bluuebunny/crossref_metadata_2025 huggingface dataset:
Febrile Neutropenia Following Autologous Stem Cell Transplantation in Patients with Lymphoma
Authors: Vrhovac Radovan , Peric Zinaida , Kalac Matko , Ostojic Alen , Kovacevic Visnja , Bukovski-Simonoski Suzana , Tambic-Andrasevic Arjana , Minigo Hrvoje , Jaksic Branimir
Published: April 2020

Background: Infections remain a major cause of morbidity and mortality in patients with lymphoma treated with autologous hematopoietic stem cell transplantation. Knowledge of local microbiological epidemiology and patients’ risk factors are essential for optimal management of these infections.

Aims: to determine incidence of febrile neutropenia (FN) in patients with lymphoma treated with autologous peripheral blood stem cell transplantation (PBSCT) and to investigate correlations of a number of variables available on the day of FN onset with the severity of infection.

Patients and Methods: 102 consecutive patients (median age 44, range 19–68 yrs, 58M/44F) with relapsed or refractory Non-Hodgkin’s Lymphoma (NHL, n=78) and Hodgkin’s Disease (HD, n=24) treated with PBSCT in a single center have been evaluated for infectious complications following transplantation.

Results: Febrile neutropenia occurred in 67 (65.7%) patients at a mean of 6 days after transplantation (range 1–9, SD 1.71). Microbiological work-up was done and empirical antibiotic therapy with piperacillin-tazobactam was initiated in all but 15 patients who received a carbapenem. Empirical therapy was modified in 31 (46.3%) patients. Vancomycin was added in 22 patients (32.8%), a sistemic antifungal in 4 (6.0%) and both in 6 (9.0%) patients. Three patiens died as a consequence of sepsis (TRM=2.9%). Twenty two patients (32.8%) had proven bacteremias while 27 (40.3%) had other microbiologically documented infections (MDIs). Gram+ microorganisms were responsible for the majority of all documented infections (63.6% of bacteremias and 54.5% of other MDIs). Correlations of a number of variables available at time of FN onset with the duration of febrile episode have been investigated. Patients with higher CRP levels at FN onset and those with earlier onset of fever following PBSCT had significantly longer (p=0.047 and p=0.0066, respectively) duration of fever. Other variables, including age, No. of previous lines of therapy, No. of stem cells reinfused, ANC and monocyte counts as well as peak temperature values at day of fever onset did not show statistically significant correlations with duration of fever. All patients with diabetes developed FN and, although not reaching statistical significance (p=0.09), its duration was longer in comparison to other patients.

Conclusions: Infections are serious but manageable complications of PBSCT. Gram+ microorganisms remain the major cause of documented infections. Early empirical antimicrobial therapy, tailored according to local microbiological epidemiology is essential for their optimal treatment.
Reduced-Intensity Conditioning Regimen in Allogeneic Haematopoietic Stem Cell Transplantation for Follicular Lymphoma and Chronic Lymphocytic Leukemia
Authors: Vaz Carlos Pinho , Ferreira Rosa Branca , Silva Joao , Barbosa Isabel , Roncon Susana , Carvalhais Alzira , Martinho A. , Campilho Fernando , Campos Antonio
Published: April 2020

Reduced-intensity conditioning (RIC) regimens is being increasingly used for allogeneic haematopoietic stem cell transplantation (HSCT) in recent years for follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). The lower risk of transplant related toxicity associated with RIC regimens makes allogeneic transplantation applicable to patients (pt) with haematological malignancies resistant to conventional therapy and/or in poor medical condition, while preserving the putative graft-versus-tumour effect (GVT). From May 99 to January 07, 31 recipients were studied. Twenty two pt (71%) with FL: 14pt CR2; 4pt PR; 2pt resistant relapse; 1pt graft failure; 1pt advanced disease and 9pt with CLL in PR, underwent to allogeneic HSCT from HLA-matched (31) sibling donors, after treatment with fludarabine 30 mg/m2/d iv × 5 + busulfan 4 mg/Kg/d × 2 ± anti-T lymphocyte globuline (10 mgKg/d) × 4 or fludarabine 30 mg/m2/d iv × 5 + cyclophosphamide (1g/m2/d) × 3 + alentuzumab 20mg/d × 5 (pt with graft failure). Stem cell source was unmanipulated peripheral blood progenitor cells. Median number of CD34+ cells infused: 6,0 × 106/Kg (3,3–12,0). CsA 3 mg/kg/d was given from day-1 until day +90 then tapered progressively, associated with Micophenolate mofetil d0 until d+84.

Results: In 31 evaluable pt hematological recovery have a median time to neutrophils ≥0.5×109/L of 13d. (3–37+) and platelets ≥20 × 109/L of 11 d. (9–105+). Transfusion requirements: median: 4 RBC units and 2 platelet transfusions per pt. Median day of discharge: d.15. Hepatic veno-occlusive disease and severe mucositis did not occur. Chimerism analysis (day +28) showed donor engraftment in all pts–mixed in 5 and full in 26 (full in graft failure pt after second transplant). 36±9,2% pts developed clinical grade 2–4 aGvHD and 68,9±10,8% developed cGvHD. Four pt had relapsed/progressive disease. Non relapse mortality occurred in 3 pts with low performance status and chemotherapy resistant disease. At 3,6 years median overall survival (OS) is 85.2±6.9%. Twenty eight pts are alive with a median follow-up of 3.6 years. Progression free survival (PFS) at 3 years is 84,1±7,1 %. Aimed to induce GvT effect, one pt with progressive disease, without GvHD was assigned to receive donor lymphocyte infusions (DLI) at regular intervals achieved stable disease. These results show that RIC regimen are well tolerated, have a low risk of transplant related mortality and are able to ensure a sustained engraftment. RIC is becoming the standard approach in allogeneic HSCT for FL/CLL and the increased risk of late disease progression might be manipulated with GvT effect associated with posterior DLI.
Should All Patients with Acute Myelogenous Leukemia (AML) Other Than Good Risk Cytogenetics Receive Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) in First Remission?
Authors: Alpdogan Onder , Lenox Raymond , Barile Amy , Foss Francine , Shlomchik Warren , Reuben Christy , Cooper Dennis , Seropian Stuart
Published: April 2020

