bluuebunny/crossref_metadata_2025 huggingface dataset:
Favorable Influence of Pretransplant Rituximab but Not of High-Dose Ara-C in Upfront Autologous Stem Cell Transplantation (SCT) for Mantle Cell Lymphoma (MCL).
Authors: Rieger Michael , Hensel Manfred , Seyfarth Baerbel , Sitter Soeren , Martin Simona , Haas Rainer , Kneba Michael , Schmitz Norbert , Ho Anthony D. , Dreger Peter
Published: October 2019

Recent data suggests that the prognosis of MCL might be improved by administration of rituximab and high-dose ara-C (HA) prior to upfront SCT. In order to investigate the relative contributions of rituximab and HA, respectively, we retrospectively analyzed 98 patients who received SCT as part of 1st-line treatment of advanced-stage MCL. Patients (median age 55 (29–67)) with stage II–IV MCL were treated with a sequential therapy comprising 2–8 cycles of CHOP or CHOP-like regimens +/− rituximab for remission induction, DexaBEAM (49%) or HA-containing chemotherapy (39%) for further consolidation and mobilization of stem cells, and high-dose therapy with SCT. 22% were mobilized with CHOP alone. High-dose regimens were TBI/CY (38%), TBI/CY + rituximab (R-TBI/CY; 35%), or BEAM (27%).

Results: With a median follow-up of 35 (1–135) months, estimated median progression-free survival (PFS) of all 98 patients was 95 months from diagnosis and 82 months from SCT(median overall survival not reached). Pretransplant rituximab was given to 40 patients and improved PFS (3-year PFS from SCT 93% (95%CI 83–100%) vs 67% (54–79%) although this was still not statistically significant (hazard ratio (HR) .37; p .07). In contrast, pretransplant HA had no impact on PFS even after adjusting for rituximab (HR 1.9; p .61). PFS after R-TBI/CY tended to be superior to that after TBI/CY only if the 58 patients without pretransplant rituximab were considered (HR .39; p .07). Age, sex, time from diagnosis, and remission status after cytoreduction or immediately prior to SCT did not affect PFS.

Conclusions: Pretransplant rituximab but not high-dose ara-C seems to improve the outcome of early SCT for MCL. Addition of rituximab to myeloablative treatment with TBI/CY might be beneficial only in the absence of pretransplant rituximab. Pretransplant rituximab with upfront SCT provides long-term disease control in a large proportion of patients with MCL.
Morphine Exaggerates Retinopathy in Transgenic Sickle Mice.
Authors: Gupta Kalpna , Chen Chunsheng , Lutty Gerald A. , Hebbel Robert P.
Published: October 2019

