bluuebunny/crossref_metadata_2025 huggingface dataset:
GATA-1 Binding to Exon 1 Directs High Level Erythroid-Specific Expression of the Human Protoporphyrinogen Oxidase Gene.
Authors: de Vooght Karen M.K. , van Wijk Richard , van Solinge Wouter W.
Published: October 2019

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX. Like other heme biosynthetic proteins, PPOX is involved in synthesizing heme for red cells (erythroid-specific expression) and as a cofactor for the respiratory cytochromes (housekeeping expression). To date, little is known about transcriptional regulation of the human PPOX gene (PPOX). We established the molecular basis for erythroid-specific expression of PPOX. Using transient in vitro transfection assays in human erythroleukemic K562 cells we studied tissue-specific expression of PPOX. We found that reporter constructs lacking exon 1 showed a 75% reduction in promoter strength in K562 cells (Figure, no 1 and 2). Hence, in vitro high-level erythroid-specific expression of PPOX is dependent on the presence of exon 1. Examination of erythroid-specific regulatory elements in exon 1 revealed two GATA-1 sites, one consensus (A/T)GATA(A/G) site (GATA-1_II AGATAA) and one non-consensus site, deviating at the first nucleotide (GATA-1_I, CGATAG). To study the relative contribution of these two GATA-1 sites to erythroid-specific transcriptional regulation, we performed in vitro transfections of wild-type and mutant (GATA → GTTA) reporter plasmids in K562 cells. We found that the highest level of transcription depended on the integrity of both sites (Figure, no 5). The consensus GATA-1_II site contributed the most to promoter strength (Figure, no 4). Subsequent electrophoretic mobility shift assay and supershift experiments using K562 nuclear extracts demonstrated that both GATA sites were able to bind GATA-1 in vitro. Our experiments showed that exon 1 was dispensable for PPOX promoter activity in human hepatoma HepG2 cells. Interestingly, in HeLa human cervical carcinoma cells the presence of exon 1 decreased promoter activity. Conclusively, exon 1 of the human PPOX gene contains two GATA-1 binding sites, which are required for high level erythroid-specific expression of PPOX and, in addition, bind GATA-1 in vitro. Our results contribute to a better understanding of the molecular mechanisms involved in differential regulation of the human PPOX promoter in erythroid and non-erythroid cells.

Figure Figure
Human Mesenchymal Stem Cells Injected Via Portal Vein Are Differentiated into Human Hepatocytes in Regenerating Rat Liver.
Authors: Won Jong-Ho , Choi Dong-Ho , Kim Jung-Hoon , Kim Sook-Ja , Cheung Hee-Jeong , Lee Nam-Su , Park Hee-Sook
Published: October 2019

Objectives: Human mesenchymal stem cells (MSCs) possess versatile differentiation potential ranging from mesenchyme-related multipotency to neuroectodermal and endodermal competency. Evidence has been accumulated to indicate that certain compartments of bone marrow cells are capable to differentiating into hepatocytes in vitro. In this study we attempted to examine the differentiation ability of human MSCs into hepatocytes in vitro and in vivo by injected them into rat portal vein in partially resected rat liver model.

Materials and Methods: MSCs were isolated from human bone marrow and induced differentiation with our protocol containing hepatocyte growth factor in vitro. Four - to - 5 week-old female Sprague Dawley rats were used for xenotransplantation model. Culture expanded MSCs (5 X 106 cells/rat) were injected into the portal vein and 70% hepatectomy was performed on the subsequent day. All rats were immunosuppressed with a daily intraperitoneal injection of cyclosporine A.

Results: The morphology of the MSCs was changed into hepatocyte-like cells after in vitro culture for 28days and expression of hepatocyte specific genes also confirmed with RT-PCR and immunohistochemical stain. Transplanted MSCs differentiated into hepatocytes and they surprisingly composed hepatic cords with expression of the human albumin and human hepatocyte specific genes at 21 days after infusion.

Conclusion: We have demonstrated that human MSCs can differentiate into functional hepatocyte-like cells in vitro and in vivo. Therefore, human MSCs may become an alternative source to hepatocyte regeneration or liver cell transplantation.
Clinical Efficacy and Safety of Wilate®, A New Generation, High-Purity VWF/FVIII-Concentrate with Optimized Pharmacokinetic Properties in the Treatment of Von Willebrand Disease.
Authors: Windyga Jerzy , Zarkova Antoaneta , Walter Olaf , Auerswald Guenter
Published: October 2019

Purpose: Wilate® represents a new generation VWF/FVIII-concentrate for the treatment of von Willebrand patients with high purity and two independent virus inactivation steps. The high purity of the product was developed to preserve the functionality of Wilate®’s VWF/FVIII complex at a physiological ratio of about 1:1.

Methods: A prospective multicenter study is conducted in 47 VWD patients to investigate the pharmacokinetics (PK) and clinical efficacy and safety of Wilate® in acute bleedings and surgical prophylaxis. For PK studies, plasma levels of VWF:RCo, FVIII:C, VWF:Ag were measured up to 72 hrs after injection of 50 IU of FVIII/kgBW. Clinical efficacy and tolerability was rated on a four-point scale. Final decisions on dosing were based on the investigators’ clinical judgment.