There has been little improvement in the standard therapy of AML in the last two decades. Allogeneic HSCT remains the most effective curative therapy for patients with AML; however, risks associated with this therapy have limited its use for patients of advanced age or requiring alternate donors. Improvements in supportive care, donor selection and conditioning protocols have decreased the risk of transplant related mortality and allogeneic HSCT now appears as a feasible option for elderly patients. We reviewed the outcome of 99 patients with AML and MDS who received allogeneic HSCT in our center from 6/98 to 12/07. 85 patients were diagnosed with AML and 14 patients had MDS. 25 patients with AML (29%) had secondary AML including chemotherapy-related (6 patients) and progression from MDS or myeloproliferative disease (19 patients). Median age was 51 (20–72). Patients received allografts from related donors (n=56) and unrelated donors (URD) (n=43). All patients who received URD in first remission had poor prognostic features. 56 patients received myeloablative conditioning protocols (MA) whereas 43 patients were transplanted with reduced intensity protocols (RI) primarily due to age >55. Overall probabilities of 4-year and 8-year survival are 51% and 41% respectively. The median follow-up is 30 months for surviving patients. RI-group (Median age: 62 (37–72) had a similar 4-year survival with MA-group (50% and 54% respectively). 4-Year probability of survival in elderly patients (age>60) after RI-HSCT (n=27) is 48%. Patients who received an URD allograft and RI conditioning (n=21) had a similar survival to those receiving MA conditioning and URD graft (n=22) (4-year survival 52% and 54% respectively). As expected, AML patients in first complete remission (CR1) had better survival than patients who were in second or higher complete remission (CR2) (4-year survival; 53% vs 29%). We conclude that HSCT for AML and MDS from related or unrelated donors, with age and comorbidity-appropriate conditioning, results in a favorable outcome compared to historical experiences with standard therapy. Allogeneic HSCT should be tested in first remission for all patients with AML excluding younger patients with good risk features.
Long Term Survival in Refractory Leukemia Patient Treated with Related HLA Haploidentical Stem Cell Transplantation
Authors: Wu Bingyi , Kunyuan Guo , Wu Jing
Published: April 2020

Objective: To assess the long term survival of patients with refractory leukemia treated by HLA haploidentical stem cell transplantation.

Methods To analysis the outcomes of 48 cases patients with refractory leukemia underwent HLA haploidentical stem cell transplantations from August, 1998 to May, 2008. The median age was 16 years old (7–52 years old). Patients received stem cells from their parants, daughter, son, and sibling donors. Twelve patients received three HLA locus mismatched stem cells and twenty patients received two HLA locus mismatched donors stem cells. Sixteen patients were grafted one HLA mismatched donors stem cells. The conditioning regime consisted of fludara (25mg/m2 × 5d), busulfan (4mg/kg × 4d) and cyclophosphamide (60mg/kg × 2d). Median dose of rabbit anti-human lymphocyte globulin (5mg/kg × 5d) was added. CSA combined with short course of MTX were used for prophylaxis GVHD. A median dose of 6.0 × 108/kg(3–9 ×108/kg) mono-nucleated cells was grafted. The mean CD34+ cells number was 5.5 × 106/kg (3–6.5 × 106/kg)

Results Forty-seven patients were successfully to be engraftment and one failed to be engraftment. The median time of white cells and platelet reconstitution was 14 days (11–20 days) and 18 days (14–20) respectively. Severe acute graft versus host disease occurred in seven patients, and six died. Seven patients suffered from intensive chronic graft versus host disease and four died with fungus infection. Seven patients relapsed and died. The median relapse time was 6 months (3 months to 24 months). Three patients died from severe diarrhea with CMV infection. Four patients died from intensive chronic graft versus host disease. Twenty patients are still survival and disease free with high karnofsky performance scores. The disease free survival is 45 percent as follow up 3 years (1 to 7 years).

Conclusion: HLA haploidentical peripheral blood stem cell transplantation may be an effect therapy to refractory leukemia. And some refractory leukemia patients could benefit from HLA haploidentical peripheral blood stem cell transplantation.
Autologous Stem Cell Transplantation (ASCT) with Busulfan Plus Cyclophosfamide as Conditioning Regimen for Acute Myeloid Leucemia: Retrospective Experience (1991–2007) of Two Bone Marrow Transplant Unit
Authors: Irrera Giuseppe , Messina Giuseppe , Martino Massimo , Console Giuseppe , Milone Giuseppe , Iacopino Pasquale
Published: April 2020

