bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma Hemoglobin: Potential Sources.
Authors: Westerman Maxwell , Pizzey Arnold , Hirschman Jocelyn , Cerino Mario , Well-Weiner Yonit , Ramotar Prya , Eze Ada , Lawrie Andrew , Purdy Gordon , Mackie Ian , Porter John
Published: October 2019

Plasma hemoglobin(Hb) is a measure of circulating red blood cell(RBC) destruction and is considered to be the basic indicator of intravascular hemolysis. We have examined the effects of splenectomy on levels of plasma Hb and circulating RBC-derived vesicles in patients with thalassemia intermedia (TI) and compared the results to patients with sickle cell anemia (SCA). Plasma Hb levels in splenectomized patients with TI were 48.5 ± 3.7 mg/ml (5)(Mn ± SEM)(No.of patients) and vesicle levels were 11.29 ± 1.12 x 10 3 /ul blood (9). In contrast, plasma Hb levels in patients with SCA were (14.52 ± 3.29)(21) and vesicle levels were 13.2 ± 2.57)(34). Plasma Hb levels and vesicle levels are closely associated in TI and SCA (r=0.79, p=0.01[9]; r=0.58, p=0.006[21] respectively). The finding that plasma Hb levels in patients with TI and SCA, both asplenic, differ in their relationships to corresponding and similar vesicle levels, suggests that other hemolytic factors may contribute to plasma Hb levels. Of importance would be intramedullary hemolysis which is considerable in TI. Vesiculation, which may occur with intramedullary hemolysis does not appear to contribute to circulating vesicle levels. The ratio of plasma Hb levels to vesicle counts would be a marker to distinguish intramedullary hemolysis from intravascular hemolysis. Similar considerations may apply to measures of lactic dehydrogenase (LDH) which is also an indicator of RBC destruction and intravascular hemolysis. The findings suggest that the contribution of intramedullary hemolysis as well as the contribution of intravascular hemolysis should be considered in measurements of plasma Hb.
Sleep Pattern in Patients with β-Thalassemia Minor.
Authors: Cetin Turker , Yetkin Sinan , Ozgen Fuat
Published: October 2019

Introduction: Although sleep disturbances, anxiety, and sadness are some of the most common symptoms in patients with chronic anemia, there is little knowledge about sleep structures in patients with chronic anemia such as thalassemia. The aim of this study is to investigate sleep structure in patients with beta-thalassemia minor.

Methods: Seventeen drug free subjects with beta-thalassemia minor and age-matched healthy subjects were enrolled in the study. The patients were diagnosed by complete blood count including reticulocyte count and hemoglobin electrophoresis. In addition, blood smear, erythrocyte sedimentation rate, serum level of urea, creatinine, indirect bilirubin, lactate dehydrogenase, ferritin, folate, vitamin B12, and thyroid hormones were determined. The other causes of anemia were ruled out. Subjects also were screened for excessive daytime sleepiness with the Epworth sleepiness scale, and for insomnia using insomnia severity index. All subjects slept in laboratory for two consecutive nights. First night served as adjustment nights. The second night results were used for analysis. Their sleep patterns were compared with healthy normal controls. Sleep was recorded and scored according to the criteria of Rechtschafffen and Kales.

Results: Four female and thirteen male patients (mean age, 26.1 ± 5.9 years) completed both nights of sleep recordings. In patients groups, one patient complained snoring and daytime sleepiness. Nine of them complained daytime weakness. Ten patients were anemic (Hb < 13,2 g/dL for men, <11,7 g/dL for women). In polysomnographic investigation, the patients with β-thalassemia minor showed increased total sleep time, sleep period time, PLM index and the increased percentage of REM sleep. The percentage of stage 4, and REM latency are decreased. Sleep fragmentation and periodic leg movement syndrome (PLMS) have been reported.

Conclusion: The disturbances of sleep continuity was prominent finding in this polysomnographic study. They also showed decreased REM latency and the increased percentage of REM sleep, although they didn’t have major depressive disorders.

Polysomnographic variables in patients with beta-thalassemia minor Polysomnographic variables Thalassemia (n: 17) Normal Controls (n: 17) Results (P value) NS: not sinificant, REM: rapid eye movement, PLM: periodic leg movement Sleep effiency 90.8±4.6 93.7±2.1 NS Total time in bed (min) 467.6±60.1 415.1±51.1 .008 Total sleep time (min) 425.8.4±65.1 388.5±49.2 .031 Sleep period time (min) 453.2.4±62.0 404.4±52.2 .012 Sleep latency (min) 9.9±9.0 8.2±5.2 NS Percentage of stage 1 (SPT) 2.5±1.0 1.8±1.1 NS Percentage of stage 2 (SPT) 61.9±6.9 64.1±7.3 NS Percentage of stage 3 (SPT) 4.8±1.9 6.5±2.4 NS Percentage of stage 4 (SPT) 8.0±6.4 12.2±6.1 .022 Percentage of stage REM (SPT) 16.3±3.8 12.5±3.4 .006 REM latency 67.9±25.0 96.8±32.5 .016 PLM index 7.4 ± 5.7 0.0 .000
Lack of Heart Iron Overload in Patients with Thalassemia Intermedia.
Authors: Galanello Renzo , Tanner Mark , Matta Gildo , Barella Susanna , Bina Patrizio , Origa Raffaella
Published: October 2019

Iron loading in patients with thalassemia intermedia occurs slowly and is mainly due to increased gastrointestinal iron absorption secondary to chronic anemia, while in patients with thalassemia major iron overload is faster and secondary to the chronic transfusion therapy. Moreover, iron accumulation in thalassemia intermedia is prevalent in parenchymal cells, while in thalassemia major iron derived from red cell breakdown firstly accumulates in the reticulo-endothelial cells and subsequently in the parenchymal cells. Although heart disease represents the main determinant of survival in beta thalassemias, the cardiac complications are different in the two clinical forms, that are thalassemia major and thalassemia intermedia. In this study we evaluated liver and iron overload in patients with thalassemia intermedia.

