bluuebunny/crossref_metadata_2025 huggingface dataset:
Langerhans Cells and T Cells in Langerhans Cell Histiocytosis Have Distinct Cytokine and Growth Factor Gene Expression Profiles Depending on the Clinical Risk Groups.
Authors: McClain Kenneth L. , Yan Xiao-tian , Li Liunan , Hicks John , Peterson Leif E.
Published: October 2019

Langerhans cell histiocytosis (LCH) can present in several clinical forms with expected response to therapy categorized by the organ systems involved. Low Risk patients (unifocal bone, skin, or lymph nodes disease) are nearly always cured of the disease, patients with multifocal bone involvement are more likely to have relapses, and High Risk patients (involving liver, lung, spleen, or bone marrow) have only a 50% chance of cure. The etiology of LCH is unknown and controversy exists whether this is purely an immunologic dysfunction or a neoplastic disease since the Langerhans cells have been identified as a clonal proliferation. Understanding interaction of Langerhans cells (LC) with T lymphocytes and macrophages in the lesions of Langerhans cell histiocytosis (LCH) is critical to elucidating LCH pathophysiology. We have used laser capture microdissection to isolate CD1a+ LC and CD3+ T cells from frozen LCH lesions. RNA from the dissected cells was enzymatically amplified, hybridized to a cytokine/growth factor array and analyzed for quantity of gene expression after normalizing to b-actin. A complex expression profile has emerged:

Gene Expression in LCH Clinical Groups

Low Risk Multifocal Bone High Risk * to ***= low to high expression of a gene, VEGF:vascular endothelial growth factor, FGF: fibroblast growth factor CD1a Cytokines IL-9**, -11***, -17**, Leptin*,TGF- β IL-10**, IL-11**,TGF- β IL-9*,11*,Leptin** CD1a Growth Factors FGF-6*, VGEF*** FGF-6**, VEGF** FGF-6***, VEGF* CD3 Cytokines IL-9**, Lymphotoxin- α IL-9*,-10 IL-9*** Lymphotoxin- α** CD3 Growth Factors GM-CSF* GM-CSF** FGF-5* GM-CSF*** FGF-5**

Growth factor and cytokine interactions from these LCH patients with different clinical presentations illustrates the importance of both LC and T-cells as well as the known influence of macrophages on the type of LCH. Although previous studies have shown TNF-a is an important factor, our data illustrates that other cytokines and growth factors may play critical roles, because of their relative higher gene expression when compared with TNF-a. These data will facilitate development of in vitro systems to test our hypotheses that a combination of cytokines/growth factors may be important in varying LCH clinical presentations.
Primary Plasma Cells Expressing CD52 Are Efficiently Targeted In Vivo by Alemtuzumab.
Authors: Carlo-Stella Carmelo , Di Nicola Massimo , Longoni Paolo , Cleris Loredana , Milanesi Marco , Lavazza Cristiana , Milani Raffaella , Carrabba Matteo , Formelli Franca , Gianni Alessandro M. , Corradini Paolo
Published: October 2019

Salvage therapy options for multiple myeloma (MM) patients relapsing following autologous stem cell transplantation remain limited. New treatments targeting the malignant plasma cell (PCs) are therefore needed. Alemtuzumab is a humanized monoclonal antibody to CD52 capable of destroying CD52+ cells by antibody-mediated cellular cytotoxicity and complement fixation. In order to evaluate the therapeutic potential of alemtuzumab for MM patients, we initially examined CD52 expression on primary malignant marrow PCs as well as a panel of MM continuous cell lines (RPMI-8226, KMS-11, OPM-2, U-266, LP-1, ARH-77). In addition, the anti-myeloma activity of alemtuzumab was evaluated in vivo in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. PCs were enriched from the marrow of MM patients (n = 40) according to CD138 expression. By using an immunomagnetic technique (Miltenyi Biotec, Germany, EU), highly purified CD138+ cells (median purity = 93%; median recovery = 55%) were obtained and further characterized by 3-color (CD138/CD45/CD52) flow cytometry. Expression of CD52 on CD138-enriched cells was detected in 28/40 (70%) of MM patients, with a median of 95% PCs expressing CD52. Detection of CD52 was equally evident on CD138+CD45+ and CD138+CD45− PCs (P ≥ 0.05). Similarly to what observed for primary CD138+ cells, MM cell lines showed a rather heterogeneous CD52 expression, ranging from dim (KMS-11, OPM-2) to bright positivity (LP-1, ARH-77). Both on primary PCs and MM cell lines, expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in NOD/SCID mice. Mice were inoculated intravenously with KMS-11 cells (0.5 x 105 per mice) and were treated with alemtuzumab (3 x 1 mg/mouse, subcutaneously, days 2, 5, 7). All placebo-treated mice (n = 15) died, whereas mice treated with alemtuzumab (n = 15) showed a significant prolongation of median survival (P ≤0.009 by log rank test) and 27% of them were alive and well at 120 days. No mice experienced any apparent treatment-related toxicity. According to these data, we conclude that: (1) CD52 is expressed on the plasma cells of a significant proportion of MM patients; (2) alemtuzumab has a strong antitumor activity in vivo on CD52-positive MM cell lines; (3) alemtuzumab might have therapeutic potential in a subset of MM patients.
SDX-101 Is Cytotoxic and Overcomes Drug Resistance in Multiple Myeloma.
Authors: Hamasaki Makoto , Hideshima Teru , Ishitsuka Kenji , Yasui Hiroshi , Shiraishi Norihiko , Kumar Shaji , Raje Noopur , Roccaro Aldo M. , Tai Yu-Tzu , Shiringarpure Reshma , Leoni Lorenzo , Kanekal Sarath , Elliott Gary , Munshi Nikhil C. , Anderson Kenneth C.
Published: October 2019