We report our results of ASCT in patients with Acute Myeloid Leukemia (AML) during the last 16 years. Between December 1991 and december 2007, 90 patients with AML received an ASCT. The main characteristics were reported on table 1. The median patient age was 46 years (range17 – 67 years). The conditioning regimen employed for all patients was Busulphan + Cyclophosfamide. The Overall Survival (OS) (Figure1) was 53,5% with a median follow up of 91,6 months. The majority of patients (81) was transplanted in first complete remission (1st CR), 8 in 2nd CR and 1 >2nd CR. The OS considering the disease phase at transplant was different: 55,7% vs 16,7%, p < 0,01 (one pts with > second CR was excluded from analysis). If we considering also the age stratified in two groups: 17–45 vs 46–67 years, all patients in second CR, included into II group died. We have calculated OS stratified for age in these groups that was 46,2% versus 59,6%, respectively, without statistical differences. We also analyzed the OS distributed for sex and cell source without statistical difference. We have documented a statistically significant correlation between FAB group and survival (Figure 2). In fact, the patients with FAB M2 and M4 (excluded M3) had a superior OS than those with other FAB (60,7% vs 40%, p < 0.019)). In 32/40 (80%) patients, the relapse has been documented within 24 months from transplant. The analysis for cytogenetic risk has been performed, but considering only 46 patients assessable. The OS cytogenetic risk-related was 87% for 8 patients with Low Risk, 47% for 36 patients with Intermediate Risk, 2 patients with High Risk died within 11 months from transplant (data not shown). We conclude that autologous bone marrow transplantation is an effective treatment in AML with the possibility of long survivorship, particularly in patients with FAB M2 and M4. In our experience, first complete remission of disease at transplant play an important role and correlates with the longest survival. The analysis of cytogenetic risk reflects the impact of karyotype on OS. Transplant Related Mortality (TRM) has been documented in 6/90 patients, all died before the years 2000.

Figure 1 Figure 1.

Table 1

Number of patientss 90 Sex (M/F) 42/48 Age (range y) (mean ±SD y) 17–67 46,3±11,4 FAB classification: M0 2 (2,2%) M1 16 (17,8%) M2 28 (31,1%) M3 4 (4,4%) M4 28 (31,1%) M5 11(12,3%) M6 Phase of disease: 1 (1,1%) 1st Complete Remission 81 (90%) 2nd Complete remission 8 (8,9%) > 2nd Complete Remission 1 (1,1%) Citogenetic risk (pts evaluable: 46) Low 8 (17,3%) Intermediate 36 (78,2%) High 2 (4,5%) Transplant Related Mortality 6/90 (6,7%)
MicroRNA Expression in Childhood Acute Lymphoblastic Leukemia (ALL)
Authors: Yang Yung-Li , Yu Sung-Liang , Chen Hsuan-Yu , Yang Pan-Chyr , Chen Jiann-Shiuh , Lin Dong-Tsamn , Lin Shu-Rung , Jou Shiann-Tarng , Yen Ching-Tzu , Lin Kai-Hsin , Hu Chung-Yi , Chang Sheng-Kai , Chiou Rong-Jing , Lu Meng-Yao , Chang Hsiu-Hao , Lin Shu-Wha
Published: April 2020

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are regulators of gene expression. A role for microRNAs in leukemia, such as chronic lymphoblastic leukemia, acute myelogeneous leukemia has recently been recognized. However, little is known about the role of microRNAs in childhood acute lymphoblastic leukemia (ALL). To determine whether miRNAs are associated with the clinical features of childhood ALL and its association with cytogenetic abnormalities, we analyzed 60 untreated childhood ALL cases for their miRNA expression using a microarray platform. Leukemic samples were collected from the ALL children between 1–18 years of age. Of the 365 miRNA analyzed with a training group of 40 patients, a miRNA signature was derived that was associated with event-free survival. The signature was tested in a validation group of 20 patients. For the latter, a miRNA compound covariate predictor (i.e., a miRNA risk score) was computed on the basis of weighted levels of the miRNAs forming the outcome signature. The signature identified from the training group contained 5 miRNA highly associated with event-free survival (P<0.05). The summary value of the signature miRNA was also highly associated with event-free survival (P <0.05) in the validation group. After adjustment for the gender, age, initial white counts, immunophenotypes, and cytogenetics, the miRNA risk score remained associated with event-free survival (P <0.05) in multivariable analysis. In conclusion, we have identified a miRNA signature of 5 miRNAs which is highly associated with the clinical outcome, cytogenetic and immunophenotypic subtypes of childhood ALL.
Mir-15a and Mir-16-1 Induce Apoptosis of Raji Cell Line
Authors: Qin Chen , Dongmei He
Published: April 2020

MicroRNAs are endogenous small noncoding RNAs that regulate gene expression negatively at posttranscriptional level. Mir-15a and mir-16-1 are deleted or downregulated in the majority of chronic lymphomatic leukemia(CLL), which is the most common human leukemia and overexpresses the antiapoptotic B cell lymphoma2 (Bcl-2) protein. Here we investigated the effect of mir-15a and mir-16-1 on human lymhpomatic cell line Raji in induction of apoptosis. We transfected chemically synthesized mir-15a and mir-16-1 oligonucleotide into Raji cells using Lipofectamine 2000 reagent. At 24,48,72 hours after transfection, the growth inhibitory effect of Raji cells was measured by trypan blue dye exclusion method and CCK8 assay. Apoptosis was determined by morphological observation and flow cytomertry analysis after AnnexinV/PI double labeling. The expression levels of Bcl-2 protein were detected by immunofluorescence assay. Trypan blue dye exclusion method and CCK8 assay showed that transfection of mir-15a or mir-16-1 decreasesd the cell growth at 24, 48 and 72 h, which were significantly lower than those cells transfected with control oligonucleotide and untransfected cells, respectively (P<0.05). Using Hoechst staining, cells treated with mir-15a or mir-16-1 oligonucleotide displayed changes of apoptosis at 48,72h after transfection. AnnexinV/PI double dyeing assays showed early and late apoptotic cells at 48 and 72 h post-transfection. At 48,72h after transfection with mir-15a or mir-16-1 oligonucleotide, early and late apoptotic cell rate were obviously higher than untransfected cells and control miRNA group. Indirect immunofluorescence assay demonstrated that the expression of Bcl-2 was degraded at post-transfection, suggesting that Bcl-2 is a direct target of mir-15a and mir-16-1. These results show that mir-15a and mir-16-1 can induce apoptosis of human lymhpomatic cell line Raji. Our results suggest that mir-15a and mir-16-1 could be used for therapy of Bcl-2 overexpression tumors.
Case Report on a Female Patient with Congenital Haemophilia a and An Acquired Type-2 Inhibitor to FVIII
Authors: Kurnik Karin , Bidlingmaier Christoph
Published: April 2020