We have studied 8 patients with thalassemia intermedia with a mean age of 36 ± 12 years. All these patients were homozygotes for the beta zero 39 non-sense mutation (C→T) and were never transfused or had received only sporadic transfusions (less than 10 blood units throughout their life). Myocardial iron (heart T2*, Anderson et al 2001), echocardiographic left ventricular ejection fraction, serum ferritin (mean of the last 5 years) and hemoglobin (mean of the last 2 years) have been evaluated in each patient. Hepatic iron content was determined in 5 patients with atomic absorption after liver biopsy. Six patients were on chelation therapy with subcutaneous desferrioxamine (mean 2 ± 1 grams/week).

Mean ferritin was 637 ± 497 ng/ml and mean hemoglobin 8.0 ± 1.0 g/dl. Heart T2* was normal in all patients (mean 46 ± 11 msec, range 36 – 62 msec). The mean left ventricle ejection fraction was 61 ± 6 % (range 51 – 70 %). Echocardiogram showed in all the patients a mild enlargement of both ventricles. Three patients had pulmonary hypertension and two had an extrasystolic arrhythmia. To our knowledge this is the first study reporting the results of heart T2* in thalassemia intermedia. As a consequence of the mechanism and rate of accumulation patients with thalassemia intermedia do not have heart iron overload, while liver iron concentration is quite relevant. Cardiac complications in thalassemia intermedia are mainly due to the hyperdynamic circulation associated with chronic anemia and to pulmonary hypertension.
Free Versus Neutrophil-Bound Antibodies in 100 Consecutive Italian Children with Chronic Neutropenia.
Authors: Porretti Laura , Lopa Raffaella , Cattaneo Alessandra , Portararo Paola , Ghilardi Roberta , Farruggia Piero , Martire Baldassarre , Corti Alessandra , Rebulla Paolo
Published: October 2019

Chronic neutropenia (CN) is defined as a blood absolute neutrophil count (NC) below 1500/ml, lasting for at least 6 months. We report the results of serological screening for anti-neutrophil antibodies, performed in our laboratory during 2002–2005, in 100 consecutive children affected from CN and followed in 4 Italian pediatric centers.

The patients, 65 males (m) and 35 females (f), had a median age of 18 months (range: 1–180). Both IgG bound to patient neutrophils (CD16+ cells) and the patient serum reactivity against healthy donor granulocytes (Direct and Indirect Granulocyte-Immunofluorescence Test, D-GIFT and I-GIFT) were assessed by flow-cytometry. The results are shown in the table, grouped according to the pattern of reactivity observed with D- and I-GIFT:

A NC above 1500/ μl on the day of serological screening was found in 5.5%, 22.4%, 0% and 35% of patients in groups 1, 2, 3 and 4, respectively. Secondary CN, usually in association with other autoimmune disorders or malignant diseases, was reported in 5.5%, 10.3%, 0% and 50% of patients in groups 1, 2, 3 and 4, respectively. Infections were present in 72%, 79%, 25% and 8% of patients in groups 1, 2, 3 and 4, respectively. In group 2, 50 of the 58 patients (86%), who didn’t show anti-neutrophil antibodies in their sera, presented neutropenia with concomitant infections. In these patients only D-GIFT was able to detect neutrophil antibodies.

Patients testing positive for I-GIFT had more severe neutropenia. Patients negative by both techniques were mostly found to have non-immunomediated neutropenia at clinical follow up. Although many laboratories perform only an indirect test to detect antineutrophil antibodies, our results suggest that direct test is more sensitive in identifying immunomediated chronic neutropenia.

table group GIFT no. of patients m/f NC (cells/μl)* WBC (x102/μl)* * Mean ± SD 1 D+ I+ 18 15/3 757 ± 520 7.58 ± 3.1 2 D+ I− 58 36/22 968 ± 818 7.29 ± 5.3 3 D– I+ 4 3/1 525 ± 369 6.71 ± 2.3 4 D- I− 20 11/9 1693 ± 1510 8.28 ± 3.2
Zoledronic Acid Inhibits the Function of Toll-Like Receptor 4 Ligand Activated Dendritic Cells.
Authors: Bringmann Anita , Schmidt Susanne M. , Brauer Katharina M. , von Schwarzenberg Karin , Werth Daniela , Kanz Lothar , Brossart Peter
Published: October 2019