Summary of results: Mean half-life of VWF:RCo in type 3 patients (n=9) was 17.1 hrs. Mean recovery for VWF:RCo was 1.5 [% per IU/kg] and 2.0 [% per IU/kg] for FVIII:C. The clearance for VWF:RCo was 2.5 [ml/h/kg] for all and 3.9 [ml/h/kg] in type 3 patients. 33 patients have been treated for a total number of 658 bleeds. Of these, 31 patients (69%) were type 3 patients. The average dose/kgBW/ED was calculated to 29.3 IU FVIII:C. Furthermore efficacy and safety was studied in 16 patients undergoing 24 surgical procedures. The overall efficacy (achievement of haemostasis) of Wilate® was rated as excellent or good for 23/24 procedures (96%). One patient with a laparoscopic cholecystectomy required two additional blood transfusions. The tolerability was assessed as very good/good by investigators and patients in all surgical cases.

Conclusion: Wilate® documented favourable PK-properties in VWD patients. The interim results of these prospective clinical studies demonstrate the safety and efficacy of the double-virus-inactivated Wilate® for the treatment of acute bleeding episodes and surgical procedures in patients with VWD.
Amino Acid Regions 572–579 and 657–666 of the Spacer Domain of ADAMTS13 Provide a Common Antigenic Core Required for Binding of Antibodies in Patients with Acquired TTP.
Authors: Luken Brenda M. , Turenhout Ellen A.M. , Kaijen Paul H.P. , Fijnheer Rob , Voorberg Jan
Published: October 2019

Inhibitory antibodies against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura. Epitope-mapping studies revealed that antibodies binding to the cysteine-rich/spacer domains of ADAMTS13 are present in plasma of all patients analyzed so far. We have previously shown that a major antigenic determinant located within the spacer domain. To confirm these results we constructed a recombinant fragment consisting of the disintegrin/TSR1/cysteine-rich domains of ADAMTS13 and the spacer domain of ADAMTS1. The resulting ADAMTS13/ADAMTS1 chimera did not react with IgG present in plasma of a panel of patients with acquired TTP. To identify the amino acid residues that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of 15 hybrids (designated A–O) in which 5–10 amino acids of the ADAMTS13 spacer were exchanged for the homologous sequence of ADAMTS1. Plasma from 6 patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of residues 572–579 (hybrid C) and 657–666 (hybrid M) of ADAMTS13 for the corresponding sequence of ADAMTS1 completely abolished the binding of antibodies from all 6 patients. Other regions of the ADAMTS13 spacer were also involved in binding of antibodies from patient plasma. Regions 580–587 (D), 602–620 (G,H), 629–638 (J), and 667–767 (N) of the ADAMTS13 spacer domain contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). IgG derived from patient 1 required region 602–620 (G,H) for binding to the ADAMTS13 spacer (CGHM-epitope). For antibodies of patient 3, residues 564–571 (B), 580–587 (D), and 629–638 (J) were required (BCDJM-epitope), whereas replacement of residues 602–610 (G) and 629–638 (J) greatly diminished binding of antibodies derived from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of the patients, our results suggest that residues 572–579 (C) and 657–666 (M) comprise a common antigenic core region within the spacer domain that is crucial for binding of anti-ADAMTS13 antibodies in all six patients. Amino acid residues derived from other regions that spatially surround this common antigenic core further modulate binding of antibodies to the spacer domain.
Leukoreduced Packed Red Cells: Do They Have a Role in Decreasing the Incidence of Heparin Induced Thrombocytopenia?.
Authors: Atluri Prashanti , Kujawa Jennifer , Hindenburg Alexander
Published: October 2019

It has been demonstrated that neutrophils activate macrophages via the heparin binding protein, thus possibly contributing to the incidence of heparin induced thrombocytopenia (HIT).

It would follow that the incidence of thrombocytopenia and its sequelae may be reduced by using leukoreduced blood in patients undergoing cardiac bypass surgery. We had a unique opportunity to study this hypothesis at our institution. Due to a policy change all patients undergoing cardiac bypass surgery received LR blood intra-operatively prior to April 2005.

Subsequent to this period non-LR blood was utilized in all patients. A retrospective chart review of 197 non-LR and 316 LR patients was done. The platelet count on admission, the first five days post- op, the day of discharge, the percent drop in platelet count post-op and heparin antibody (HA) test were obtained. Thrombocytopenia was defined as an overall drop in platelet count by > 50%. The CABG and the non-CABG patients were looked at separately.

The results are as follows:

Percent of patients with > 50% drop in platelet count: all patients CABG patients non-CABG patients leukoreduced arm 64% 62.4% 73.3% non-leukoreduced arm 64.6% 61.7% 82.1% Fisher’s exact test p = 0.92 p = 0.92 p = 0.57 Percent of patients with heparin antibody positivity: all patients CABG patients non-CABG patients leukoreduced arm (n=81) 24.7% 23.3% 28.6% non-leukoreduced arm (n=39) 30.8% 35.5% 12.5% Fisher’s exact test p = 0.51 p = 0.23 p = 0.63

In both the LR and non-LR populations there is only a 0.6% difference in the number of patients with a > 50% platelet drop. The relative risk is 1.01 with the 95% confidence interval of (0.88, 1.15). This is a relatively narrow confidence interval reflective of the relatively large sample size.

Heparin antibody testing was not done on all the patients. Only those with persistent or profound thrombocytopenia had the test ordered. Overall heparin antibody positivity in both cohorts was not statistically different.