SDX-101 is an oral antineoplastic agent, has been evaluated in a phase I/II study in B-cell malignancies. It induces cytotoxicity at least in part, by significantly inhibiting expression of Mcl-1, an anti-apoptotic Bcl-2 family protein which is highly expressed in CLL cells. Here, we examined the cytotoxicity of SDX-101 against multiple myeloma (MM) cell lines. SDX-101 significantly inhibited growth in MM.1S, U266, RPMI8226 MM cell lines using MTT assays in a time- and dose-dependent fashion, with IC50s of 0.6mM, 1.0mM, and 0.4mM, respectively. In contrast, SDX-101 did not induce cytotoxicity in normal peripheral mononuclear cells (PBMCs) at these concentrations. Importantly, SDX-101 induced cytotoxicity even in dexamethasone (MM.1R)-, doxorubicin (RPMI-Dox40)-, and melphalan (LR5)- resistant MM cell lines. SDX-101 (0.3–1.25mM) triggered apoptosis associated with pro-caspase-3, pro-caspase-8, and PARP cleavage, as confirmed by immunoblotting. Although, interleukin-6 (IL-6) and insulin-like growth factor (IGF)-1 completely abrogates Dex-induced MM cell apoptosis, neither protects against SDX-101-induced apoptosis in MM.1S and RPMI8226 cells. Moreover, dexamethasone, Melphalan, and AS2O3 augment apoptosis induced by SDX-101. Importantly, SDX-101 downregulated both b-catenin and cyclin D1 expression in RPMI8226 cells. Finally, our recent studies demonstrate that the bone marrow (BM) microenvironment promotes MM cell growth, survival, and drug resistance while SDX-101 inhibits viability even if MM cells adherent to BM stromal cells; an environment in which cytotoxic potency is lost with other anti-neoplastic agents. Our data therefore demonstrate that SDX-101 induces apoptosis in MM cells via a mechanism different from conventional MM drugs, and support clinical trials of this agent, alone or in combination with conventional and/or novel agents to improve therapeutic outcome in MM.
Randomized Comparison of Weekly Administration and Daily Courses of Cladribine in Patients with Hairy Cell Leukemia - Updated Results.
Authors: Robak Tadeusz , Kasznicki Marek , Gora-Tybor Joanna , Dwilewicz-Trojaczek Jadwiga , Dmoszynska Anna , Wojtaszko Maria , Zdziarska Barbara , Calbecka Malgorzata , Hellmann Andrzej , Stella-Holowiecka Beata , Sulek Kazimierz , Skotnicki Aleksander , Zawilska Krystyna , Kloczko Janusz , Warzocha Krzysztof , Konopka Lech
Published: October 2019

Cladribine is the drug of choice in the treatment of hairy cell leukemia (HCL). However, its optimal method of administration is still unknown. There are suggestions that weekly administration is less toxic than daily schedule. We, therefore, compared these two methods of 2-CdA administration in a prospective randomized study. One group was treated with 2-CdA 0.12 mg/kg/d in 2 h i.v. infusion for 5 consecutive days and the second group received the same doses once a week for six weeks. Inclusion and response criteria were those recommended by Grever et al (J. Clin. Oncol 1995, 13, 974). From January 1998 to December 2003 118 patients with previously untreated symptomatic HCL were included into the study.

Both complete response (CR) and overall response (OR) rates are similar in compared groups. Response duration has not reached median yet. There is no statistically significant difference in toxicity between groups except for thrombocytopenia. It seems, however, that daily administration of 2-CdA may more frequently induce neutropenia and lead to more frequent infections.

In conclusion, the updated results of our study suggest that weekly administration of cladribine in HCL may be equally effective but less toxic than daily courses.

Patients (n=118) OR CR Thrombocytopenia grIII/IV Neutropenia grIII/IV Infections Relapsed Died Daily (n=59) 50 (85%) 43 (73%) 8 (13.6%) 21 (35.6%) 14 (24%) 5 (8.5%) 6 (10.1%) Weekly (n=59) 49 (83%) 44 (76%) 2 (3.4%) 12 (20.3%) 6 (10.2%) 5 (8.5%) 4 (6.8%) p value 0.8 0.8 0.04 0.06 0.05 1.0 0.5
POKEMON Is a Proto-Oncogene Which Plays a Key Role in Lymphomagenesis.
Authors: Maeda Takahiro , Hobbs Robin , Merghoub Taha , Guernah Ilhem , Zelent Arthur , Teruya-Feldstein Julie , Pandolfi Pier Paolo
Published: October 2019

POKEMON (for POK, Erythroid, Myeloid ONtogenic factor, also known as LRF) was originally isolated as a homologue of PLZF (Promyelocytic Leukemia Zinc Finger) which can physically interact with the BCL6 (B Cell Lymphoma 6) proto-oncogene.