Zoledronic acid (ZA; Zometa®) is a cyclic nitrogen-containing bisphosphonate with anti-tumor activity used to treat patients with malignant diseases. As a side effect, ZA induces inflammatory responses which are accompanied by expansion of gd T cells that probably mediate its anti-tumor activity. In our study we analyzed the effects of ZA on the function and differentiation of monocyte-derived dendritic cells (DC). We investigated the differentiation of immature and LPS-stimulated DCs incubated with different ZA concentrations which are usually achieved in treated patients. Expression of DC-specific marker molecules was assessed by FACS. Furthermore, the activation state and involvement of different signal transduction pathways were evaluated by Western blot. To monitor DC function we used a migration assay and analyzed the cytokine secretion. We found that DC activation with TLR4 ligand LPS is modulated by ZA. We show that the expression of the surface maturation markers CD80, CD86, CD40, CD54, CD83 and DC-SIGN is diminished with increasing ZA levels upon LPS activation. The migratory capacity of LPS stimulated DCs towards the chemokine CCL19/MIP-3beta and the secretion of the activatory cytokine IL-12 are reduced at higher ZA levels. Increasing ZA concentrations lead to the down-regulation MyD88 and diminish the nuclear localization of NF-kB family members RelB and c-Rel as well as interferon-regulatory factors IRF-3 and IRF-8 in LPS activated DCs. Hence, ZA concentrations used to treat patients have inhibitory effects on DC differentiation and activation. This might lead to immuno-suppression and negatively affect tumor rejection or result in infectious complications.
Genetic Screening of Familial Mediterranean Fever in Japan.
Authors: Miyahara Yasuko , Yamashita Kouhei , Miyoshi Takashi , Takaori Akifumi , Sasada Masataka , Uchiyama Takashi
Published: October 2019

There have been few reports of the patients with Familial Mediterranean Fever (FMF) in Japan, probably because FMF patients are preoccupied to be rare in Japan. We experienced 10 cases likely to be clinically diagnosed as FMF with periodic fever of unknown origin. FMF is an autosomal recessive disease resulting from the genetic mutations in the FMF gene (MEFV), which codes for a protein named Pyrin. Pyrin is expressed in mainly polymorphonuclear cells and monocytes and it is proposed that it regulates inflammation. The MEFV gene is located on chromosome 16p13.3 and comprises 10 exons. Several mutations in the MEFV gene have been identified, however the mutations are mostly located in exon 2 and 10. Therefore, we performed genetic screening of exon 2 and 10 in the 10 patient samples. The median age of the patients was 34 (17–49) years old. They are four males and six females. DNA was isolated from polymorphonuclear cells of the patients and PCR was performed with selective primers of exon 2 and 10 of MEFV gene, respectively. Thereafter, direct sequence of exon 2 and 10 of the PCR products was performed. As a result, the mutations of E148Q in exon 2 and M694I in exon 10, which are commonly observed in previous reports, were identified in seven and six out of 10 patients, respectively. All patients had either E148Q or M694I mutation. Three patients have both E148Q and M694I mutations. One 50-year-old female patient, who had a homozygous M694I mutation, suffered from severe renal AA amyloidosis. The mutation at M694 has been mostly reported as M694V, particularly in Mediterranean countries, however interestingly, all of the mutations of M694 were M694I, not M694V in our 10 Japanese patients. It is reported that healthy carrier frequency of the E148Q mutation was about 16% in Japan, so it is suggested that E148Q mutation may be profoundly involved in cause of disease, because E148Q mutation is observed among 70% of our patients. We herein report the unique genetic features of FMF patients in Japan. A further large scale of investigation would be necessary for confirming the significance of E148Q and M694I mutations in FMF patients in Japan.
Variation of Serum Lipid Profile during Enzyme Replacement Therapy, in Patients with Type 1 Gauchers Disease.
Authors: Symeonidis Argiris , Hughes Derralynn , Richfield Linda , Mehta Atul
Published: October 2019

Enzyme Replacement Therapy (ERT) has dramatically changed the clinical course and prognosis of patients with Gaucher disease. We have assessed the extent to which the recently defined Goals of Therapy (Pastores et al, Semin Hematol 41(suppl 5):4–14) in 9 major fields (anemia, thrombocytopenia, hepatomegaly, splenomegaly, skeletal pathology,biomarkers, pulmonary manifestations, growth and functional health and well being) were achieved in our cohort of patients with type I Gaucher disease. Fifty-seven patients (27 females, 30 males, median age at diagnosis 30 years (range 2–77 years) and at start of ERT 39 years (range 5–79 years) were assessed. The median period of ERT was 94 months (range 24–155 months). Patients on ERT for less than 2 years were excluded. Twenty five specific goals are defined within the 9 fields. The median overall success rate was 20 of 25 goals or 80% per patient (range 12–25 goals or 60–100%). The success rate for 9 goals was 100% ( normalization of hemoglobin 1 year post-splenectomy, no major bleeding 1 year after ERT start, doubling of baseline platelet count in severely thrombocytopenic patients 2 years after ERT start, maintenance of platelet count in non-thrombocytopenic patients, prevention of bone crises, reversion of hepatopulmonary syndrome and dependence on oxygen, amelioration of pulmonary hypertension and prevention of rapid deterioration of pulmonary disease and sudden death). Higher success rates were achieved for the goals related to thrombocytopenia (97.9%) and pulmonary manifestations (97%) although in the latter case the applicability was low. Lower success rates were achieved for the goals related to bone manifestations (78.7%). There was no significant difference in the success rate between males and females, splenectomised and non-splenectomised patients, and between patients treated less or more than 6 years. However patients started on ERT before the 40th year of age exhibited significantly higher success rate as compared to patients started after the 40th year (p=0.047) and there was a significant inverse correlation between overall success rate and patient’s age at treatment start (r = − 0.437). Finally, the success rate was higher among double heterozygotes, carrying the N370S mutation, as compared to patients homozygous for the N370S mutation (89.2±9.6% vs. 80.2±12.9%, p=0.016).
Identification of Specific Chemokines and Apoptosis Molecules in Pediatric Idiopathic Neutropenia.
Authors: Callejas Angel , Nadeau Kari , Bakshi Kokil , Wong Wendy , Carroll Tanya , Lau Ken , Yang Ya-Wun , Schilling Jim , Clayberger Carol , Krensky Alan , Jeng Michael
Published: October 2019