The LR arm had a slightly higher positivity rate overall. The risk ratio is 1.25 indicating that the non-LR arm is 25% more likely to have a positive antibody test compared to the LR arm. The higher positivity rate in non-CABG patients may be due to the more complex nature of the surgery relative to CABG surgery.

The non-CABG group has a higher percent of heparin antibody positivity in the LR arm.

This inconsistency may be secondary to the smaller sample size of this group. In both the CABG or non-CABG groups, there was no statistically significant difference in the percent platelet drop or heparin antibody positivity.

One can conclude that leukoreduction of red blood cell products during cardiac surgery does not significantly impact on thrombocytopenia or the development of HIT.
Two "Fast" Hemoglobin Components in Liberian Blood Samples
Authors: ROBINSON ABNER R. , ZUELZER WOLF W. , NEEL JAMES V. , LIVINGSTONE FRANK B. , MILLER MAX J.
Published: October 2019

Two abnormal hemoglobins, at least one of which has not previously been described, were encountered with appreciable frequency in Liberian blood samples. Some properties of these components are described.
Disseminated Neuroblastoma in an Adult Presenting the Picture of Thrombocytopenic Purpura
Authors: CHRISTENSON WILLIAM N. , ULTMANN JOHN E. , MOHOS STEVEN C.
Published: October 2019

A case of neuroblastoma in an adult with extensive metastatic lesions is presented. The initial symptoms and findings suggested idiopathic thrombocytopenic purpura. The blood picture and changes in the clinical picture later led to a diagnosis of acute leukemia. Autopsy disclosed the correct diagnosis, which would have been possible antemortem had the implication of pseudorosette arrangement of immature cells in the bone marrow and the possible occurrence of neuroblastoma in an adult been fully appreciated.
Histochemical Demonstration of Phosphorylase in Blood and Bone Marrow Cells
Authors: TAKEUCHI TADAO , KINOSHITA KENJI
Published: October 2019

Phosphorylase activity has been demonstrated histochemically in blood and bone marrow cells. The polysaccharide newly synthesized from glucose-1-phosphate by the enzyme of these cells gave a blue or violet blue iodine staining reaction. The reaction occurred in cytoplasm and the nuclei did not stain.

This was shown in the neutrophilic, eosinophilic or pseudoeosinophilic leukocytes of man, dog, rabbit and pigeon peripheral blood. It was likewise demonstrated in the bicolored leukocytes of snake, fish and frog. The reaction also appeared in the neutrophilic, pseudoeosinophilic or eosinophilic leukocytes, metamyelocytes, myelocytes and promyelocytes of human and rabbit bone marrow smears.
Immunization of Rabbits Against Human Anti-Hemophilic Factor (AHF)
Authors: RICHARDS MARVIN D. , SPAET THEODORE H.
Published: October 2019

Antibodies against human AHF were produced in the rabbit. These antibodies acted as anticoagulants in vitro by inactivating AHF and preventing normal thromboplastin formation. Species specificity and physical properties of the anticoagulant are described. It is postulated that similar anticoagulants occurring in humans are also antibodies produced by AHF immunization.
The Present Status of Treatment of Autoimmune Hemolytic Anemia with ACTH and Cortisone
Authors: DAMESHEK WILLIAM , KOMNINOS ZACHARIAS D. , DÉSY LOUISE
Published: October 2019

Forty-three patients with autoimmune hemolytic anemia (both "idiopathic" and "symptomatic") were treated with ACTH, Cortisone, or Compound F. In more recent cases not reported here, Prednisone (Meticorten) was used. Intensive and presumably adequate hormonal therapy resulted in sustained and complete clinical and hematologic responses in 65 per cent of the cases. Complete and sustained remissions were more commonly obtained in the "idiopathic" group (80 per cent remissions) than in the "symptomatic" variety. In 28 per cent of the cases, although the response was definite, it was not so striking. Complete failure was obtained in 3 cases (7%) of this series. When therapy was discontinued, following a remission, some degree of relapse developed in approximately two-thirds of the cases. In the remaining one-third, full clinical and hematologic remission continued well after discontinuance of therapy, although the persistently positive Coombs test indicated the possibility of future recurrence. Splenectomy was performed as the final therapeutic maneuver in 9 of the 43 cases and resulted in complete remissions in 6 without the further use of steroid hormones. In two cases splenectomy was followed by the development of outspoken disseminated lupus, previously undiscovered and presumably latent.

It is apparent from these studies that the steroid hormones in adequate dosage represent a remarkably effective form of therapy for at least the initial control of most cases of autoimmune hemolytic anemia. Their use for a lengthy period is often followed by a lasting remission, and the end-results of continued steroid therapy, either used alone or in some cases combined with later splenectomy, are definitely better than with splenectomy alone. Thus, control of this rather unpredictable disease has become a distinct possibility in most cases, and its management simply a matter of varying the dosage of steroid hormone.
Composite Nodular Sclerosis Hodgkin and Diffuse Large B Cell Non-Hodgkin Lymphoma Arising from the Thymus.
Authors: Sumoza Luis D. , Jorgensen Jeffrey L. , Sumoza Ilia R.
Published: October 2019