Proliferative retinopathy is a complication of sickle cell disease (SCD). Promotion of angiogenesis by opioids (Cancer Res62: 4491, 2002) suggests that opioid analgesics used to treat pain may exaggerate retinopathy in patients with SCD. To examine the hypothesis that morphine augments retinopathy, we used sickle mice with transgenes for human α and βS globin (NY1DD model) and their wild type (WT) C57/BL6 controls. Simulating the analgesic doses used in humans, we injected morphine subcutaneously (50 mg / day / 70 Kg with 10 mg increments every 2 weeks and / or equimolar amount of naloxone) for 6 to 15 months into 9 – 11 months old mice. Retinal vasculature was visualized with darkfield illumination using adenosine diphosphatese reaction. Morphine induced neovascularization, corkscrew formation resembling microangiopathy in human, arterio-venous anastomoses and several vascular tufts in the inner 1/3 and whole retina after 10 and 15 months of treatment, respectively in all NY1DD but not in WT mice. Only 1/5 or 2/7 PBS-treated NY1DD mice showed early neovascular changes at 20 (treated for 10 mo) or 26 (treated for 15 mo) months of age, respectively. Retinopathy scores by ICROP system used in neonatal intensive care units for abnormal vessels, blood vessel tortuosity and tufts in NY1DD mice were: 10 mo treatment, PBS, 1.5±0.5; morphine, 10±0.7 (p<0.0001 vs PBS); morphine + naloxone, 4±1; naloxone, 3.8±1.2 and 15 mo treatment, PBS, 5.9±2 and morphine, 13±0.5 (p<0.003 vs PBS). Naloxone significantly inhibited (p<0.005, morphine vs morphine +naloxone) morphine-induced retinopathy, suggesting an opioid receptor-mediated mechanism. To elucidate the mechanism of morphine-induced retinopathy we isolated retinal endothelial cells (REC) by panning with anti-CD31 from WT and NY1DD mice. Similar to VEGF induced proliferation and survival, morphine stimulated 5–6 fold proliferation and increased survival by about 75% in both WT and NY1DD REC, in a dose dependent manner. Both VEGF164 and morphine stimulated time-dependent MAPK/ERK and STAT3 phosphorylation in WT REC. VEGF and morphine stimulate nitric oxide synthase pathway and stimulate angiogenesis. This raised the possibility of a cross-talk between VEGF receptor(s) and opioid receptor(s). We found that morphine stimulates the phosphorylation of VEGFR2/Flk1 in WT REC which co-immunoprecipitated with mu opioid receptor (MOR) using anti-MOR antibodies. Both, morphine- or VEGF-induced phospho STAT3 also co-immunoprecipitated with MOR, suggesting that activation of either MOR or Flk1 transactivates the other. Thus, addition of morphine to the VEGF replete microenvironment in SCD can have an exaggerating effect on the progression of retinopathy. Since patients with SCD receive opioids for long periods of time, we speculate that retinopathy in these patients may in part be due to opioid use.
Autologous Stem Cell Transplantation and Rituximab for Mantle Cell Lymphoma.
Authors: Capote Francisco Javier , González-Barca E. , Bergua J.M. , Pascual M.J. , García-Boyero R. , Ribera J.M. , Jiménez A. , Romero A. , Giraldo P. , Fernández-Calvo J. , Ramírez J. , Amigo M.L. , Muñoz M.I. , Caballero D. , Guinot M. , Oriol A. , Queizán J.A. , Cantalapiedra A. , Moraleda J.M. , Grande C. , Palomera L. , Almagro M. , Leon A. , Fernández-Sevilla A. , Gil J.L.
Published: October 2019

Introduction: Mantle cell lymphoma (MCL) is a mature B-cell lymphoma comprising up 5% of non-Hodgkins lymphomas. Although the prognosis for MCL patients has improved in recent years, the outlook for those with advanced or recurrent disease remains poor and the role of hematopoietic stem cell transplantation is unclear. The HyperCVAD-M/A regimen (fractionated high-dose cyclophosphamide, vincristine, doxorubicin and prednisolone alternated with methotraxate and cytarabine) has yielded encouraging results when combined with autologous stem cell transplantation (ASCT). In an effort to improve these results further, we have combined rituximab in vivo purging and post-transplant consolidation with HyperCVAD-M/A plus ASCT.

Methods: Patients aged <65 years with previously untreated or relapsed MCL were treated with four courses of HyperCVAD-M/A followed by four once-weekly doses of rituximab 375mg/m2 as purging prior to stem cell mobilization and harvesting, high-dose chemotherapy (ICT-CY or BEAM), stem cell reinfusion and four further doses of rituximab immunotherapy post-transplant.

Results: Of the 40 patients enrolled so far, 20 (15 male, 5 female; 18 previously untreated) have been transplanted. The median age was 50 years (range 38–63 years). After the final post-ASCT immunotherapy all 20 patients were in clinical complete remission. With a median follow-up of 36 months from diagnosis (range 7–64 months), 18 patients remain alive with 13 in complete remission. One patient died 15 months post-ASCT without evidence of disease recurrence. Kaplan-Meier estimates of 5-year overall and event-free survival are 90% and 65% respectively.