Analysis of Pokemon−/− mice demonstrates that this transcriptional repressor plays a key developmental role whose function is required for cellular differentiation in multiple tissues such as skin, adipose tissue and hematopoietic lineage cells. Taking advantage of Pokemon−/− MEFs (Mouse Embryonic Fibroblasts), we found that Pokemon is indispensable for cellular transformation mediated by oncogenes (such as BCL6, E1A, H-RasV12, c-Myc, or T-Ag) and Pokemon overexpression overcomes oncogene induced premature senescence and apoptosis leading to oncogenic transformation by repressing p19ARF tumor suppressor. Furthermore, Pokemon−/− MEFs showed a premature senescence phenotype in early passage which was rescued by inactivation of p19ARF. To explore the oncogenic role of Pokemon in vivo, we generated Lck/Eμ Pokemon transgenic mice in which Pokemon is over expressed in T and B lineage cells under the control of Lck/Eμ promoter/enhancer. Strikingly, about 50% of the mice developed T lymphoblastic lymphoma (CD4+, CD8+, CD44 + and TdT+) after a latent period of 2 to 4 months. As Pokemon expression proved essential for cellular transformation and is oncogenic when overexpressed in vivo, we analyzed the levels of POKEMON expression in human cancers, performing Tissue Microarray Analysis (TMA). We first analyzed human B-cell lymphomas as POKEMON is normally co-expressed with BCL6 in the B-cell within the germinal center, and may therefore cooperate with BCL6 in lymphomagenesis. We analyzed and scored a total of 123 cases of diffuse large B-cell lymphoma (DLBCL) and 290 cases of follicular lymphoma (FL) for POKEMON protein expression levels. POKEMON was highly expressed in 25–35% and moderately expressed in 40–50% of both DLBCL and FL clinical samples. Strikingly, as previously shown for BCL6, POKEMON positivity predicted a better prognosis of overall survival in DLBCL. Interestingly, the vast majority (approximately 80%) of BCL6 positive DLBCLs and FLs were found to be POKEMON positive. By contrast, not all the POKEMON positive lymphomas expressed BCL6. Importantly, coexpression of POKEMON and BCL6 in DLBCL predicted an even better clinical outcome, identifying a clinically distinct DLBCL subtype.
The Seasonal Incidence of Venous Thromboembolism (VTE): A Population-Based Cohort Study.
Authors: Kobbervig Catie E. , Heit John A. , Petterson Tanya M. , Christianson Teresa J. , Bailey Kent R. , Melton L. Joseph
Published: October 2019

If the incidence of VTE varies by season, then the etiology of VTE could be related to seasonal exposures. However, the few studies that estimated VTE incidence by season reached conflicting conclusions.

Objective: To estimate the incidence of VTE by month and by season of the year.

Methods: Using the resources of the Rochester Epidemiology Project, we identified the inception cohort of Olmsted County, MN, residents with a first lifetime VTE over the 30-year period, 1966–1995 (n=2761). For each case, we reviewed the complete medical records in the community for the date of VTE onset, date of birth, gender and 48 baseline clinical characteristics commonly accepted as risk factors for VTE. We categorized cases as idiopathic (n=305) if no such characteristics were present; remaining cases were categorized as secondary. Incidence rates were calculated using the number of overall, idiopathic or secondary VTE cases by month of onset as the numerator, and age- and sex-specific estimates of the monthly population of Olmsted County as the denominator (yearly population estimates divided by 12).

Results: The incidence of VTE did not vary appreciably by month of the year for overall, idiopathic or secondary VTE (all p-values >0.2). However, the overall incidence of VTE was higher in the 3 summer months (June, July, August) compared to the 3 winter months (December, January, February; 132.6 vs. 116.9 per 100,000 person-years, respectively, both age- and sex-adjusted to year 2000 U.S. Whites; p-value = 0.026). The same general pattern was true for both idiopathic and secondary VTE, but separately neither was statistically significant (p-value=0.48 and 0.15, respectively).

Conclusions: The incidence of VTE is higher in the summer compared to the winter months, suggesting the hypothesis that vector-borne (e.g., mosquito, tick) and/or enteroviral infection may play a role in the etiology of VTE.
Staphylococcus Epidermidis Induced Platelet Aggregation Is Mediated by the Fibrinogen Binding Surface Protein SdrG.
Authors: Brennan Marian P. , Devocelle Marc , Foster Timothy J. , Cox Dermot
Published: October 2019

S. epidermidis is normally present on the surface of the skin, however in a hospital setting, it is responsible for many infections involving implanted medical devices resulting in potentially fatal complications such as infective endocarditis and septicemia. S. epidermidis has previously been shown to induce platelet aggregation (Ohshima et al. Microbiol Immunol 1991) through an unknown mechanism. The surface protein SdrG (serine-aspartate repeat G protein) is important in the initial colonization of the device, as it binds fibrinogen, which is one of the first proteins to coat foreign devices. We thus postulated that this SdrG fibrinogen complex may mediate the S. epidermidis interaction with platelets. In order to investigate the specific effects of SdrG on platelets, we expressed SdrG in Lactococcus lactis which does not activate platelets. L. lactis SdrG was able to support platelet adhesion in the absence of fibrinogen, and to a greater extent in the presence of fibrinogen suggesting both a direct interaction with the platelet and an indirect interaction via a fibrinogen bridge. Fibrinogen dependent adhesion was inhibited by both abciximab and tirofiban confirming an interaction with GPIIb/IIIa. Furthermore, SdrG when expressed in L. lactis can induce platelet aggregation with a lag time of 1.5 ± 0.4 min. This aggregation was inhibited by abciximab suggesting that it is true aggregation rather than agglutination. We have also shown this to be dependent on cyclooxygenase signalling, as it is inhibited by aspirin. SdrG binds to the C-terminus of the beta chain of fibrinogen (Ponnuraj K et al. , Cell 2003). We synthesized a 15-mer peptide mimicking the binding region of the fibrinogen beta chain in order to determine whether fibrinogen binding was important for this aggregation. The native fibrinopeptide NEEGFFSARGHRPLD increased the lag time for aggregation to 9.2 ± 1.4 min, however, the control peptide NEEGFFAARGHRPLD did not affect aggregation (1.4 ± 0.3 min). The lag time did not increase further with saturating concentrations of peptide concentrations suggesting instability of the peptide, or a second mechanism involved in the aggregation which is consistent with the fibrinogen independent interaction seen in the adhesion assay. This work shows that SdrG alone is sufficient to support platelet adhesion and aggregation. SdrG mediates platelet activation through a novel mechanism via GPIIb/IIIa, and an as yet unknown direct interaction. Thus SdrG appears to be the dominant virulence factor for S. epidermidis induced thrombosis, therefore, inhibition of these interactions have potential for treatment of thrombotic complications associated with S. epidermidis infections.
Both 5′ and 3′ Distal Regulatory Elements Are Required for Human CD34 Gene Expression.
Authors: Okuno Yutaka , Levantini Elena , Tatetsu Hiro , Mitsuya Hiroaki , Tenen Daniel G.
Published: October 2019