We report the first case of composite Nodular Sclerosis Hodgkin and Diffuse Large B-Cell Non-hodgkin’s lymphoma of the mediastinum. We present a case of an inmunocompetent patient operated on for a mediastinal tumor similar to a Thymoma, which the histological examination morphological, and immunophenotyping were performed and confirmed the existence of 2 independent, unrelated tumors. The pathology blocks submitted show a composite lymphoma, with components of both classical Hodgkin lymphoma (Hodgkin’s disease), nodular sclerosis type, grade 2 of 2 in all three blocks. One block also shows non-Hodgkin lymphoma, namely diffuse large B-cell lymphoma, with a distinct immunophenotype. The Hodgkin-Reed-Sternberg cells were positive for CD15, CD20, CD30, PAX-5 (weak/partial), and EBV (EBERs), and negative for CD45. In contrast, the large B-cells are positive for CD20, PAX-5 (strong), CD45, and CD30 (very focal), and negative for CD15 and EBERs. The large B-cell area had an increased mitotic rate. Taken together, these data indicate that these Hodgkin and the Non-Hodgkin’ lymphomas arose as a consequence of independent malignant transformation events.
Rapid Infusion of Rituximab (RIR) Is Well Tolerated and Can Safely Be Delivered in Patients with Lymphoproliferative Diseases.
Authors: Milone Jorge , Prates Virginia , Lucia Zoppegno , Ana Basquiera , Sebastian Yantorno , Marta Gelemur , Juan Garcia
Published: October 2019

It has been proved that Rituximab is a useful drug for the treatment of patients with lymphoma. Being a humanized antibody, it presents high specificity. Rituximab is a well tolerated medicine, although a slow infusion rate given in many hours is recommended to avoid reactions derived from the liberation of cytokines. As a consequence, patients need admissions for a treatment that can take hours. The recent demostration of its usefulness in the maintenance of the response of patients with follicular lymphomas has concluded in the revision of hospitalization time, proposing a faster infusion. We are presenting our experience with RIR, on a group of patients with lymphoproliferative diseases. We used RIR with the aim to observe the toxicity and to reduce the time of hospitalization in patients with lymphoma. We evaluated the adverse events and tested the feasibility of a 90 minutes infusion schedule for rituximab (20% of the dose administered in the first 30 minutes, remaining administered over 60 minutes). Premedication included oral paracetamol and intravenous hidrocortisone and diphenhydramine. Sixty seven RIR were used to treated 31 patients (27 non-Hodgkin Lymphoma and 4 Chronic Lymphocitic Leukemia). All of them had previously received one or more rituximab conventional infusion without grade 3 or 4 toxicity. Twenty nine patients received also CHOP or CHOP-like chemotherapy and the other two Rituximab alone as maintenance therapy. Overall RIR was well tolerated. There were 5 adverse events. Four patients developed grade one adverse events: one patient had an hypotension episode, one presented chest pain and the other two had abdominal pain. Only one patient developed a grade 3 adverse event (abdominal pain) and withdrew the RIR schedule. We conclude that rapid infusion rituximab is safe and well tolerated. The adoption of this schedule in clinical practice reduces in many hours the time of hospitalization for patients with lymphoma and may be a cost-effective strategy.
Additon of Low-Dose Interferon-alpha (LD-IFN-a) and Lenalidomide after Failure to a Combination of Thalidomide and Prednisone in Myelofibrosis with Myeloid Metaplasia (MMM).
Authors: Reale Lisa , Besa Emmanuel C.
Published: October 2019

Primary myelofibrosis with myeloid metaplasia (MMM) is an uncommon myeloproliferative disease with limited effective treatment options. Therapy using thalidomide and prednisone (TP) or lenalidomide (L) show variable objective response of improvement of 22% in L to 62% in TP for anemia, 50% in L to 75% in TP for thrombocytopenia, and decrease in splenomegaly in 19% with TP to 33% with L and was well-tolerated. Additional clinical benefits show resolution of leukoerythroblastosis, and a decrease in medullary fibrosis and abnormal angiogenesis. Prolonged therapy with alpha-interferon (IFN-a) can improve hematopoiesis and reverse marrow fibrosis but doses used were poorly tolerated.

Methods and Patients: Here we present five patients treated with combination T at 50 mg daily, P at 0.5 mg/kg po for 2 weeks then to 20 mg daily every other day for 3 months with no response. If this was tolerated, low-dose IFN-a (LD-IFN-a) at 1.5 million units sc. three times a week was added. The clinical and hematologic criterion of the European Myelofibrosis Network (EUMNET) were used as response criteria for our study. Three males, 2 females, age ranged from 60 to 85 years who had failed previous treatments from EPO, hydrea and transfusion support. White blood cell counts ranged from 4.3 to 29.7 B/L. Hemoglobin levels ranged from 7.3 to 11.6 g/dL. Platelets ranged from 13,000 to 283,000 B/L. Three patients developed iron overload from rbc transfusions and required iron-chelation therapy.

Results: Of the five patients, one had a CR, one had a PR and two had regression that did not meet criteria for response and one unresponsive with no splenomegaly but stable disease. We noted that the addition of LD-IFN-a decreased the elevated WBC counts and Improvement in spleen size was seen about three months after the start of treatment. One patient had a CR in his WBC count approximately a month after starting therapy. The patient with CR had normal hemoglobin but had to discontinue after a year of T due to grade 2 neuropathy and was started on 10 mg/day of L while staying on IFN-a and P. She developed anemia and required transfusions for 3 months and achieved CR with shrinkage of her spleen size from 15 to 4 cm. The patient who was unresponsive to the combination of IFN-a- TP changed from T to L was stopped after the patient developed hemolytic anemia and splenomegaly.