Only rare cases of female haemophilia have been reported. Moreover, no case of congenital haemophilia A associated with an acquired type-2 inhibitor to FVIII in a female child has been described in literature yet. We report the case of a 15-year old girl, diagnosed with severe haemophilia A (FVIII:C <1%) at the age of 13 months. Her father suffered from severe haemophilia A (FVIII:C <1%) due to an intron 22 inversion. In the girl both an intron 22 inversion and a non-random X-inactivation were found. At 3 years the girl received prophylactic treatment, and recombinant FVIII (3 × 40 IU/kg/week) from 6 years of age. Bleeding episodes were rare. Aged 13 she developed two large spontaneous haematomas. In-depth analysis confirmed decreased FVIII-recovery, –half-life and an inhibitor titre of 7.6 BU following type-2 kinetics (haemophilic autoantibodies). All of the few reported children with an aquired FVIII inhibitor presented with other autoimmune diseases or had used penicillin. Our patient showed none of this, but interestingly experienced menarche 4 weeks after inhibitor detection. This lets us speculate that the ethiology of the inhibitor formation in our patient at the time of hormonal change might be similar to that in pregnant or postpartum women.

Taking into account the recommendations of Ma et al. (2006) regarding inhibitor development during pregnancy in healthy women, we initiated a modified ITI with 75 IU/kg rFVIII concentrate (4,000 IU per infusion) every other day without additional immunosuppressive therapy. After 6 weeks of ITI, inhibitor titres declined and became negative after 8 months, resulting in a normal FVIII half-life till now.
Treatment Patterns and Splenectomy Failure Rates in Patients with Chronic Immune Thrombocytopenic Purpura in Canada: A Retrospective Chart Review
Authors: Mikhael Joseph , Tinmouth Alan , Merali Tazmin , Amin Mo , Lam Wendy
Published: April 2020

Background: In clinical practice there is little consensus on treating patients with chronic immune thrombocytopenic purpura (ITP) beyond first line therapy with steroids.

Objectives:

Describe the demographic and disease characteristics of adult ITP patients who receive treatment; Obtain the treatment approach for patients with ITP beyond first line treatment with steroids, with an emphasis on splenectomy; and Evaluate adverse bleeding outcomes associated with ITP.

Methods: A retrospective chart review of ITP patients after initial therapy with steroids was conducted. Ten physicians (oncologists and hematologists) were recruited from across Canada and each physician provided at least 5 ITP cases for review. A total of 51 cases were reviewed and patients (> 18 years old) were required to have had more than 1 course of steroids as treatment to be eligible.

Results: The average age of patients at diagnosis was 42 (range 3 to 82 years); 37 (73%) of the patients were female, and 37 (73%) were Caucasian. The median platelet count upon presentation was 5×109/L. Median lines of therapy after initial therapy was 3 (range 0 to 6). Second line therapies varied, but most commonly patients underwent splenectomy (43%), followed by continued steroid treatment (16%), steroids plus IV Ig (16%), IV Ig alone (14%), immunosuppressant alone (2%), and anti-D plus steroids (2%). Of the patients reviewed, 40 (78%) eventually underwent splenectomy. Of the 40 splenectomized patients, 27 were splenectomized within the first year of diagnosis and 35 underwent splenectomy during the first 5 years. In addition, 62 of the splenectomies were laparoscopic, and the median hospital stay for all procedures was 5 days (range 1 to 60 days). Immediate failure of splenectomy occurred in 18% of patients, and the one- and five- year relapse/failure rates (defined as platelet count < 30×109/L) were 38% and 55%, respectively. Other therapies following splenectomy included IV Ig, azathioprine, cyclosporine, danazol, mycophenolate, rituximab, vincristine, cyclophosphamide, and anti-D. Only 6% of IV Ig use was for chronic maintenance therapy and 74% of use was for urgent therapy. A total of 61 bleeding events occurred, of which 10 were major (upper GI, mouth, rectal, and nose). Major bleeding events required hospitalization of the patient with an average length of stay of 5 days (range 2 to 14 days).

Conclusions: Notwithstanding several limitations, the retrospective chart review suggests that there is wide variation in long-term therapy for patients with chronic ITP in Canada. Splenectomy was the most widely used second line therapy, although 18% of the patients were non-responders and the five-year relapse/failure rate was 55%.
Refractory Severe Thrombocytopenia Associated with Intravenous Treprostinil Use: a Case Report
Authors: Kuo Sherry , Tandon Rajive , Boggio Lisa
Published: April 2020

Background: Intravenous epoprostenol was the first drug approved by the U.S. Food and Drug Administration for the treatment of pulmonary arterial hypertension. Treprostinil, a tricyclic benzidene prostocycline analog of epoprostenol has been favored over epoprostenol due to its more advantageous mode of administration and similar efficacy. All prostocyclines are known to cause thrombocytopenia, but severe platelet reductions are rare below 100,000/μL.