Conclusions: This approach seems safe and feasible and produces durable remissions; longer follow-up of a more patients will be required to assess the effect of the procedure on survival.
Nucleotide Insertions and Deletions in Chronic Lymphocytic Leukemia. A CLL Specific Deletion among IGHV3-21 Expressing Cases with Stereotyped Receptors.
Authors: Belessi Chrysoula , Davi Frédéric , Stamatopoulos Kostas , Degano Massimo , Andreou Thanassis , Moreno Carol , Merle-Beral Hélène , Crespo Marta , Laoutaris Nikolaos , Montserrat Emili , Caligaris-Cappio Federico , Anagnostopoulos Achilles , Ghia Paolo
Published: October 2019

Insertions/duplications and deletions (I/D/Ds) in IG variable region genes are infrequent sequelae of the somatic hypermutation process. In the present study, we document the occurrence of nucleotide I/D/Ds in rearranged IGHV genes in CLL patients. Among 809 IGHV-D-J sequences amplified in 760 CLL cases, 9 (1.11%) and 15 (1.85%) sequences exhibited IGHV sequence changes consistent with nucleotide insertions/duplications or deletions, respectively. I/D/Ds were found in genes of the IGHV1, IGHV3 and IGHV4 subgroups, always within mutated IGHV-D-J rearrangements. In 21/24 cases, the inserted/duplicated or lost nucleotides occurred in multiples of 3; therefore, the original reading frame was maintained and a potentially intact receptor was coded. I/D/Ds were located completely or in part within CDRs in 21/24 cases; sequence motifs (AGY, AGA, AAC trinucleotides) that resemble intrinsic hotspots for somatic hypermutation were identified in 21/24 sequences. Short stretches with high homology (misalignment feet) that would offer the DNA polymerase an alternative template for re-annealing in cases of replication slippage were identified in all CLL sequences carrying I/D/Ds. I/D/Ds were generated somatically since (i) they always occurred in cases with somatic mutations, (ii) they could not be found on sequencing analysis of the corresponding germline IGHV genes amplified on DNA isolated from selected patients’ neutrophils, thus excluding a possible genetic polymorphism. The incidence of I/D/Ds in CLL is consistent with previous reports in normal, autoreactive and neoplastic human B cells, thus seemingly indicating that these modifications generally arise without any particular disease-specific associations. A striking exception to this rule was identified in the case of CLL IGHV3-21 expressing cases: one aminoacid was deleted from the CDR2 region in 16/74 (21.6%) IGHV3-21 CLL sequences (database-derived IGHV3-21 CLL cases + present series) vs. only 2/340 (0.59%) non-CLL IGHV3-21 sequences; 15/16 CLL IGHV3-21 sequences carrying this deletion belonged to a subset with unique, shared HCDR3s and light chain CDR3 motifs. The effect of the CDR2 deletion on the structure of the IG molecules in this subset of CLL patients was studied with molecular modelling and dynamics simulations. The models suggested that the deletion could be accommodated without significantly affecting the local structure. The close association of deletions in CDR2 with the homologous subset of IGHV3-21 expressing CLL cases provides further evidence for the importance of an antigenic drive in malignant transformation and/or maintenance of the neoplastic clone in at least some CLL cases.
Interaction between B-CLL Cells and Lymphoid Microenvironment Leads to Activation of PI3-K/Akt Pathway and Inhibition of Apoptosis.
Authors: Shehata Medhat , Schnabl Susanne , Demirtas Dita , Schwarzmeier Josef D. , Hilgarth Martin , Duechler Markus , Hubmann Rainer , Jaeger Ulrich
Published: October 2019

Inhibition of apoptosis and long survival leads to accumulation of the leukemic cells in B cell chronic lymphocytic leukemia (B-CLL). This could be due to activation of anti-apoptotic cascades in CLL cells through interaction with their lymphoid microenvironment. Therefore, we investigated the role of tumor microenvironment in prolongation of survival of B-CLL cells and activation of the potent anti-apoptotic PI3-K/Akt pathway.

