multiple myeloma (MM), which adversely affects their quality of life. The administration of recombinant human erythropoietin (r-HuEPO) improves anemia in about 2/3 of patients. However, it has not been clarified if r-HuEPO has any beneficial or adverse impact on the overall survival, particularly in a clear population of MM patients. The aim of this study was to examine the impact of r-HuEPO administration on the overall survival of newly diagnosed patients with MM. Two hundred forty-six newly diagnosed symptomatic MM patients, 139 males and 107 females, with a median age of 67 years (range 29–90) were studied. R-HuEPO was administered according to standard criteria, when Hb levels were less than 10.5g/dl and it was titrated and discontinued when Hb level reached 13g/dl. The parameters evaluated for predicting survival were: Age, sex, Hb, platelets, bone marrow infiltration, serum creatinine, serum ferritin, ISS score, B2-microglobulin and r-HuEPO administration. Cox regression was used for the univariate and multivariate analysis. One hundred forty-two patients received r-HuEPO and 105 did not. The median duration of r-HuEPO administration was 6 weeks (range 4–10) and the median hemoglobin level of patients who received r-huEPO, was 9.2g/dl (range 7.3–10.5 g/dl). The median follow up was 31 months (range 1–231). The univariate analysis showed that, age, Hb, platelets, serum creatinine, serum ferritin, ISS score, B2-microglobulin and r-HuEPO administration predicted for survival (p<0.05). The multivariate analysis demonstrated that, age, ISS score and r-HuEPO administration were statistically significant for predicting overall survival (p<0.05). The median survival of patients in the r-HuEPO group was 22 months (SD 22.7mo) whereas in the group without r-HuEPO administration it was 40 months (SD 35.8mo) (p=0.02). These results, suggest that r-HuEPO administration may negatively influence overall survival in newly diagnosed patients with MM and therefore, within this context, it should be used with caution. The large number of exclusively MM patients with a long follow up included in this study, highlights the importance of these results.