Aim: We report the first case of severe thrombocytopenia associated with intravenous treprostinil with subsequent recovery of a normal platelet count following transition to epoprostenol.

Case Report: A 55 year-old Caucasian woman diagnosed with idiopathic pulmonary artery hypertension noted significant improvement in symptoms and exercise tolerance following initiation of intravenous treprostinil. However, she developed new onset severe thrombocytopenia with her platelet count falling to 51,000/μL (150,000–399,000/μL) and recurrent anterior epistaxis after receiving intravenous treprostinil for six months. She had no known hematologic disorders or history of recurrent bleeding and was not receiving any other new medications. There was no evidence of disseminated intravascular coagulation, hemolysis, coagulation factor deficiency, or splenic sequestration. Empiric treatment with corticosteroids was started for positive antiplatelet IgG direct antibody, but the platelet count further declined to 25,000/μL. The patient was subsequently transitioned from intravenous treprostinil to epoprostenol under the assumption that her severe thrombocytopenia was an idiosyncratic reaction rather than a class effect of prostocyclines. Her platelet count had returned to normal two weeks after the transition.

Conclusion: In practice, patients developing thrombocytopenia from epoprostenol are frequently switched to treprostinil hoping to attenuate the degree of thrombocytopenia. This case is the first report of severe thrombocytopenia associated with intravenous treprostinil, as well as its successful resolution following transition to another prostocycline analog, epoprostenol. Although the exact mechanism of thrombocytopenia is unknown, it raises a question if the cyclohexane ring on treprostinil compared to the lactone ring on epoprostenol can potentially lead to more severe thrombocytopenia. Although at this point, it is uncertain if severe thrombocytopenia is an idiosyncratic reaction to treprostinil, this report will hopefully draw attention to this potential adverse effect and identify the mechanism of this reaction.
Successful Management of Refractory Severe Thrombocytopenia Associated with Antiphospholipid Syndrome Using rituximab
Authors: Jaouni Soad Al
Published: April 2020

Background: Rituximab, a monoclonal antibody against the pan B-cell antigen CD20, that induces a rapid in vivo depletions of normal B lymphocytes. Although this agent was originally developed for use in patients with B-cell-NHL, its use has been successfully extended to many autoimmune disorders. Thrombocytopenia associated with antiphospholipid syndrome can be mild to severe. Severe cases can be associated with significant morbidity and mortality if refractory to the usual therapy for autoimmune thrombocytopenia.

Aim: To report an effective regimen in treating a case of severe bleeding thrombocytopenia associated with antiphospholipid syndrome.

Patient and Methods: A 49 year old female was diagnosed with thrombocytopenia associated with antiphospholipid syndrome and antibodies to double-stranded DNA, controlled over the last 15 years. Unfortunately, over the last two years the patient has had frequent admissions for uncontrolled nose bleed, soft palate petechiae and generalized bruising.

Many treatments have been tried for this case of difficult refractory bleeding with platelets in the range 2.0–6.0 K/uL. These include systemic treatments such as corticosteroids, high pulse therapy of methylprednisolone, Imuran, i.v. immunoglobulin infusion and anti Rh-D intravenous therapy.

We report an adult female patient with severe refractory bleeding thrombocytopenia associated with antiphospholipid syndrome and successful treatment with Rituximab. Her platelets count have been maintained above 200.0K/uL over a 14 months period. Unfortunately, this patient has had side effects of steroid therapy.

In conclusion: Rituximab may be effective treatment on patients with refractory, severe thrombocytopenia associated with antiphospholipid syndrome and significant bleeding complications. Rituximab is a promising alternative option for the eradication of the autoantibodies and restoration of normal hemostasis while avoiding the use of high-dose steroid in refractory bleeding thrombocytopenia.
Assessment of Bone Marrow Mesenchymal Stem Cells Biological Characteristics in Patients with Polycythemia Vera
Authors: Zhang Xiaoyan , Li Jianyong , Lu Hua , Wu Yujie , Xia Jun , Duan Limin , Zhang Yaping
Published: April 2020

Objective: To isolate and culture bone marrow mesenchymal stem cell (MSCs) from polycythemia vera (PV) patients and identify their in-vitro biological characteristics.

Methods: Bone marrow was extracted from 9 patients with PV and 6 normal individuals. MSCs were isolated by density gradient centrifugation. The adherent cells were cultured and passaged. The cultured cells harvested from the third passage were assessed. The morphology of the cells was observed by Wright’s staining and the growth curve of the cells was plotted by cell counting. The ultrastructure of MSCs was observed with electron microscopy. The cell cycle and immunophenotype of the expanded MSCs were detected by flow cytometry (FCM). Different agents were used to induce MSCs to differentiate into osteocyte and adipocyte. Von Kossa staining and oil-red staining were used to examine the differentiation. Conventional cytogenetics (CC) was carried out to detect karyotype. Alleles specific polymerase chain reaction (AS-PCR) was used to detect JAK2V617F mutation. RT-PCR were used to analyze the expression of multiple hematopoietic growth factors at mRNA level for MSCs.