Conclusion: Addition of LD-IFN-a to the reported TP or L to replace T show clinical activity in patients with refractory MMM and is well tolerated with minimal toxicity. The use of lenalidomide may alleviate neuropathy but may induce hemolysis as an undesirable side effect. Further studies using low doses of these agents with varying activities to MMM in combination is needed to define their clinical use in the treatment of this disease entity in a much larger number of patients.
Response Predictive Factors to Anagrelide in Essential Thrombocythemia Diagnosed Patients.
Authors: Besses Carlos , Giralt Manuel , Hernandez-Nieto Luis , Burgaleta Carmen , Navas Victor , Ramirez Gema , Vicente Vicente , Carbonell Felix
Published: October 2019

We performed a retrospective analysis of our Spanish database of patients with ET in order to assess the role of different response predictive potential factors to anagrelide treatment. 411 ET patients from 54 sites from February 2005 to August 2006 were included in a retrospective chart review. ET was diagnosed according to PVSG criteria (1997). All patients had started treatment with anagrelide before December 2004, either as a first line or as a second line therapy. The population was divided itself in three groups of risk at diagnosis:

of high Risk: those patients older than 60 years or with previous history of thrombosis (40.6%); of low Risk: patients younger than 60 years with thrombocytosis lower than 1.5· 109/L, without cardiovascular risk factors and previous history of thrombosis (35.8%); intermediate risk: those patients who did not reunite the criteria of previously mentioned groups (23.6%).

The response to the treatment was defined as complete remission (CR), when a reduction of platelets′ count were equal or less than 400×109/L, or a reduction higher than 50% with respect to the basal numbers; partial remission (PR), when the platelets′ count was between 400× 109/L and 600×109/L with respect to the basal numbers; and no response, those with a lower reduction than PR or increasing their platelets′count. CR was obtained in 219 patients (53.6%; 95%CI = 48.6–58.5) and PR in 113 (27.6%; 95%CI = 23.4–32.2), giving an overall response (OR) rate of 81.2% [95%CI = 77.0–84.9]. The influence of certain factors (such as age, gender, risk group, platelets levels before treatment with anagrelide, previous cytoreductive untreated patients and maximum anagrelide dose), in the objective response was explored. A chi-square test and a multivariate analysis (logistic regression) were performed. A worse response (p = 0.021) was associated with a higher dose (≥ 2.5 mg per day). A trend (p = 0.103) to better response in patients without previous treatment (anagrelide as a first line) was detected. Additionally a significant association (p = 0.02) between the previously treated with hydroxyurea and the presence of evolutive hematological transformations (myelofibrosis and acute myeloid leukemia/myelodysplastic syndrome) was observed. It is difficult to set a cause effect relation in all these findings due to the retrospective design of the analysis. As a conclusion, the response to anagrelide in ET patients was demonstrated to be independent of the age, gender, platelets′ count and level of previous risk.
CD38 as a Prognostic Factor in Chinese Patients with Chronic Lymphocytic Leukaemia.
Authors: Jianyong LI , Xu Wei , Wu Yujie , Fan Lei , Qiu Hongxia , Ruan Changgeng
Published: October 2019

B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western world, however, infrequent in the Eastern. Although the median survival is around 10 years, CLL patients have a highly variable clinical course and prognosis. While some patients show an indolent disease and never require treatment, others suffer from a much more aggressive course requiring intensive treatment shortly or immediately after diagnosis. Identification of these subgroups and insight into the prognosis for each individual patient will be important to determine individualized treatment strategies. To explore the prognostic significance of CD38 expression in Chinese patients with CLL, multi-parameter flow cytometry was used to detect the expression of CD38 on CD5+CD19+ cells of 147 patients with CLL. CD38 expression and its association with some other prognostic factors such as Binet stage, lymphocyte count in peripheral blood, serum lactate dehydrogenase (LDH), β2-microglobulin (β2-MG), ZAP-70 expression and cytogenetic abnormalities were analyzed. The Kaplan-Meier method was used to construct survival curves, and results were compared using the log-rank test. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. Out of the 147 CLL patients, positive expression of CD38 was found in 45 (30.6%) cases. CD38-positivity identified a subgroup of CLL patients with aggressive disease of Binet stage at the time of the test (P=0.036). Furthermore, the presence of higher serum LDH and β2-MG levels at diagnosis was strongly correlated with CD38-positive (P=0.016 and P=0.025, respectively). Prognostically unfavorable cytogenetic abnormalities, including 17p13 deletions and 11q22 deletions were significantly more frequent in CD38-positive patients than in CD38-negative ones (P=0.047 and P=0.001, respectively). There was no significant difference between CD38-positive and CD38-negative subgroups in terms of age, sex, or lymphocyte count. In addition, in CD38-positive patients, the percentage of leukemic cells expressing ZAP-70 protein was not significantly higher than in CD38-negative ones (P=0.120). There was no significant difference between CD38-positive and CD38-negative groups in molecular cytogenetic aberrations of del(6q23), del(13q14), 14q32 translocation, or trisomy 12. CD38 expression was associated with poor outcome. Patients with positive expression of CD38 had significantly shorter overall survival (mean, 81 months) than patients without CD38 expression (mean, 179 months) (P=0.015). Univariate and multivariate analysis showed that serum levels of LDH and β2-MG, del(17p13) and CD38 expression were strongly associated with survival. It was showed that the patients with high level of CD38 expression had poorer outcome; CD38 was a good predictor of survival time in Chinese patients with CLL.
Analysis of CD4+CD25hi Regulatory T Cells in the Peripheral Blood of Patients with B Cell Chronic Lymphocytic Leukemia (CLL).
Authors: Pappa Vassiliki , Giannopoulou Vassiliki , Spyridaki Katerina , Karayanni Vassiliki , Liakata Elisabeth , Kontsioti Frieda , Giamarelos Evangelos , Papageorgiou Efstathios , Dervenoulas John , Economopoulos Theofanis
Published: October 2019