In order to explore possible mechanisms for idiopathic neutropenia, molecular and functional analyses on neutrophils and plasma of pediatric neutropenic patients were performed. 22 subjects, 8 acute and 14 chronic neutropenia, aged 5 months to 12 years, were recruited through Lucile Salter Packard Children’s Hospital hematology clinics with an IRB-approved consent. Patients with known neutrophil elastase mutations were excluded. We first examined whether the neutrophils of neutropenia patients had increased expression of molecules associated with cell death. QT-PCR was performed and transcript levels in neutrophils of neutropenia subjects were compared against those of normal subjects. In addition, to assess if a factor(s) in the plasma of neutropenia patients induced neutrophil cell death, neutrophil survival assays were done by incubating neutrophils isolated from normal healthy controls with 200 ul of undiluted plasma from chronic neutropenic subjects. Neutrophil death was then analyzed via Annexin V-PI staining. Finally, to identify specific plasma proteins, which may have a potential role in neutrophil-mediated cell death, 2D gels were run on neutropenic and control plasma.

Results: Due to limited sample size, assays were not run on all subjects. Acute and chronic neutropenic patients (n=10) experienced an increase in FAS, an apoptotic marker and protein known to be associated with neutrophil cell death. There were no significant differences in fold expression between acute and chronic patients. Neutrophil survival assays (n=9) revealed a median of 6.5 and mean 6.8 fold (range 2–24 fold) higher level of plasma-mediated cell death with neutropenic plasma. Preliminary studies of 2D gels on plasma revealed differential expression of inflammatory proteins in neutropenia patients compared to normal healthy controls. Further investigation on these proteins is ongoing. In summary, rapid cell death can be induced in normal neutrophils through neutropenia plasma incubation. Further identification of these apoptosis-associated plasma proteins is in process. In addition, the increase in certain apoptosis-associated chemokines and cytokines could lead to further target identification for diagnostic and/or treatment purposes of neutropenia.

Table 1 Patient Number Trascripts Expressed in Neutrophils (Fold Over Trancripts from Neutrophil of Normal, Healthy Control *= acute neutropenic patient TNF-α R1 FAS 1 2 13 2* 1.5 12.5 3 2 10 4 1 14 5* 2 12 6 2 13.5 7* 1 11 8* 2 10.5 9 2 7 10 1.5 10
Proliferative Function of Bone Marrow Hematopoietic Stem Cells of Patients with Systemic Lupus Erythematosus.
Authors: Fu Rong , Deng Jizi , Yuan Shang , Gong Lu , Sun Jun , Shao Zonghong
Published: October 2019

Objective:To investigate the bone marrow function of stem cells in patients with systemic lupus erythematosus (SLE), and to explore the pathogenesis of cytopenia in patients with SLE.

Methods:Sixteen patients with SLE and 10 healthy controls were studied for in vitro cultures of CFU-E, BFU-E and CFU-GM from bone marrow mononuclear cells.

Results:49.43±34.65 of CFU-E colonies per 105 BMMCs, 49.2±39.61 of CFU-GM colonies per 105 BMMNCs and 3.01±4.54 of BFU-E colonies per 105 BMMCs were seen in the group of SLE patients with normal blood count. 143.33±152.8 of CFU-E colonies per 105 BMMCs, 122.2±169.87 of CFU-GM colonies per 105 BMMCs, and 2.76±3.28 of BFU-E colonies per 105 BMMCs were seen in the group of SLE with hemocytopenia. 66.3±12.95 of CFU-E colonies per 105 BMMCs, 36.7±11.95 of CFU-GM colonies per 105 BMMCs, and 36±11.66 of BFU-E colonies per 105 BMMCs were seen in the group of normal controls.The quantity of BFU-E colonies per 105 BMMCs in SLE patients were significant lower than that in normal controls. There were no significant differences of the quantities of CFU-GM and CFU-E colonies between the SLE patients and normal controls.

Conclusions: 1. There were no significant differences of CFU-GM and CFU-E colonies between the SLE patients and normal controls, that suggested that SLE patients have normal proliferate function of bone marrow hematopoietic stem cells.2 There were significant lower BFU-E colonies per 105 BMMCs in SLE patients than that in normal controls.
The Frequency of Occurrence of Koxakie-Viruses at the Patients with Hematological Malignancies and Pulmonary Infections.
Authors: Domnikova Natalia P. , Bryakotnina Elena V. , Evstropov Alexandr N.
Published: October 2019

Aim: To determine frequency of occurrence of Koxakie-viruses at the patients with hematological malignancies and pulmonary infections.