Results: MSCs from patients and normal individuals displayed a fibroblast-like morphology adhering to the culture plate and the doubling time of expand MSC was about 43h. The cells were CD73, CD90 positive, and CD34, CD45, CD133, HLA-DR negative. Under suitable conditions, these MSCs could differentiate into osteocytes and adipocytes. MSCs from patients and normal individuals showed normal karyotype. And JAK2V617F mutation was negative in both MSCs. The same expression pattern was seen in patients and normal individuals that they expressed mRNA of SCF, Flt3-ligand, TPO, LIF, IL-6, IL-11 and did not express G-CSF, GM-CSF and IL-3.

Conclusion: MSCs from patients with PV are similar to that from normal individuals.
Thiotepa in the Conditioning Regimen Decreases Rejection after HLA Identical Allogeneic Marrow Transplantation in Children with Beta Thalassemia Major Aged Less Then 4 Years
Authors: Polchi Paola , Gaziev Javid , Lucarelli Guido , Sodani Pietro , Alfieri Cecilia , De Angelis Gioia , Gallucci Cristiano , Isgro Antonella , Marziali Marco , Roveda Andrea , Paciaroni Katia
Published: April 2020

BMT in children with beta thalassemia major is the only form of definitive cure with correction of the genetic disease in a relevant proportion of patients, and long term thalassemia free survival. Lucarelli and coll. showed since 1986 that transplant performed early in the course of disease gives high chance of cure, and a system of class definition was reported in 1990 identifying as in class 1 patients without any evidence of organ damage, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. Majority of children aged less then four years were then in class 1 and occasionally in class 2. Thiotepa has been introduced in the conditioning regimen of children less then 4 y.o. in the attempt to decrease the rejection rate in this category of good prognosis patients. Out of 519 children affected by not advanced Beta Thalassemia Major, either in class 1 or 2 of risk, with an HLA identical sibling donor, 25 patients aged 1 to 4 yo received an intensified conditioning regimen including thiotepa 10 mg/kg given in one day, in addition to standard BUS-CY regimen called PC 6. This was based on the observation that children aged below 4 transplanted after the standard PC 6 had significantly higher relapse rate with return to thalassemic pre transplant condition (OS, TFS, REJ were 87%; 86%; 4% vs 87%; 73%; 14% in children aged above or below 4 yo respectively (n=398 =>4; n=96 < 4). The increased relapse rate was more relevant in children treated with standard PC 6 and GVHD prophylaxis performed using short MTX plus CSA versus those who received cyclosporine alone. The GVHD prophylaxis protocol including sMTX + CSA +Pred, used after 1999 for all patients in our practice significantly improved OS, lowered GVHD rate, but had a trend toward increased rejection rate. This trend was more evident in children aged less than 4 despite an increased dose of Busulfan (16 mg/kg total) was already in use for younger children. Therefore, since 2003 we introduced Thiotepa for children less then 4 yo, maintaining the GVHD prophylaxis with sMTX and CSA. In the last group of 25 children aged below 4 receiving the intensified protocol, 1 patient rejected the graft and had thalassemia recovery, one died of GVHD and infection, and 23 are alive and cured. Actuarial probability of OS, TFS, Rejection were 96%; 91%, 5%. These results favourably compare to the previous results in this category of patients, confirming that Thiotepa exerts additional immunosuppressive and eradicating action, counteracting the effect of GVHD prophylaxis with sMTX+CSA, that exerts an action favouring rejection.
Optimization of a BCR-ABL-Specific DNA Vaccine against Ph+ ALL Using a Nonviral Vector System and Nanoparticle Delivery
Authors: Rott Yvonne , Arndt Stefanie , Green Jordan , Anderson Daniel , Stripecke Renata , Wittig Burghardt , Schmidt Manuel , Köchling Joachim
Published: April 2020

Although 40–50% of adults and 70–80 % of children with acute lymphoblastic leukemia (ALL) can be cured by poly chemo therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing leukemia cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the immunization of mice with DNA-based vaccines subsequently challenged by the cell line BM185. Combinations of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide, GM-CSF, IL12, IL27 or CD40L were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. In order to increase transfection efficacy, MIDGE-vectors were microencapsulated into poly(β-aminoester) nanoparticles with diameters of 200 nm.

Mice immunized with the BCR-ABL/GM-CSF/dSLIM vaccine showed a significant longer mean tumor-free (p=0.019) and overall survival (p=0.008) compared to nonvaccinated mice. BCR-ABL specific sequences were required to prevent Ph+ acute lymphoblastic leukemia. Furthermore, CTL assays showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GMCSF/dSLIM (p<0.05) and to naïve mice (p<0.005). The vaccine efficacy was clearly dosedependent. Microencapsulation of MIDGE vectors increased the efficacy of the vaccine compared to the naked DNA-vaccine. Mice immunized with the microencapsulated vaccine BCR-ABL/GM-CSF/dSLIM showed a significant longer mean tumor-free (p<0.0001) and overall survival (p<0.0001) compared to non-vaccinated mice and 70% survived and never developed leukemia. Cotransfection with IL27 or IL12 lead to significant longer tumor free (IL27: p=0.02; IL12: p<000.1) and overall survival (IL-27: p=0.03; IL12: p<000.1) compared to the vaccine BCR-ABL/GM-CSF/dSLIM. The best protection with a survival rate of 91% was observed in mice which received the vaccine BCR-ABL/GMCSF/IL12/dSLIM. We have shown previously in T-cell depletion studies that CD8+ T cells were the effector cells in the BM185 cell-based vaccine model and currently we evaluate whether CD8+ T cells also play a major role in the BM185 DNA-based vaccine model. In conclusion, we provide survival and functional data that show immunization and protection of mice with optimized leukemia specific DNA-vaccines.
Efficient Gene Transfer into Human CD34+ Peripheral Blood Progenitor Cells Using Pseudotyped Recombinant Adeno-Associated Viral Vectors
Authors: Sellner Leopold , Veldwijk Marlon , Stiefelhagen Marius , Kleinschmidt Jurgen A. , Laufs Stephanie , Topaly Julian , Zeller W. Jens , Wenz Frederik , Fruehauf Stefan
Published: April 2020