Stromal fibroblasts of bone marrow (BMFs), spleen (SF) and lymph gland (LGF) were used as an in vitro model for lymphoid microenvironment and we tested their ability to inhibit spontaneous apoptosis of B-CLL cells. Co-culture of B-CLL cells with human BMFs, LGF, and SF significantly inhibited apoptosis and prolonged survival of the leukemic cells in comparison to suspension cultures and to co-cultures with fibroblasts obtained from non-lymphoid organs. Trans-well culture experiments indicated that cell-cell interaction and soluble mediators are essential for this supportive effect. To explore the involvement of PI3-K/Akt pathway in the anti-apoptotic effect of stromal fibroblasts, co-cultures were performed in presence of PI3-K inhibitors (wortmannin or Ly294002) or siRNAs against PI3-K (p110ß subunit) and Akt1. These inhibitors significantly reduced the supportive effect of stromal fibroblasts and induced apoptosis in B-CLL cells. Interestingly, the leukemic cells were far more sensitive to PI3-K inhibition than T cells, monocytes and fibroblasts. Induction of apoptosis was associated with a significant decrease in the intracellular PIP3, PI3-K, PDK1 and Akt1, NF-kappa B, IKK and de-phosphorylation/activation of tumor suppressor protein PTEN. Studies using phosphospecific anti-PTEN antibody demonstrated that PBMC of CLL patients (n=40) highly express a phosphorylated form of PTEN. The results demonstrate that the PI3-K/Akt pathway is involved in inhibition of apoptosis of B-CLL cells and suggest that interaction of the leukemic cells with lymphoid microenvironment maintains the activation of this pathway. The data also suggest that targeting this pathway represents a new option for designing novel therapeutic strategies in B-CLL.
Hypoxia Inducible Factor-1α Is over Expressed in CLL B Cells Because of an Impaired Proteasome Pathway Associated with Defective Interaction with von Hippel-Landau Protein.
Authors: Lee Yean K. , Strege Ann K. , Bone Nancy D. , Wellik Linda E. , Chan D. A. , Giaccia A. J. , Mukhopadhyay Debabrata , Kay Neil E.
Published: October 2019

We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.
FCR-3 as Frontline Therapy for Patients with Chronic Lymphocytic Leukemia (CLL).
Authors: O’Brien Susan , Wierda William G. , Faderl Stefan , Ferrajoli Alessandra , Bueso-Ramos Carlos E. , Browning Mary , Kantarjian Hagop M. , Keating Michael J.
Published: October 2019

The combination regimen of Fludarabine, Cyclophosphamide, and Rituximab (FCR) has produced high overall response (OR) rates and complete remission (CR) rates in patients receiving this as initial treatment for CLL. Earlier studies have shown that dose-dense or dose-intensified regimens of single-agent Rituximab produced higher response rates in patients with CLL. The current regimen, FCR-3, was based on the hypothesis that increasing the amount of Rituximab in the combination might improve response rates and remission duration. Doses of chemotherapy were Fludarabine 25 mg/m2/Dx3, and Cyclophosphamide 250 mg/m2/Dx3 given monthly. Rituximab was given at 375 mg/m2 as the first dose and 500 mg/m2 for all subsequent doses. On each day of the chemotherapy a dose of Rituximab was given so 3 doses were given monthly. With the previous FCR regimen 45% of patients achieved a CR or nPR and had <5% CD19+ 5 positive cells in the marrow at the completion of 3 cycles of therapy (rapid response, RR). The median time to progression (TTP) in that group has not been reached; it was 3 years in patients who did not achieve that endpoint (p<.001). The current protocol aimed to increase that response rate from 45% to 60%. Sixty-five patients were treated. Eighty percent were men. The median age was 59 years (27–82). Rai Stage 3–4 disease was present in 25% of patients. Median WBC count was 92.4 x 103/ul (7.9–363). Median B2-microglobulin was 3.8 (1.6–10.1). Unfavorable FISH abnormalities were present in 35% of 52 evaluable patients. Somatic hypermutation status was available for 44 patients; 61% were unmutated. ZAP-70 expression analysis performed by immunostaining or flow cytometry was positive in 62% of 47 evaluable patients. Results of FCR-3 in comparison to FCR are shown in the table.