In order to understand the controls of gene expression in hematopoietic stem cells (HSC), we have studied the regulation of human CD34, a cell surface glycoprotein used in isolation of HSC. We previously reported that regions 10 to 18 kb 5′ upstream of the transcription start site (TSS) and/or 17 to 26 kb 3′ downstream of the transcription termination site (TTS) of the human CD34 gene contain critical elements for expression in vivo. Mice transgenic for a human CD34 PAC clone (PAC54A19) that includes 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequences expressed human CD34 antigen in the majority of HSC. Because these transgenic mice also demonstrated downregulation of human CD34 gene expression with maturation of hematopoietic cells, we concluded that these critical cis-elements are necessary not only for human CD34 expression in immature cells, but also for downregulation in mature cells. To further delineate the distal cis-elements which regulate the human CD34 gene, we generated two deletion constructs which removed sequences either upstream from −12.8 kb (PmeI deletion) or downstream of +18.2 kb (FseI deletion) of the human CD34 gene. We used a murine myeloblastic cell line, 416B, to test these constructs. We first generated stable clones of 416B cells with PAC54A19; these stable clones expressed human CD34 RNA and protein. In contrast, neither PmeI nor FseI deletion constructs conferred human CD34 mRNA or protein expression in these 416B stable cell lines. Therefore, we conclude that both 5′ upstream and 3′ downstream distal cis-elements are necessary for proper human CD34 expression in cell lines. We also performed DNaseI hypersensitivity assays to localize open chromatin structures in the human CD34 gene in several human myeloblastic cell lines. We detected one DNaseI hypersensitive site (DHS) −12.5 kb upstream of the TSS and several DHS in the region located between +17 kb to +26 kb 3′ downstream of the human CD34 TTS in the human myeloblastic CD34+ KG1a cell line. In addition, we performed similarity searches of the −18 kb to −10 kb human CD34 5′ flanking sequence and +17 kb to +26 kb human CD34 3′ flanking sequence, comparing with the murine CD34 genomic sequence. We identified one conserved region in the −11.5 kb 5′ upstream region of the TSS and one highly conserved region located +19 kb 3′ downstream region of the TTS. To further localize distal cis-elements, we generated several deletion constructs in the parental PAC54A19 PAC clone using the ET cloning system, which facilitates manipulation of PACs by homologous recombination in bacteria. Using this method, we showed that deletion of a 600 bp region located 15 kb 5′ of the TSS or a 1000 bp sequence located 19 kb downstream of the human CD34 gene led to loss of human CD34 expression in 416B stable lines. Our current efforts are focused on identifying the transcription factors that bind to these regions to regulate the expression of human CD34 in stem cells.
Steady State Haemoglobin Level Increases with the Proportion of Erythrocyte Membrane n-3 Fatty Acids in Sickle Cell Anaemia.
Authors: Okpala Iheanyi , Ren Hongmei , Ibegbulam Obike , Ugochukwu Cynthia C. , Ghebremeskel Kebreab , Crawford Michael
Published: October 2019

Previous studies indicate that perturbation of erythrocyte and/or plasma lipids occurs in homozygous sickle cell disease (SCD), and treatment with n-3 fatty acids (FA) is beneficial to affected individuals (Tomer et al, Thromb Haemost2001; 966–974). Phosphatidylserine has a role in sickle erythrocyte-endothelial adhesion, which contributes to vessel occlusion in SCD (Setty et al, Blood2002; 1564–1571). Considering that n-3 and n-6 FA are essential structural and functional components of the red cell membrane, the objective of this study was to find out if abnormalities of these FA affect the degree of anaemia in SCD. We recruited 43 HbSS patients and 43 racially-matched, healthy, HbAA controls living in the same environment; analysed the fatty acid composition of erythrocyte membrane choline (CPG), serine (SPG) and ethanolamine (EPG) phosphoglycerides and sphingomyelin (SPM); and sought for relationship between steady-state Hb level in SCD and the proportion of n-3 FA in the red cell membrane. HbSS individuals had high levels of adrenic and docosapentanoic acids in CPG, EPG, SPG (p<0.001), and SPM (p<0.05). Arachidonic acid (AA) was increased in CPG (p<0.001) and EPG (p<0.005) of the patients. In contrast, linoleic acid (LA) was low in patients’ CPG, EPG, SPG and SPM (p<0.001). Both LA and AA were reduced in plasma CPG, triglycerides and cholesterol esters of SCD patients. There was significant positive correlation between steady-state Hb level and n-3 FA content of erythrocytes: docosahexaenoic (DHA, p<0.01, r = 0.63) and eicosapentanoic (EPA) acids in red cell CPG, and EPA in EPG (p<0.05, r = 0.60); figs. 1&2. The observed high AA with low DHA/EPA levels favour red cell adherence to vascular endothelium and vaso-occlusion. The data suggest that in SCD: synthesis of AA and/or membrane uptake of LA and AA may be abnormal; increased proportions of erythrocyte membrane DHA and EPA confer some resistance to haemolysis.
A Novel Mechanism of ADAMTS13 Deficiency in Mice.
Authors: Zhou Wenhua , Dong Lingli , Bouhassira Eric E. , Tsai Han-Mou
Published: October 2019

Background. ADAMTS13, a circulating metalloprotease that cleaves conformationally altered von Willebrand factor (VWF), is critical for preventing microvascular thrombosis. Deficiency of the protease, due to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura. In the course of investigating the regulation of ADAMTS13, we noted that mice differed widely in their plasma ADAMTS13 activity levels. In order to understand the factors affecting plasma ADAMTS13 levels, we examined the molecular basis of ADAMTS13 variation in different strains of mice.