Due to their pluripotency, human CD34+ hematopoietic stem cells are targets of interest for the treatment of many acquired and inherited disorders of the hematopoietic system using gene therapeutic approaches. Human CD34+ peripheral blood progenitor cells (PBPC) can be readily mobilized into the blood and harvested by leukapheresis, providing an easily accessible source of hematopoietic progenitor cells. Unfortunately, for gene transfer into CD34+ PBPC, most current vector systems either lack sufficient transduction efficiency or an acceptable safety profile. Standard adeno-associated virus-based vectors have an advantageous safety profile, yet lack the required efficiency. Therefore a panel of pseudotyped recombinant adeno-associated viral (rAAV2/1 - rAAV2/6) vectors expressing the eGFP gene was screened on human G-CSF-mobilized CD34+ PBPC to determine their efficacy. In addition, the difference in transgene expression between conventional single-stranded rAAV and self complementary rAAV (scAAV) vectors was determined. For each vector n≥6 was performed and data are shown as mean ± SD. Of all screened conventional rAAV vectors, rAAV2/6 proved to be the most efficient (13.5% ± 9.8% GFP+ and CD34+ PBPC; p<0.001 vs other vectors) on human CD34+ PBPC, followed by rAAV2/2 (2.6% ± 2.0% GFP+ cells) and rAAV2/1 (1.4% ± 1.2% GFP+ cells). For rAAV2/3, rAAV2/4 and rAAV2/5 no relevant gene transfer efficiency (<1% GFP+ cells) was observed. Furthermore, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using scAAV vectors. scAAV2/6 and scAAV2/2 (both p<0.001) showed significantly higher gene expression (38.4% ± 12.2% and 11.8% ± 5.7% GFP+ cells, respectively) compared to their conventional counterparts in this cell entity. Similar results were observed for scAAV2/1 vectors (2.8% ± 1.9% GFP+ cells), though the difference was not significant. Of note, as previously observed using AAV peptide library-derived rAAV vectors (Sellner et al., 2008, Exp Hematol. 36), also here inter-patient variances in CD34+ PBPC susceptibility were found. For the first time we were able to obtain clinically relevant gene transfer and expression levels (>10%) with expression rates up to 60% in human CD34+ PBPC using an AAV-based vector system, thereby providing an efficient alternative vector system for gene transfer into this clinically important target cell.
Effects of Proteoglycans on the Maturation of Dendritic Cells Derived from Human Peripheral Blood Monocytes
Authors: Yoshino Hironori , Takahashi Kenji , Kashiwakura Ikuo
Published: April 2020

Dendritic cells (DCs) are a type of antigen-presenting cell which play an essential role in the immune system. The transition from immature DC (iDCs) to mature DCs (mDCs) requires maturation stimuli, such as pro-inflammatory cytokines or pathogen-derived components. Proteoglycans (PGs) are one of main components of the extracellular matrix and are composed of core proteins and glycosaminoglycans that bind to the core proteins. PGs are also constituent elements of bacteria and the role of PGs in the stimulation of DCs has not been elucidated. This study investigated the effects of PGs extracted from the nasal cartilage of a salmon head (S-PG) and the nasal septum cartilage of a whale (W-PG) on the maturation of DCs derived from human peripheral blood monocytes. This study was approved by the Committee of Medical Ethics of Hirosaki University School of Medicine. The human peripheral blood mononuclear cells (PBMCs) were separated from the buffy coat. Furthermore, the monocytes were separated from the PBMCs by allowing them to adhere to a plastic dish. To prepare iDCs, the monocytes were cultured in the presence of 50 ng/ml rhGM-CSF and rhIL-4 for 5 days. The iDCs were stimulated by S-PG or W-PG for 4 days to investigate whether the PGs alone were able to induce the maturation of DCs. In addition, other iDCs were stimulated by a cytokine mixture (rhTNF-α, rhIL-1β, rhIL- 6 and PGE2: MIX) or a combination of MIX+S-PG or W-PG for 48 hours. The surface phenotype of the DCs was analyzed by flow cytometry and the matrix metalloproteinase-9 (MMP-9) activity in the culture supernatants was assayed by zymography. Furthermore, the functions of DCs stimulated by a combination of MIX+S-PG or MIX+W-PG were examined. When the iDCs were stimulated by either S-PG or W-PG alone, the PGs-stimulated DCs did not express the DC-maturation marker CD83, thus indicating that S-PG and W-PG alone could not induce the maturation of DCs. However, the CCR5 expression on DCs stimulated by W-PG was down-regulated. When DCs were stimulated by MIX + 100 μg/ml W-PG, an up-regulation of CCR7 expression was observed. In association with the up-regulation of CCR7 expression, the stimulation by MIX+W-PG actually enhanced the chemotactic responsiveness of DCs to CCR7 ligand MIP-3β. These effects were not observed in the combination of MIX+S-PG. The MMP-9 activity was next examined by zymography, because the degradation of extracellular matrix by MMPs is required for DCs migration. However, neither S-PG nor W-PG promoted MMP-9 secretion. The present study therefore demonstrates that W-PG not but S-PG can selectively regulate the chemotactic activity of DCs in vitro. Further understanding of the mechanism and studies using human PGs is therefore expected to provide valuable insight into the migration of immune cells, including DCs both in vitro and in vivo.
A134T, G156A and G1333A SNPs in the CD177 Gene Are Associated with Atypical Expression of Human Neutrophil Antigen 2a (HNA-2a)
Authors: Moritz Elyse , Chiba Akemi Kuroda , Albuquerque Dulcinéia M , Costa Fernando F , Yamamoto Mihoko , Bordin Jose Orlando
Published: April 2020