Methods: A total 53 patients with hematological malignancies and pulmonary infections, admitted of the Novosibirsk Regional Clinical Hospital were examined. The analysis of serum in first three days was carried out at occurrence of attributes of pulmonary infection (1-st serum) and in dynamics - in 3 weeks from a beginning of an infection (2-nd serum). Definition of antibodies to a virus Koxakie-B3 (strain “Nancy”) was made in reaction of neutralization in 3-daily cultures Hep-2. Results took into account after approach of cytopathogenic effect in cultures Hep-2, containing 100 dozes of a virus in control titration. Limiting cultivation of serum, neutralizing of 100 cytopathogenic dozes of a virus, was considered as its titer.

Results: At 43 of 53 examined patients on admission or in a debut of an infection (at the patients with the developed infectious complication) the neutralized antibodies to a virus Koxakie-B3 were found out in serum. Thus the titer of antibodies from 1:4 up to 1:8 was revealed at 28 patients, at 14 it has made 1:16 – 1:32 and only at one patient on admission the titer of antibodies to a virus Koxakie B3 was 1:64. The total titer of virusneutrolized antibodies on admission or in a debut of an infection was accordingly 2,03 log2 and 2,84 log2. At research of serum taken after three weeks of stay in hospital of the patients, at which the infection was not developed (after a beginning of an infection at the patients with the developed infectious complication), in both groups the increase of a titer of antibodies in 1,97 and 1,5 times was marked, accordingly, and the total titer of antibodies in log2 has made 4,0 and 4,3. It is necessary to take into account, however, that at an estimation of serologic status at virus infections the absolute parameters of antibodies titers, how many their growth in pair serums matter not so much. Thus, authentic confirmation of the current virus infection is the increase of a titer of antibodies in dynamics in 4 times and more. The essential distinctions between two chosen groups were here again revealed. And, in group with hematological malignancies without infectious complications the four-multiple increase of a antibodies titer was marked only at 4 of 26 patients, and in group with infectious complications the authentic growth of a level of antibodies to a virus Koxakie-B3 was marked more than at half of surveyed patients (15 of 27), OR=6,88; 95%CI=1,61–31,87.

Conclusions: On the basis of the received results it is possible to conclude, that Koxakie B3 - virus infection is the factor enough frequently accompanying clinical current of pulmonary infections in patients with hematological malignancies.
GERD, Helicobacter pylori and MGUS-Is There a Relationtionship?.
Authors: Singh Jaswinder , Malani Ashok , Pabla Maninder , Patel Vijay , Gupta Chakshu , Kambhampati Suman
Published: October 2019

Background: The prevalence of clinically significant gastro esophageal reflux (GERD) in the general population varies between 3–6%. We have observed GERD to be one of the major complaints of our patient population with monoclonal gammopathy of unknown significance (MGUS). There is no literature available on the incidence of GERD in MGUS. However, there have been conflicting reports on the prevalence of helicobacter pylori (H. pylori) and MGUS.

Objective: To study the incidence and relationship of GERD, H. pylori and MGUS.

Methods: We identified all patients diagnosed with MGUS at the Kansas City VA Medical Center over the last 20 years. Out of the 417 patients identified with MGUS, 394 patients fulfilled the inclusion criteria. All patients with GERD were evaluated by gastroenterologists at our institution who determined their diagnostic and therapeutic management. Charts on these patients were then reviewed for endoscopic findings, pathological biopsies, H. pylori staining on histology, serum protein electrophoresis with immunofixation (SPEP/IFE), immunoglobulin levels, and bone marrow biopsies.

Results: In this study, 41% patients with MGUS were noted to have moderate to severe symptoms of GERD (162/394). The median age of patients with and without GERD was similar (72 versus 71 years). Endoscopies were performed on 60% of the patients with GERD (97 of 162). Histology was positively stained for H. pylori in 27.8% patients (27/97). We did not find any correlation between H. pylori positivity and the type or level of monoclonal protein. In addition, we observed no change in immunoglobulin levels following treatment and eradication of H. pylori.

Conclusion: The incidence of GERD is significantly higher in patients with MGUS than in the general population. In our study, no correlation was seen between H. pylori infection and MGUS.
Analysis of the T-Cell Receptor Vα Gene Repertoire and Clonal Expansion in the Benzene-Exposed Group.
Authors: Li Bo , Li Yangqiu , Chen Shaohua , Yang Lijian , Chen Jiayu , Liu Weiwei
Published: October 2019

Benzene is a potent human leukemogen, but its mechanism of hematotoxicity is uncertain. It is well know that benzene inhibit T cells proliferation. Several reports revealed that clonal expansion TCR Vβ T cells could be found in workers exposed to benzene. In this study we observe the distribution of TCR Vα gene repertoire and clonal expansion in peripheral blood mononuclear cells from 9 donors and 16 workers exposed to benzene. Complementarity determining region 3 (CDR3) of TCR Vα subfamily genes were amplified using RT-PCR. The PCR products were further analyzed by genescan to evaluating clonality of T cells. 29 Vα subfamily could be detected in 9 donors.1~11 Vα subfamilies were identified in all but one of the workers studied. The most frequently expressed Vα subfamily were Vα3, Vα12 and Vα19 (68.8%), Vα14(56.3%), with a lower expression rate found in Vα5,Vα15, Vα16, Vα 22, Vα 23 and Vα 24 (6.3%). Clonal expansion T cells in one or more Vα subfamily were found in 12 out of all workers studied, including oligoclonal, oligocolonal trend and bioclonal patterns. The frequency of clonal expansion T cells in Vα12, Vα14 and Vα19 subfamilies were higher than others. In conclusion, skewed distribution and clonal expansion of TCR Vα subfamily T cells could be found in workers exposed to benzene. Vα12, Vα14 and Vα19 subfamilies may be highly sensitive to benzene exposed. This is the first report of clonal expansion TCR Vα T cells in the benzene-exposed group. The bias pattern of TCR Vα T cells may be due to the immune cytotocicity from benzene. However, whether the oligoclonality in some Vα subfamilies reflect the phenomenon of clone absense or may be a response clone to benzene-related impairment during exposed to benzene, remains an open question.
Fate Discrimination of Normal B Cells When Cross-Linked with Anti-IgM Antibodies of Different Affinities and Valence.
Authors: Paul Santanu , AbuHelu Rasmi F. , Mongini Patricia K. , Steinberg Bettie , Rai Kanti R. , Chiorazzi Nicholas
Published: October 2019