Methods. Plasma ADAMTS13 activity level was determined by previously described SDS PAGE and immunoblotting. ADAMTS13 transcripts were analyzed by RT PCR, RACE, nucleotide sequencing, and real-time RT PCR. Plasmids containing the cDNA of mouse ADAMTS13 were constructed for transfection of mammalian cell lines.

Results. The mouse strains FVB/NJ and 129X1/SvJ differed from C57BL/6J and DBA/2J by more than 10 folds in their plasma ADAMTS13 activity levels (3.09 +/− 0.45 vs 0.24 +/− 0.11 U/mL for FVB/NJ and C57BL/6J respectively, P < 0.0001). To determine the causes of the difference, we analyzed the ADAMTS13 transcripts by using RT PCR and RACE, which showed that the FVB/NJ mice contained the predicted full-length ADAMTS13 transcript with a domain structure similar to human ADAMTS13, while the C57BL/6J mice contained at least 4 isoforms: the full-length transcript, one internal splicing isoform, and two truncated forms that ended with an extraneous sequence homologous to the long-terminal repeat (LTR) of an retrotransposone of the IAP type. Comparison of the genomic sequences showed that the ADAMTS13 gene of C57BL/6J mice contained in its intron #23 an IAP-type retrotransposone sequence whose LTR sequence with a stop codon was included in the mouse transcripts. The IAP retrotransposone sequence, which contained one base substitution at the 5′-end 4-base repeat (tgtt>g) and was flanked at both ends by a 6-base repeat (cactag), was also present in the DBA/2J but not the 129X1/SvJ strains of mice. Real-time RT PCR showed that the FVB/NJ and C57BL/6J mice had similar levels of ADAMTS13 transcripts in the liver. Nevertheless in the C57BL/6J mice the IAP-truncated forms accounted for >90% of the ADAMTS13 transcripts. Expression of mouse ADAMTS13 cDNA in mammalian cell lines revealed that the both the full-length and the IAP-truncated forms of the ADAMTS13 protease were similar in VWF-cleaving activity.

Conclusion. This study shows the presence of intragenic retrotransposone in the ADAMTS13 gene of some mouse strains. The presence of an IAP-retrotransposone within the ADAMTS13 gene of C57BL/6J mice affects the splicing of the ADAMTS13 transcripts, creating truncated forms of the protease that lack the last two TSP-1 and the CUB domains but remain proteolytically active in cleaving VWF. The lower plasma ADAMTS13 activity level of C57BL/6J may result from abnormal intravascular clearance of the protease or other post-secretory events.
The Effect of Dietary Factors on Hemochromatosis Disease Expression in C282Y Homozygotes.
Authors: Phatak Pradyumna D. , Bundy Brain , Andrews Caroline , Braggins Laura , Sham Ronald L.
Published: October 2019

Background. Hereditary hemochromatosis (HHC) is a common inherited disorder and the vast majority of cases are associated with HFE gene mutations; about 80% are homozygous for the C282Y HFE mutation. Although homozygosity for C282Y is relatively common among individuals of northern European descent, the penetrance, as measured by clinically relevant iron overload, is low. Both environmental and genetic factors have been implicated in modulating disease expression. Most clinicians and patients attribute at least some of the observed variation to differences in dietary patterns.

Methods: 28 C282Y homozygous subjects who had completed de-ironing therapy at our center, and were on stable maintenance phlebotomy treatments, participated in a previously validated dietary survey. We examined the influence of dietary iron, heme iron, fiber, ascorbic acid and alcohol intake on disease expression as measured by liver iron, phlebotomy-mobilized iron and maintenance phlebotomy requirement.

Results. We developed a maintenance iron score (Maint Fe) which was the iron in milligrams removed per month by phlebotomy treatments in order to maintain serum ferritin in the 25–75 μG/L range. Maint Fe ranged from 36.8–203 mg/month in our patients (87.8 ± 37.7). Liver iron ranged from 30.5 to 505.4 micromoles per gram dry weight (192.7 ± 123). Phlebotomy-mobilized iron ranged from 2.35–18.03 G (6.43 ± 4.08). The Spearman’s Rank Correlation was used to compare dietary parameters with each of the above measures of disease expression. No significant correlation was found.

Conclusions. Disease expression varied considerably in our C282Y homozygous subjects as measured by liver iron, phlebotomy-mobilized iron and Maint Fe. The observed variation is not explained by variations in dietary iron, fiber, ascorbic acid or ethanol intake. Our data suggests that other unknown environmental variables or alternatively, modifier genes, may play a role in modulating disease expression in these patients. Contrary to popular belief, dietary variation does not appear to play a major role.
Chronic Aluminum Toxicity Due to Intravenous Substance Abuse Managed with Deferoxamine Chelation Therapy: Case Report.
Authors: Sreenivasan Gayatri M. , Yong Raymund L.M. , Holmes Dan
Published: October 2019