Introduction: The HNA-2a has gained clinical importance for being involved in immune neutropenias, febrile transfusion reactions and transfusion-related acute lung injury (TRALI). The HNA-2a shows the unique feature of being expressed only on a neutrophil subpopulation. The gene encoding the HNA-2a, CD177, is located on chromosome 19 at 19q13.2 and the cDNA consists of 2364 bp (RefSeq: NM 020406) coding for 437 amino acids including a signal peptide of 21 amino acids. In this study we investigated the presence of SNPs in the CD177 and its association with atypical expressions of HNA-2a.

Methods: After evaluating the expression of HNA-2a in 135 randomly selected Brazilians by flow cytometry (FACSCalibur, BD) using two pure monoclonal antibodies (TAG4 and 7D8), we observed that some subjects revealed an atypical expression of the antigen (a non reactive cell population plus two distinct reactive cell populations). Subjects were grouped according to cytometric results as having high (≥80%), low (≤50%) or atypical expression of HNA-2a. We isolated granulocyte RNA from the selected individuals and reverse transcription was performed according to kit protocol (SuperScript III First-Strand Synthesis, Invitrogen). Full-length cDNA was amplified and then sequenced according the manufacturer’s protocol (Mega BACE, GE). Data were analyzed using the Bioedit program.

Results: The flow cytometry analysis revealed that 131 (97%) subjects were HNA-2a positive, 4 (3%) HNA-2a negative, while 8 subjects (5.9%) showed an atypical expression of HNA-2a with levels ranging from 49 to 80%. Sequencing analysis of subjects with high (n=14), low (n=22) or atypical (n=8) HNA-2a expression identified six polimorfic sites (C49G, A134T, G156A, A793C, G1084A and G1333A), five of them predicting amino acid exchanges (except the SNP G156A). Comparing the cDNA sequences between individuals with typical (n=36) or atypical expression (n=8), we observed three polimorphisms (A134T, G156A and G1333A) that showed a significantly higher frequency in the group with atypical expression comparing to the group of individuals with typical expression (P= 0.004; 0.006; and <0.0001, respectively; Fisher exact test).

Conclusions: The analysis of individuals with two distinct positive cell populations revealed three SNPs strongly related to this atypical HNA-2a expression, but do not seem to be related to the size of the HNA-2a-expressing neutrophil subpopulations. Matsuo et al (Transfusion2000;40:654) and Caruccio et al (Transfusion2003;43:357) have described atypical reactions with HNA-2a-specific MoAbs, however this is the first study in the literature that gives some clues to the molecular basis of the atypical expression of HNA- 2a in otherwise healthy individuals.
Primary Prophylaxis Using Peg-Filgrastim Is Useful Preventing Severe Infections and Neutropenia in Patients Diagnosed with Non Hodgkin Lymphoma (NHL). Report of Latin America International Multicenter Study
Authors: Villela Luis , Villalobos Alberto , Reyes-Brena Gustavo , Duque Jorge , Solís-Poblano Juan Carlos , Reynoso Eduardo , Ovilla Roberto , Camacho Alejandro , Vazquez Norberto , Garces Oscar , Pedrasa Enrique , Silva Salvador
Published: April 2020

Background: Severe infectious events after chemotherapy (CHT) are potentially life-threatening. This is directly related to neutropenia. Granulocyte colony-stimulating factor is used as supporting therapy to prevent neutropenia. Peg-filgrastim has been available in Mexico since 2005 and there is not any report about the use of this drug in Latin America.

Aim: To evaluate grade 4 neutropenia and infections related to neutropenia while using peg-filgrastim.

Methods: Patients between the ages of 18–65, with NHL (de novo or relapsed) with ≤ 30% bone marrow involvement were eligible. CHT was selected by each institution. Complete blood counts were obtained on days 0 (day before chemotherapy), 7, and 14.

Outcomes: Fifty three patients with NHL were included (one withdrew from study after the 2nd cycle of CHT). Three hundred and fourteen cycles were analyzed. Clinical characteristics: Age (mean ±SD): 51.8(±13.5), male: 47%, ECOG ≥ 2:32%), Advance stage (III/IV Ann Arbor): 53%, adjusted IPI (Intermediate/High):60.4%, First-line CHT 72%, second-line CHT 28%. Median neutrophil count (ANM) on day 0 was 8.69 ×109/L (IC95%: 6.44 to 10.94, previous each CHT), ANM on day +7 was 7.9×109/L (IC95%: 5.34 a 10.45) and day +14 ANM was 8.61 ×109/L (IC95%: 6.74 a 10.48) see figure 1. Neutropenia grade 4 was observed in 12.8% of the cycles, being more frequent on day 7 (90%) it means chemotherapy nadir, see figure 2. Severe infectious events were registered in 0.63% of the global cycles (two pneumonias, one of them when ANC was over 2 ×109/L) and non-severe in 1.91% being more frequent superior tract infections. No delays in CHT administration or deaths were reported.

Conclusion: Peg-filgarstim could decrease the frequency of grade 4 neutropenia the day before the next administration of CHT as well as the incidence of severe and non-severe infections in young patients.