The B-cell receptor (BCR) is a key signaling molecule that engages antigens and triggers pathways that induce survival, proliferation, differentiation, anergy, or apoptosis. The “choice” of which functional fate the B cell makes is decided upon by a series of receptor and cellular interactions. The potential for ligand-initiated signal transduction through the BCR is assessed in terms of ligand binding affinity for membrane Ig. We have used a series of anti-IgM H chain mAb that differ in their affinities (HB57 − Ka = 5x108 “high”, Mu53 − Ka = 2x107 “intermediate”, and P24 − Ka = ~2x106 M−1 “low”) in both soluble and immobilized forms as models for antigenic stimulation of the BCR, and assessed the response of normal B cells isolated by negative selection from peripheral blood. Proliferative responses were observed upon cross-linking the BCR with antibodies of all affinities at 10 ug/ml of immobilized antibodies for 72 hours, with a modest stimulation at 1ug/ml and no stimulation at either 0.01 or 0.1 ug/ml. Interestingly, the low affinity antibody P24 showed the highest proliferative response (3.3 fold), while stimulation with the moderate affinity antibody was intermediate (2.2 fold) and the response of the high affinity antibody, was negligible. Soluble antibodies did not stimulate proliferation at any concentration tested. When B cells were co-incubated with soluble anti-IgM antibodies plus IL 4,10ug/ml of either high or intermediate affinity induced proliferation with stimulation indices of 2.5 and 1.9, respectively, while the low affinity antibody still had no effect. Co-incubation with IL 4 and immobilized anti-IgM antibodies induced the same pattern of responses as seen with antibody alone, slightly increasing proliferation induced with 1 ug/ml low and intermediate and markedly increasing stimulation by all three antibodies at 10ug/ml. Our results suggest that even in the absence of T cell help, normal B cells can be activated via the BCR upon cross-linking and the efficiency at which this is accomplished is largely dependent on affinity for the BCR.
Absolute Lymphocyte Count Predicts Overall Survival in T Cell Lymphomas.
Authors: Behl Deepti , Ristow Kay M. , Markovic Svetomir , Witzig Thomas E. , Habermann Thomas M. , Colgan Joseph P. , Micallef Ivana N.M. , Johnston Patrick B. , Ansell Stephen , Yasenchak Christopher A. , Manchanda Rajnish , Porrata Luis F.
Published: October 2019

Background: Absolute lymphocyte count (ALC) recovery is an independent prognostic factor for survival for different hematological malignancies treated with autologous stem cell transplantation. Recently, we reported superior overall survival (OS) in patients with follicular lymphomas that presented with a higher ALC (ALC ≥ 1 x 109/L) at diagnosis. The role of ALC at diagnosis on survival in T cell lymphomas is not known.

Methods: All patients evaluated at the Mayo Clinic for T cell lymphomas from October 1984 until April 2005 were analyzed in the study. The primary objective of the study was to assess the role of ALC at diagnosis and survival in T cell lymphomas. ALC at diagnosis was obtained from the standard complete blood cell count (CBC).

Results: 223 patients with T cell lymphomas were included in the study. The histology included: peripheral T-cell lymphoma-unspecified (40%), anaplastic large cell (13%), angioimmunoblastic (10%), angiocentric (4%), intestinal T-cell (2%), and other (31%). The median follow-up was 18.6 months (range: 1 month – 229 months [19 years]). The sample population included 60% male and 40% females with a median age of 60 years (15–90 years). The median ALC at diagnosis was 1.2 x 109/L (range: 0.04–6.7 x 109/L). ALC as a continuous variable was identified as predictor for OS in the univariate analysis (HR= 0.690, 95%CI: 0.542–0.862, p < 0.0006). ALC ≥ 1 x 109/L was selected as the dichotomized value because it yielded the maximum difference in survival by c2 analysis looking at different cut-point from 0.6 to 1.7 x 109/L that were included between the 25% and 75% quartiles. Univariately, ALC ≥ 1 x 109/L was also identified as a predictor for OS (HR = 0.682, 95%CL= 0.578–0.804, p < 0.0001). Patients (N = 122) with an ALC ≥ 1 x 109/L experienced superior survival compared to patients (N = 101) with an ALC < 1 x 109/L [median OS: 76.3 vs 10.6 months, respectively; and 5 years OS estimates of 53% vs 23%, respectively, p < 0.0001]. When ALC ≥ 1 x 109/L was compared to the international prognostic index (IPI), ALC ≥ 1 x 109/L remained an independent predictor for survival in the multivariate analysis (HR = 0.779, 95%CL: 0.655–0.925, p < 0.004).