Aluminum toxicity first came to attention in the medical literature as a sequela of peritoneal or hemodialysis, where accumulation of aluminum in body tissues results from the use of aluminum-containing dialysate and phosphate binders. The most common clinical presentations of aluminum toxicity include anemia, bone disease, and a syndrome of encephalopathy known as the dialysis encephalopathy sydrome (DES). We present the unique case of a 42-year-old man who developed chronic aluminum toxicity from the intravenous injection of a methadone preparation intended for oral consumption. The methadone solution was concentrated for intravenous injection by heating in aluminum cookware. Experiments performed in our laboratory reproducing this technique, using weakly acidic solutions similar to popular drink mixes, confirmed that significant amounts of elemental aluminum can be leached from aluminum cookware when heated. The patient presented to medical attention with generalized seizures, profound cerebellar dysfunction, myoclonus, and speech apraxia, all of which are characteristic of DES. The peripheral blood film revealed bizarre red cell morphology, with clear features of iron deficiency and prominent coarse basophilic stippling, suggesting impaired iron utilization, which is at least one mechanism of aluminum related hematologic toxicity. A bone marrow biopsy demonstrated extensive osteosclerosis and myelosclerosis, compatible with significant metabolic bone disease. Subsequent investigations revealed a serum aluminum level of 7200 nmol/L, eighteen times the upper limit of normal (400 nmol/L). Chelation therapy with a continuous intravenous infusion of deferoxamine was started, and over a course of ten months the serum aluminum level was reduced to 2390 nmol/L with few side effects. The patient made a significant neurologic recovery and returned to life in the community. This is the first detailed account of the successful use of deferoxamine to chelate aluminum in a patient without dialysis-dependent renal insufficiency. This case also highlights heavy metal exposure as a diagnostic consideration in intravenous substance abusers presenting with multiorgan dysfunction, and the importance of performing a thorough inquiry into injection drug practices when evaluating these patients.
γδT Cell Is Impacted to Erythropoiesis Suppression in Patients with Acquired Pure Red Cell Aplastic Anemia.
Authors: Liu Ting , Liu Min , Dai Yang , Zhu Huanling , Cui Xu , Meng Wentong , Xu Caigang
Published: October 2019

Acquired pure red cell aplastic anemia(PRCA)is a group of heterogeneneous disorders characterized by erythropoiesis failure or disorder in bone marrow. PRCA is often associated with thymoma, large granular lymphocytic leukemia, and also some autoimmune diseases. It has been well documented that T cell subsets from the patients with PRCA often show some abnormalities either in quantum or in the effects to suppress erythropoiesis, and anemia symptom of the patients usually have a good response to cyclosporine treatment. So it suggested that activated T cell might play a key role in the pathogenesis of PRCA. This study is to explore the abnormalities of γδT cell subsets and the effects of immunosuppressive therapy in the PRCA patients.

Methods: 12 patients, whose age ranged 37~72 years old, were diagnosed as PRCA based on bone marrow smear and biopsy, and were treated with cyclosporine, and 24 healthy adults were matched as normal controls. The three-color flowcytometry technique was used for lymphocytes subsets and γδT cells analyses. Furthermore, peripheral mononuclear cells (MNC) isolated from PRCA patients were cultured in RPMI1640 medium (106 cells/ml) with 10% FCS, phytohemagglutinin(PHA, 10μg/ml), and recombinant human interleukin-2(rIL-2, 50U/ml)for two weeks, then γδT cells were isolated with the TCRγδ Microbead Kit from cultured cells. The collected γδT cells were incubated with normal control bone marrow MNC in RPMI1640 medium (37°, 5% CO2 atmosphere) for CFU-E, CFU-GM, and BFU-E clones assay.

Results: Comparing with control group, CD3+/CD8+ cells were increased significantly in the patients group (p<0.05), and the CD4+/CD8+ ratio was decreased and reversed. Furthermore, an increased Tc2 cells percentage and a decreased Tc1/Tc2 ratio were observed, and γδT cells were overrepresented in patients group(P<0.05). After treatment with cyclosporine, 10 of 12 patients got very good response (Hb recovered to over 100g/L), and in the responding patients, CD3+/CD8+ cells, Tc1 and Tc2 cells were decreased, and the ratio of CD4+/CD8+ and Tc1/Tc2 were return to normal respectively, and γδT cells were also decreased to normal range. Moreover, in vitro culture, the γδT cells isolated from PRCA patients show an inhibiting role to CFU-E and BFU-E but not to CFU-GM in a dose-dependent manner.

Conclusion: Our study suggested that γδT cells might impacted to pathogenesis of acquired PRCA, a significant improvement of anemia symptoms had been shown after cyclosporine treatment, meanwhile, the percentage of γδT cells recovered to normal range, and it is very interesting, the γδT cells isolated from PRCA patients show an inhibiting role to CFU-E and BFU-E but not to CFU-GM in vitro culture. We think that it is an effective way to treat the patients with acquired PRCA by to rectify the abnormal cellular immunity.
Mycophenolate Acid Has a Negative Effect on the Maturation and Immune Function of the Murine Bone Marrow-Derived Dendritic Cells.
Authors: Cai Zhen , Huang Wenye , Sun Wenji
Published: October 2019