Introduction: Naturally occurring regulatory T cells (Treg) prevent autoimmune and inflammatory diseases and represent one important factor contributing to the inhibition of antitumor immune response in cancer. CLL is a disease that is characterized both by immunodeficiency and sometimes by autoimmunity. We analyzed the frequency of Treg population in the peripheral blood of patients with B-CLL and correlated them with clinical and laboratory characteristics.

Methods: We analyzed prospectively 49 patients with B-CLL (29 males, 20 females) with median age of 63 and 24 normal healthy volunteers. Freshly isolated peripheral blood mononuclear cells were analysed by flow cytometry using the EPICS XL /MSL cytometer (Beckman Coulter Co). We determined the level of apoptosis by the method of annexin-V and the frequency of Treg as cells positive for CD4, CD25hi and intracellular staining of Foxp3 using the PE anti-Human Foxp3 staining set protocol. We recorded clinical information regarding Rai and Binet staging, hematological and biochemical parameters and the presence of autoimmune hemolytic anemia.

Results: The level of apoptosis as determined by the annexin V method was significantly lower on CD19+ cells of patients compared to normal controls (4.7 vs11.34 p=0.02). Moreover patients under treatment had significantly lower apoptosis level vs untreated (4vs6.4, p=0.023). The mean and median values of Treg cells in patients with CLL were higher but not significantly compared to controls. However the log10 values of the Treg frequencies were significantly higher in the group of CLL (0.6287vs0.1021, p=0.03). There was not any statistically significant association of Tregs with age, Rai and Binet staging, LDH values, and the level of apoptosis. Patients with levels of Treg cells>mean control value presented lower median expression of CD38 antigen of borderline significance (3.8vs7, p=0,06). 5/26 patients with Treg frequency >median control value presented autoimmune hemolytic anemia compared to 0/18 with Treg <median control value (p=0.06)

Conclusion: The log10 values of Treg frequencies in patients with B-CLL were significantly higher compared to normal controls in accordance with previously published data. We did not observe any significant association with any laboratory or clinical characteristics or the level of apoptosis. Patients with Treg levels above the mean control values had a lower although not significantly expression of CD38 antigen indicating an association with more indolent disease. Functional study of these cells will help the interpretation of the data and will shed more light on the their exact role in the pathogenesis of the disease.
Expression of MAGE-A1 and MAGE-A3 in Bone Marrow from Monoclonal Gammapathy to Myeloma Patients.
Authors: Necasova Jana , Kadlecova Jitka , Spesna Renata , Penka Miroslav , Hajek Roman
Published: October 2019

Multiple myeloma (MM) is an incurable tumor characterized by multifocal clonal expansion of malignant plasma cells within bone marrow (BM), with sometimes extramedullary location, i.e. mainly in peripheral blood (PB), pleural effusion or ascites. Cancer/testis antigens (CTA) are a category of tumor antigens with normal expression restricted to a male germ cells in the testis but not in adult somatic tissues. CTA are promising candidates for cancer immunotherapy. Presently, 44 distinct CT-antigen families have been identified. Presence of RNA transcripts encoding members of the MAGE gene family, as well as NY-ESO-1, CT7/MAGE-C1 in myeloma tumor cells and cell lines has been documented. In MM, cancer germ-line specific genes are preferentially expressed in advanced disease (up to 50%) and in almost all cell lines. Most of the genes of the MAGE-A family were found to be expressed in myeloma cells, although with various incidences. The aim of this study was to evaluate the posibility of using these genes as molecular markers of the progression MGUS to MM and the early relapse of the MM. Due to rare extramedullary location of malignant plasma cells we tried to undercover if expression is detecable in PB simultaneously with expression in the BM. Total of 151 samples from BM were evaluated: 83 samples from advanced myeloma, 12 samples of patients with early stage of MM who did not required treatment (smoldering MM 4x, stage IA 8x), 41 samples of MGUS patiens and 15 samples of normal healthy donors served as control group. In 15 advanced myeloma patients we simultaneously assessed BM sample and PB sample. Total RNA was evaluated by RT-PCR and then by real-time PCR using FRET probes. For relative quantification we used G6PDH housekeeping gene as external standard. As positive control we used myeloma cell line U266. Only 4,8% (2 /41) samples from MGUS patient showed expression of MAGE-A1 or MAGE-A3. 50% (6/12) patients with early stage of MM (IA and smoldering) showed expression of MAGE-A1 or MAGE-A3. 39,7% (33/83) of the samples from the advanced myeloma patients expressed at least 1 of these genes, or both (20 cases). We tested 15 BM from normal donors. All were negative. In 15 advanced myeloma BM samples we have observed expression in 8/15 cases but no expression in PB sample. We have confirmed that expression of MAGE is not present in samples of healthy donors. There is an obvious correlation between expression of the MAGE genes and early-late stage of the disease. Our evaluation confirmed the detection of low expression levels of MAGE-type mRNA in BM from patients with MGUS and early stage of MM. It is possible that MAGE antigen monitoring may predict the evolution towards more advanced disease. But due to need of separated myeloma cells or BM samples this metod can not be used for monitoring minimal residual disease in patients with MM.
Chemokine RANTES (Regulated upon Activation, Normal T Cell Expressed and Secreted) in Multiple Myeloma and MGUS (Monoclonal Gammopathy of Undetermined Significance).
Authors: Hayashi Kunio , Nomura Shosaku , Mugitani Atsuko , Hasegawa Minoru , Uoshima Nobuhiko , Takubo Takayuki , Kami Masahiro , Komatsu Tsunehiko , Hamaki Tamae , Shimokawa Toshio
Published: October 2019