Conclusion: This study demonstrates ALC as an independent predictor for OS in T cell lymphomas, suggesting that the host immune system affects survival in this group of patients.
Selective Lympholysis: A Unique Method Using the Double-Dye Technique.
Authors: Owens William J. , Cesario Thomas C. , Shanbrom Edward
Published: October 2019

Many methods are utilized to destroy mononuclear cells (primarily lymphocytes) either for neoplasm or immunosuppression. Neither radiation nor chemotherapy are truly selective or completely successful. The concept of “Double-Dye” treatment of blood for transfusion has been developed in order to inactivate parasites, bacteria and viruses (J Thromb Haemost2003; 1 Supplement 1 July: P1114). In recent studies, it has been observed that this same “Double-Dye” concept presents the possibility of very selectively eliminating lymphocytes (mononuclear cells) without affecting neutrophils in whole blood.

To demonstrate the selectivity of dyes for lymphocytic/mononuclear cell types, two sets of experiments were performed. In the first, 0.3% (w/v) of the “Double Dye’ solution was added to several normal citrated whole blood samples to assess the effect on normal cells, compared to an untreated control. At 24hours post treatment, the lymphocyte count in the treated sample had dropped more than 80%, while little effect on neutrophils was noted. The control counts showed little change for either lymphocytes or neutrophils.

Table 1. Lymphocyte reduction by 0.3%(w/v) Double-dye solution (units: cells/mm3). 0hr Control 0.3% Dye WBC 6850 ± 560 6920 ± 630 Neut 5030 ± 375 5100 ± 420 Lymph 1485 ± 220 1490 ± 265 24hr Control 0.3% Dye WBC 6700 ± 480 5300 ± 505 Neut 5025 ± 360 5025 ± 420 Lymph 1474 ± 240 275 ± 75

In the second series of experiments, 0.3%(w/v) “Double-Dye” solution or 0.15%(w/v) Crystal Violet or 0.15%(w/v) Methylene Blue were added to two T-cell leukemia lines (Jurkat, L1210), with a non-malignant, non-lymphocytic cell line (WISH) for the control. The combination of dyes showed the most potent activity against the lymphocytic lines, while the control was virtually unaffected.

Table 2: Viability of cell lines after 24 hour exposure to dye solutions. Jurkat L1210 WISH Control 100% ± 2% 100% ± 4% 99% ± 2% 0.3% Double Dye 37% ± 5% 12% ± 4% 88% ± 9% 0.15% Meth. Blue 58% ± 12% 52% ± 9% 100% ± 3% 0.15% Cr. Violet 90% ± 12% 92% ± 10% 99% ± 4%

The novel use of these dyes reported here coincided with the recent interest in utilizing methylene blue to increase transfusion safety, but recognizing that the concurrent need to photoactivate was too toxic to certain proteins and didn’t inactivate all pathogens (Transfusion2003; 43(9): 1238–47). Studies investigating the in-vivo efficacy of these novel immunosuppressive and chemotherapeutic methods are currently underway.
CTLA-4 A49G Polymorphism and Autoimmune Blood Diseases.
Authors: Akturk Faruk , Hancer Veysel Sabri , Guvenc Serkan , Artim-esen Bahar , Diz-Kucukkaya Reyhan
Published: October 2019

The cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is expressed on T lymphocytes, and inhibits the T cell responses. CTLA-4 A49G polymorphism was found to be associated with various autoimmune diseases in recent studies. In this study we evaluated the frequency of CTLA-4 A49G polymorphism in patients with autoimmune hemolytic anemia and autoimmune thrombocytopenia. One hundred and fifty healthy Turkish subjects, 46 patients with autoimmune hemolytic anemia (AIHA) and 62 patients with autoimmune trombocytopenic purpura (ITP) were included to our study. In healthy controls; genotype frequencies were 48% for AA, 47.3% for AG and 4.7% for GG. A-allele frequency was 71% and G allele frequency was 29%. Allele frequencies were similar to the other European countries. In AIHA group; 21 patients had AA (45.6%), 21 had AG (45.6%), and 4 had GG (8.8%) genotype. A allele frequency was 68.4%, and G allele frequency was 31.6% which are similar to healthy controls. In ITP group; 38 had AA (61.3.%), 21 had AG (33.9%), and 3 had GG (4.8%) genotype. A allele frequency was 78.4 %, and G allele frequency was 21.6%. We found no difference between ITP patients and controls for CTLA-4 A49G polymorphism. In subgroup analysis however, we found that all CLL patients with AIHA had CTLA-4 A49G polymorphism (3 had AG, 1 had GG). This may suggest that CTLA-4 A49G polymorphism is an important risk factor for the development of AIHA in CLL patients. Further genetic and clinical studies are required to understand the exact role of CTLA-4 A49G polymorphism in the development of autoimmune diseases.
Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.
Authors: Brischwein Klaus , Hammond Scott A. , Parr Larissa , Bernd Schlereth , Locher Mathias , Kufer Peter , Baeuerle Patrick A.
Published: October 2019

Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells.

Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array.

Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude.

Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.
Dendritic Cells Can Be Primed to Concurrently Activate Both Unconventional Gamma/delta and Conventional alpha/beta T Cells.
Authors: Fiore Francesca , Castella Barbara , Nuschak Barbara , Bertieri Raffaello , Mariani Sara , Zappia Serena , Coscia Marta , Boccadoro Mario , Massaia Massimo
Published: October 2019

Vgamma9/Vdelta2 (gamma/delta) T cells represent the major subset of unconventional T cells circulating in the peripheral blood. Gamma/delta T cells play a major role in immune defenses against microbes, stressed cells and tumor cells. This property is based on their capability to naturally recognize phosphoantigens (pAgs), which are produced via the mevalonate (Mev) or the DOXP pathway in mammalian and nonmammalian cells, and induced self-ligands, which are de novo expressed or upregulated on the surface of stressed or tumor cells. Interestingly, gamma/delta T cells can also be activated by aminobisphosphonates (ABP)-treated monocytes. We have previously shown that ABP specifically target the Mev pathway of monocytes and induce the accumulation of phosphorylated Mev metabolites naturally recognized by gamma/delta T cells. The aim of this work was to determine whether ABP-treated dendritic cells (DC) can also activate gamma/delta T cells and whether this activation, if any, is detrimental or beneficial to the generation of antigen (Ag)-specific MHC-restricted immune responses mediated by conventional alpha/beta T cells. To this end, we have generated highly purified immature (iDC) and mature DC (mDC) from peripheral blood monocytes of healthy donors and incubated with zoledronic acid (Zol) for 24 hours. Zol is the most potent ABP currently available for clinical use. Zol treatment did not affect the phenotype and immunostimulatory properties of iDC and mDC. Zol-treated iDC and mDC induced a rapid and vigorous expansion of central memory and effector memory gamma/delta T cells. Zol-treated iDC were more potent inducers of gamma/delta T-cell activation than mDC and monocytes. Activated gamma/delta T cells displayed antitumor activity and expressed on the cell surface the appropriate antigen repertoire to target secondary lymphoid organs and exert costimulatory activity on conventional alpha/beta T cells. Indeed, an in vitro model showed that antigen-specific MHC-restricted immune responses againt the influenza matrix peptide were significantly improved by the concurrent activation of gamma/delta T cells. This is the first report showing that: 1) DC can simultaneously be primed to activate both gamma/delta and alpha/beta T cells; 2) the former act as cellular adjuvants for the development of adaptive immune responses. In conclusion, large numbers of gamma/delta T cells with effector and costimulatory activities can rapidly be generated by Zol-treated iDC/mDC. This strategy is worth of further investigation to improve adoptive cell therapy and vaccine interventions against tumors and infections.
Protease Activated Receptors Differentially Regulate Human Platelet Activation through Phosphatidic Acid-Dependent DAG Formation.
Authors: Holinstat Michael , Hamm Heidi E.
Published: October 2019

Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through PAR1 and PAR4, plays a central role in regulation of human platelet function as it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intra-cellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin’s action. In this study we show that platelet activation by PAR1 requires a signaling pathway that generates diacylglycerol (DAG) formation independent of its PLC-mediated production. Specifically, inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine (PC)-mediated phosphatidic acid (PA) formation with a primary alcohol significantly attenuated platelet activation by PAR1. Platelet activation by thrombin or PAR4 was insensitive to these inhibitors. Further, activation of Rap1 was significantly attenuated by these inhibitors following stimulation by PAR1, but not thrombin or PAR4. In the mouse, evidence indicates that Rap1 can be activated through the DAG-sensitive guanine nucleotide exchange factor CalDAG-GEF1, and our data indicate that the differential signaling of PARs may, in part, occur through formation of distinct DAG species following thrombin stimulation.
Impaired Viability, Platelet Survival, Morphology and Function in Mice Lacking Filamin A.
Authors: Falet Herve , Marchetti Michael P. , Hoffmeister Karin M. , Italiano Joseph E. , Feng Yuanyi , Stossel Thomas P. , Kwiatkowski David J. , Walsh Christopher A. , Hartwig John H.
Published: October 2019

Platelet filamin A (FlnA), encoded by the FLNA gene on the X-chromosome, cross-links actin filaments, links them to the GPIbα subunit of the von Willebrand factor (vWf) receptor in the plasma membrane, and serves as a scaffold for signaling proteins. Mice lacking FlnA were generated. FlnA deficiency is embryonic lethal in hemizygous males due to pericardial and visceral hemorrhage. Heterozygous females are born, but experience premature mortality. The distribution of FlnA-expressing hematopoietic cells in FlnA+/− females was investigated by intracellular flow cytometry. About half of all hematopoietic cells investigated express FlnA, including bone marrow megakaryocytes, suggesting a random X-chromosome inactivation in these cells. However, about 90% of all blood platelets express FlnA. Mice lacking platelet FlnA were further generated by breeding FlnA loxP females with GATA-1 Cre males. FlnA loxP/+ GATA-1 Cre female offspring outnumber FlnA loxP/y GATA-1 Cre males by a ratio of 7:1. FlnA loxP/y GATA-1 Cre males have about 25% of normal blood platelet counts and their platelets are large, lack a normal actin cytoskeleton, and have decreased GPIbα expression, which has an altered distribution by electron microscopy. FlnA-null platelets are cleared rapidly from the circulation when transfused into GATA-1 Cre recipients and have severely impaired functional and signaling responses downstream of the collagen receptor GPVI, i.e. α-granule secretion, integrin αIibβ3 activation and protein tyrosine phosphorylation. Surprisingly, thrombin-mediated GPIbα down-regulation and botrocetin-mediated vWf binding are minimally affected in FlnA-null platelets. The data show the critical nature of FlnA for platelet survival, morphology and function.