Mycophenolate mofetil (MMF) is a newly developed immunosuppressor, currently widely used in allogeneic bone marrow transplantation. Its active metabolite, mycophenolic acid (MPA) is a noncompetitive, reversible inhibitor of the enzyme inosine 59-monophosphate dehydrogenase, which plays a major role in the de novo synthesis of guanosine nucleotides. Unlike other cells that also use the salvage pathway for purine biosynthesis, proliferating B and T cells are dependent on the de novo pathway generate guanosine. Thus, MMF exerts its immunosuppressive effects of lymphocyte proliferation. Recently, some studies found that MPA could inhibit the immun immune function of antigen presenting cells. Dendritic cells (DCs), the most potent antigen presenting cells with the unique ability to prime naive T cells, play a central role in antigen processing and presentation to induce T cell response in vitro and in vivo. This study is to evaluate the effects of MPA, the in vivo active metabolite of MMF, on the maturation and immune function of murine bone marrow-derived dendritic cells, and to explore the underlying mechanisms of MMF in graft versus host disease. Bone marrow-derived dendritic cells (DC) were cultured with GM-CSF and IL-4 in the presence of MPA at doses of 0.01 and 0.1μmol/L. The ability of the allostimulatory activities of the DCs on allogeneic T cells was assessed by MLR. IL-12 production in culture supernatant and the Th1/Th2 cytokines such as IL-2, IFN-g, IL-4 and IL-10 levels in mixed lymphocyte reaction (MLR) supernatant were examined by ELISA assays. The activity of NF-κB in DCs was measured with Western blot assays. Our results showed that DCs cultured in the presence of MPA expressed lower levels of CD40, CD80 and CD86, exhibited weaker activity of stimulating the allogeneic T cell proliferation and weaker in antigen presenting function with a concurrent reduction of IL-12 production. MPA-treated DCs stimulated allogeneic T cells to secrete higher levels of Th2 cytokines IL-4 and IL-10 but lower levels of Th1 cytokines IL-2 and IFN-g than did DCs not treated with MPA. The activity of NF-κB was decreased in DCs treated with MPA in a dose-dependent manner. We conclude that MPA, and hence MMF, exerts a negative effect on the maturation and immune function of in vitro cultured DCs, and drives a shift of Th1 cytokines to Th2 cytokines in MLR. This negative effect is associated with a decrease in NF-κB activity. Say something about the significance of this finding regarding GVHD.
The Direct Cytotoxicity of Activated Human Umbilical Cord Blood Dendritic Cells to Tumor Cell.
Authors: Jun Shi , Ikeda Kazuma , Fujii Nobuhara , Kasumoto Kinuyo , Tanimoto Mitune , Xiao Li , Quan Pu
Published: October 2019

Besides their role as antigen presenting cells, human peripheral blood mononuclear cell and CD34+ cell-derived dendritic cells (DCs), now have been demonstrated to exert cytotoxicity against some tumor cells, and their tumoricidal activity can be enhanced by some stimili. However, there have been no reports concerning the tumor-cell killing activity by human cord blood cell-derived dendritic cells (CBDCs). We report here that human cord blood monocyte-derived DCs aquire the ability to kill tumor cells after activation with lipopolysaccharide (LPS) or interferon-γ(IFN-γ), associated with the enhanced TNF-α-related apoptosis-inducing ligand (TRAIL) expression in CBDC cytoplasm. The CD14-positive cells collected from cord blood from healthy volunteers were cultured with interleukin-4 and granulocyte-machrophage colony- stimulating factor for seven days to induce CBDCs, which showed no cytotoxicity. However, after activation with IFN-γ for additional 12 hours, CBDCs exhibited cytotoxicity against HL60 and Jurkat cells, while activation with LPS for 12 hours induced cytotoxicity against Daudi and Jurkat cells as we detected in human peripheral blood monocytes- drived DCs (PBDCs). IFN-γ or LPS stimulation enhanced intracellular but not cellular surface TRAIL, and neither intracellular nor cellular surface Fas Ligand as analyzed by flow cytometry. Our results suggest that activated CBDCs can serve as immunological effectors against tumor cells, through TRAIL- dependent and Fas-independent mechanisms.
Macrophage Motility Dysfunction in a Patient with Recurrent Infections.
Authors: Jubinsky Paul T. , Short Mary , Ashton Anthony , Cox Diane , Pixley Fiona
Published: October 2019

Three members of a family with normal parents had a common constellation of findings that included absent corpus callosum and recurrent bacterial infections. The older male and female siblings both died from infection at an early age. The patient’s CBC was significant for elevated numbers of monocytes that were large and vacuolated. Her T- and B-cell function was normal. These preliminary findings suggested a defect in innate immunity. Evaluation of the patient’s peripheral blood monocytes by flow cytometry showed normal size and maturity. Phagocytosis and activation of peripheral blood derived macrophages by cytokines were also similar to controls. In contrast, the patient’s cultured macrophages were significantly more spread than those from normal individuals and contained a disordered actin cytoskeleton when cultured on fibronectin. The ability of the macrophage to transmigrate across an endothelial cell barrier was impaired. However examination by time-lapse videomicroscopy showed that the mutant macrophages had increased protrusional activity and movement. This is the first report describing a genetic macrophage motility disorder that results in an increased susceptibility to infection.
Blocking the Expression of CD33 Enhances Chemotaxis of Activated T Lymphocytes Generated from AML Patients.
Authors: Zhong Rui-kun , Ball Edward D.
Published: October 2019

Autologous T lymphocytes can be employed for immune therapy of patients with acute myeloid leukemia (AML). In an earlier study (Biol Blood Marrow Transplant2002; 8:557), we found that AML cells can be used as antigen presenting cells to activate and expand T cells in culture with IL-2 and monoclonal antibody (mAb) to CD3. In this study, we found that a substantial proportion of the expanded T cells expressed CD33 (34 ± 16%; range: 8 – 58%; N=11) and CD13 (26 ± 5%; range: 22 – 33%), but not CD14. It is unlikely that the myeloid markers were absorbed onto the activated T cells, since the same expression pattern was observed in cultures with purified T cells from normal donors. A literature review disclosed two reports that also observed co-expression of CD33 on activated T cells from both AML patients and normal controls (Schmidt-Wolf et al, Br J Haematol.1995; 90:512; Nakamura et al, letter in Blood, 1994; 83:1442). We measured the functional capacity of the activated T cells in a 4-hr Cr-51 release assay. Pre-incubation of the T cells with anti-CD33 mAb and/or anti-CD13 mAb did not change the cytotoxicity against AML cells. Also, killing of AML cells was not affected by the depletion of CD33+ or CD13+ activated T cells using magnetic beads. The activated T cells migrated in a chemotactic response to a peptide analog (CTCE-0214) of Stromal cell derived factor-1 (SDF-1) when measured in a trans-well assay. When activated T cells were pre-incubated with anti-CD33 mAb, enhanced chemotaxis was observed in response to CTCE-0214 (39.9 ± 0.5% vs 26.9 ± 0.3%, p < 0.001). However, incubation with anti-CD13 mAb did not change the proportion of chemotactic cells. The results of this study confirmed that myeloid antigens can be induced on activated T cells from patients with AML and that there are differences in the mobility of CD33+ T cells in the presence of a chemotactic molecule. Further study of the biological significance of CD33 expression on activated T cells in AML and other diseases is warranted.
TEL/PDGFβR Tyrosine Kinase Fusion Specifically Activates Myeloid Differentiation Programs, and Converts Lymphoid Progenitors into the Myeloid Lineage.
Authors: Shigematsu Hirokazu , Iwasaki Hiromi , Mizuno Shin-ichi , Sternberg David W. , Huntly Brian J.P. , Gilliland D. Gary , Akashi Koichi
Published: October 2019