Introduction: Bone disease in patients with myeloma is the devastating complication. RANTES (CCR chemokine, Regulated upon Activation,Normal T Cell Expressed and Secreted) is involved in the bone remodeling that is made by coupled functions of osteoclasts and osteoblasts. Osteoclasts represent the RANTES receptor CCR1 and product RANTES. Osteoblasts express CCR1,3,4,5, and also product RANTES. Osteoblasts chemotaxicaly move to osteoclasts to repair the absorbed bone lesions (Shozo Yano et al., Endocrinology Vol.145, No.5, 2324–2335). The bone pathophysiology of multiple myeloma is the distortion of the well-balanced functions of osteoclast and osteobast. The change of RANTES may be representative of this distortion and the restoration of bone.

Method: Blood samples were collected from 54 patients with Multiple Myeloma, 15 patients with MGUS, 41 from donors (30 healthy people or 11 people who have no bone disease). Plasma samples were separated and analyzed by ELISA to determine the levels of RANTES.

Result: In healthy people, RANTES changes in proportion to ages with sexual difference. The value of RANTES is 155000pg/ml in20ys male, 51100pg/ml in 20ys female, 133000pg/ml in 40ys male and 78933 pg/ml in 40ys female. The difference of male and female becomes smaller and the value becomes nearly 60000pg/ml at sixty ages in male and female. Serum-bone specific alkaline phosphatase (BAP) is a biochemical indicator of bone turnover and urinary collagen type 1 cross-linked N-telopeptide (NTX) excretion normalized to creatinine (NTX/Cr) from urine sample is a bone resorption marker. At sixty ages, BAP and NTX/Cr become lower in accord with RANTES. In Myeloma cases, RANTES is about 20000pg/ml lower than the age-related value in female and about 75000pg/ml higher in male. After the treatment, RANRES increases in most of patients (both male and female) who have got response after chemotherapy. The rise of female is average 34566pg/ml but the rise of male is very small, average 5250pg/ml, because RANTES has already increased before chemotherapy. Beyond our expectation,RANTES of MGUS is almost the same as Myeloma.

Discussion: The bone remodeling activity of male is higher than female and may be easily induced by the chance of bone absorption. The data suggest that tumor growth suppression produces the recovery of the remodeling. The value of RANTES of MGUS indicates why some patients with MGUS have the punched-out lesions like Myeloma.

Conclusion: This study has turned out for the first time that RANTES is associated with the bone pathophysiology of multiple myeloma and MGUS.
Mesenchymal Stem Cells Promoted Long-Term Graft Survival of Xenogenic Mouse Islet Cells in Recipient Mice.
Authors: Tan Guangxiao , He Dongmei , Liu Gexiu
Published: October 2019

INTRODUCTION: In clinical islet transplantation, immunologic rejection is a major factor contributing to the poor engraftment of the islets. More recently, experimental evidence and preliminary clinical studies have demonstrated that mesenchymal stem cells (MSCs) have an important immune modulatory function. Here we described that co-transplantation of islet cells and donor MSCs from Balb/c mice promoted long-term graft survival of transplanted islet cells in recipient mice with diabetes.

METHODS: Bone marrow MSCs of adult Balb/c mice were cultured, and characterized by surface molecules, the typical spindle-shaped morphology, and the ability of differentiation, and islet cells were isolated by the collagenase digestion method. After MSCs (10 5 ) were transplanted via the tail vein into C57BL/6 recipient mice with diabetes induced by streptozotocin, mixed MSCs and islet cells (1000:500) were injected under the kidney capsules. To assess whether MSCs downregulated T- cell responses in vivo, the histological examination by light microscape was performed, and status of blood T-lymphocytes by flow cytometry and immunofluorescence. Before and after transplantation, blood insulin and glucose levels were monitored.

RESULTS: 7 days after transplantation, blood insulin and glucose levels in recipient mice injected with both islet cells and donor MSCs were (30.6±5.1)U/L and (6.9±0.4)mmol/L, respectively, whereas those in mice injected with islet cells alone were (19.8±4.9)U/L and (9.9±0.6)mmol/L, respectively. Moreover, Flow cytometry showed that expression of the activation markers CD25, CD69, and IFN-γ on CD4 or CD8 T cells increased significantly in latter. 6 wk after transplantation, blood insulin and glucose levels in the former still were normal, but not in latter. The histological examination showed that there was inflammation in latter, but not any morphological destructed evidence in former. The hormone release from islet cells-MSCs mixed grafts was a complete functional recovery.

CONCLUSION: Our results indicated that MSCs suppressed allogeneic T- cell responses in vivo, and MSCs promoted long-term graft survival of transplanted islet cells in recipient mice with diabetes.
Increased Risk of Monoclonal Gammopathy of Undetermined Significance (MGUS) and Lymphoproliferative Tumors among 14689 First-Degree Relatives of 4488 MGUS Patients in Sweden.
Authors: Landgren Ola , Kristinsson Sigurdur Y. , Goldin Lynn R. , Caporaso Neil E. , Blimark Cecilie , Mellqvist Ulf-Henrik , Wahlin Anders , Bjorkholm Magnus , Turesson Ingemar
Published: October 2019

Background. Evidence from multiply affected families, case-control and population-based registry studies implicate a role for genetic factors in multiple myeloma (MM). Previous studies based on small numbers have suggested excess risk of MGUS in relatives of MM patients. Aims of this large nationwide MGUS study were to quantify risks of developing MGUS and lymphoproliferative malignancies in first-degree relatives and to define characteristics of familial aggregation.

Methods. We identified 4488 MGUS patients diagnosed in all major hematology/oncology outpatient units in Sweden (1967–2005), with linkable relatives. Using the population-based central Multigenerational Registry, we obtained 17628 frequency-matched controls and first-degree relatives of cases (n=14689) and controls (n=58698). Relatives of MGUS cases and controls were linked with hospital-based outpatient registries and the central Swedish Cancer Registry to define occurrence of MGUS and lymphoproliferative tumors. Measures of familial aggregation were calculated by a marginal survival model using relatives as the cohort.

Results. Compared with controls, relative risk (RR) of MGUS was significantly increased (RR=2.84; 95% CI 1.45–5.57) in relatives of MGUS cases. Relatives of MGUS cases (vs. controls) also had excess risk of MM (RR=2.87; 95% CI 1.92–4.27), Waldenstrom’s macroglobulinemia (WM) (RR=4.94; 95% CI 1.32–18.46), and chronic lymphocytic leukemia (CLL) (RR=2.05; 95% CI 1.22–3.43). Risk-estimates were similar for various types of first-degree relatives (parents, siblings, offspring); the same was true when we estimated risks by age at MGUS of cases (above vs. below 65 yrs), and sex of relatives. Age at onset of MGUS and lymphoproliferative tumors was virtually the same for case and control relatives. There was no increased risk (RR∼1) of NHL or HL among relatives of MGUS cases.

Conclusions. Among first-degree relatives of a large nationwide MGUS cohort in Sweden, we found 2- to 3-fold elevated risks of developing MGUS, MM, WM, and CLL. These results suggest the operation of shared common germ line susceptibility genes in the pathway to MGUS and certain lymphoproliferative tumors. Better characterization of early genetic lesions mediating monoclonal plasma-cell proliferation, survival, and migration in the bone marrow microenvironment will ultimately enhance our understanding of MGUS, MM, WM, and CLL pathophysiology, provide clues to etiology, and allow identification of novel molecular targets.
PRL3 Is a Mediator of IL6/STAT3 Signaling and Defines a Population of Multiple Myeloma Patients Distinct from Those That Activate NFkB.
Authors: Sebag Michael , Stewart Keith , Shi Chang-Xin , Zhu Yuan-Xiao , Chng Wee-Joo , Keats Jonathan , Tiedemann Rodger E. , Fonseca Rafael , Chesi Marta , Bergsagel Peter Leif
Published: October 2019

Previous work in our laboratory has identified the importance of the NFB signaling pathway for plasma cell (PC) survival and accumulation in Multiple Myeloma (MM). Although up to 43% of patient PCs appear to have activated NFkB by gene expression profiling (GEP), the remainder must rely on an alternate signaling pathway for their survival. We examined our MM GEP datasets and supervised the clustering of patients according to an index of NFkB activity. The majority of patients not activating NFkB were associated with a high IL-6/Stat3 signature, which included the protein phosphatase of regenerating liver-3 (PRL-3). Interestingly, analysis of the PRL-3 promoter revealed competing binding sites for both NFkB and STAT3 within the first intron, suggesting that these play a role in the transcriptional regulation of this gene. PRL-3 has been previously demonstrated to be absent in most normal tissues while its expression in MM cells is quite elevated. As normal PCs have low levels of PRL-3 expression, we determined the upper limit of normal to be 3SD above the mean of these cells. Using this cutoff we report that elevated PRL-3 expression is observed in plasma cells from 1/15 normal individuals, 4/22 MGUS patients and 47/126 patients with either smoldering or overt MM (6.7%, 18.2% and 45.6% respectively). Less than 10% of patients were seen to have both an elevated NFkB index and high PRL-3 expression. Patients with higher expression of PRL-3 were found to have statistically significant higher mean values of creatinine, C-reactive protein, lower albumin levels and a lower overall survival in our Mayo Clinic MM dataset. To examine the role PRL-3 in MM biology we first stimulated MM cell lines with IL-6 and observed PRL-3 up-regulation in two cell lines (INA6, OPM1) but not in JK6L (an NFkB activated cell line). Using a luciferase reporter we have determined that STAT3 induction of PRL3 expression requires the first intron of the PRL3 gene which contains the putative STAT3 binding site. Finally we show that PRL-3 inhibition by shRNA leads to decreased cell growth and loss of IL-6 responsiveness in vitro in at least one cell line. Based on these results, we propose that the majority of MM patients depend on either the NFkB or STAT3 signaling pathways for their survival and that these are to a large degree mutually exclusive. Our experimental results suggest that PRL-3 plays a role in IL-6/STAT3 signaling and that it is a promising target for drug development.