Acute leukemias are classified into myeloid and lymphoid subtypes according to their phenotypic characteristics. Leukemic cells, however, sometimes co-express myeloid and lymphoid phenotypes, or switch their phenotype from myeloid to lymphoid lineages and vice versa. The conventional classification of leukemias is based mainly upon the concept that leukemic phenotype reflects a progenitor stage at which leukemic transformation occurs, or that it represents a stage at which transformed hematopoietic stem cells (HSCs) become incapable of further differentiation. Here we propose another possibility that phenotype of leukemias could be determined by instructive signals from the leukemic transformation mechanism itself. We found that TEL/PDGFβR (T/P), a tyrosine kinase fusion isolated from chronic myelomonocytic leukemia, can instruct myeloid lineage commitment and conversion at stem and progenitor stages of hematopoiesis. The T/P gene was transduced into purified progenitors or HSCs by using a retrovirus carrying a green fluorescent protein reporter. HSCs transduced with T/P (T/P+ HSCs) spontaneously formed GM colonies without cytokines. Furthermore, T/P+ HSCs were incapable of differentiation into B cells on OP9 stromal layer in the presence of IL-7. To test the effect of T/P on lymphoid commitment more precisely, we transduced T/P into purified common lymphoid progenitors (CLPs) that normally differentiate only into T, B and NK lineages. In a limiting dilution assay, 1 in 7 control-GFP transduced CLPs generated B cell progeny in vitro, while only 1 in 500 T/P+ CLPs differentiated into B cells. Instead, surprisingly, the majority of T/P+ CLPs and even T/P+ thymic proT cells quickly differentiated into granulocytes and monocytes in vitro. We then transplanted T/P+ CLPs into lethally irradiated congenic mice. T/P+ CLPs again differentiated into Gr-1+ granulocytes and monocytes in vivo. Gene expression analyses showed that T/P+ CLPs upregulated GM-related molecules including C/EBPα and GM-CSFRα immediately after T/P transduction, while transduction of either C/EBPα or GM-CSFRα also reprogrammed CLPs into the myeloid lineage as we reported previously. Thus, T/P signaling can activate these critical GM-related molecules in lymphoid progenitors to convert them into the myeloid lineage. These data collectively suggest that at least some types of oncogenic tyrosine kinase fusions can specify leukemic phenotypes, through activating critical signals for lineage commitment.
A Study of Effects of 2A-1-1 on Platelet Function in Hypertension Patients.
Authors: Nie Danian , Zeng Furen , Xie Shuangfeng , Li Yiqing , Yin Songmei , Ma Liping , Wu Yudan
Published: October 2019

Objective To study the effects of 2A-1-1, a purified component from Panax notoginsengs which is considered to be a blocker of ROC (Receptor-operated Ca2+ channel), on platelet aggregation in hypertension patients with Nifedipine and 1-{β-[3-(4-methoxyphenyl) propoxy]- 4-methoxyphenethyl}- 1H - imidazole hydrochloride (SK and F96365) in contrast.

Methods Platelet rich plasma (PRP) was made in routine way from venous blood samples which taken from 30 essential hypertension patients and 30 normal people. PRP was incubated with different concentrations of 2A-1-1, Nifedipine and SK&F96365 for 3 minutes. Therefore, maximal platelet aggregation rate (PAGmax) induced by ADP had been measured on TYXN-91 aggregometer.

Results 1. PAGmax in essential hypertension group was 88.48±5.36%, higher than that in normal group which was 67.54±8.46%( P<0.01). 2. Nifedipine (10μmol/L and 20μmol/L) had no effect on PAGmax in neither essential hypertension group nor normal group( P>10;0.05). 3. 2.5μmol/L, 5μmol/L, 10μmol/L and 20μmol/L SK&F96365 could reduce the PAGmax in essential hypertension group by 14.32%) 17.9%) 30.26%) 78.23% respectively; 5μmol/L, 10μmol/L and 20μmol/L SK&F96365 could reduce the PAGmax in normal group by 21.57%) 22.85%) 59.38% respectively. 4. 2.5μmol/L, 5μmol/L, 10μmol/L and 20μmol/L 2A-1-1 could reduce the PAGmax in essential hypertension group by 24.83%) 27.56%) 48.73%) 92.77% respectively; 5μmol/L, 10μmol/L, 20 μmol/L 2A-1-1 could reduce the PAGmax in normal group by 47.06%) 53.47%)71.52% respectively.

Conclusions 1. PAGmax in essential hypertension patients is higher than normal people remarkably, suggesting that the high reactive status of platelets is an important factor for the development of thrombus in hypertension patients.2. Nifedipine has no effects on PAGmax, therefore it can not inhibit platelet aggregation.3.SK and F96365 can reduce the platelet aggregation.4. 2A-1-1 can inhibit platelet aggregation, therefore, it has the definite effect of anti-platelet.