3 Cycles 6 Cycles No. RR(%) OR CR Flow<5% FCR 300 45 95 72 82 FCR-3 65 45 94 65 74

PCR negativity using consensus primer PCR was achieved in 49% of patients at the end of therapy. Median number of days between courses ranges from 29–35 per course (overall range 27–95). Eighty-five percent of patients completed 6 cycles. No patient has progressed with a median follow-up of 10 months. With limited follow-up the addition of 3 doses of Rituximab to FC chemotherapy does not appear to provide greater benefit than one dose.
The Effect of Melatonin on Thrombopoiesis in Irradiated Mice.
Authors: Yang Mo , Li Karen Kwai Har , Huang Wei Zhe , Li Xiao Jing , Pong Nga Hin , Liu Ai Guo , Li Chi Kong , Fok Tai Fai , Ng Pak Cheung
Published: October 2019

We have reported that serotonin (5-hydroxytryptamine, 5-HT) significantly stimulates megakaryocytopoiesis. It has been known that platelet count can be increased in stress conditions and serotonin has an effect on actin re-organization and promotes nitric oxide (NO) synthesis via 5-HT2A receptors. NO may induce apoptosis of mature megakaryocytes to produce platelets. Taken together, we proposed a possible mechanism of serotonin on megakaryocyte differentiation and platelet formation: serotonin enhances the differentiation and maturation of megakaryocytes via 5-HT2BR and actin re-organization; and serotonin is involved in the process of platelet formation by promoting the synthesis of NO to induce cell apoptosis via 5-HT2AR. Serotonin is the precursor of melatonin. The effect of melatonin on thrombopoiesis in irradiated mice (4 Gy) was investigated in this study. Eighteen mice were randomly divided into three groups (6 in each). Group 1 (normal control, N) received no irradiation or melatonin; Group 2 (model control, C) and Group 3 (melatonin, M) received 4 Gy total body irradiation for 3 minutes. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group M for consecutively 21 days. And Group C was administered normal saline instead of melatonin. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21. All the mice were executed to collect bone marrow cells for the assay of CFU-MK and CFU-F (fibroblastoid). Our results showed that melatonin enhanced the recovery of platelets and WBC counts. Moreover, melatonin also promoted CFU-MK (20 ± 3 vs 11 ± 2 colonies/2 x105 cells) and CFU-F formation (28 ± 10 vs 15 ± 3 colonies/2 x106 cells). We also investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0–500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, N=6) compared with the control. The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature. Our studies showed that melatonin had a promoting effect on thrombopoiesis in this model and also stimulated in vitro CFU-MK formation. Thus, melatonin could be developed as an alternative drug for the treatment of thrombocytopenia.
Tissue Factor-Driven Enhanced Platelet Aggregation in Flowing Blood in a Rabbit Model of Childhood Hemolytic Uremic Syndrome.
Authors: Grabowski Eric F. , Nestoridi Eirini , Garcia Alexander , Fox James G. , Ingelfinger Julie R.
Published: October 2019

The Dutch belted rabbit constitutes a promising animal model of the childhood hemolytic uremic syndrome (HUS; A. Garcia, J. Infect. Dis. 2002). When Shiga toxin (Stx)-producing E. coli are administered orally, these rabbits develop HUS. Platelet-fibrin thrombus formation in this model was examined on unfixed renal tissue (5 μm sections) after mounting on 150-μm glass slides in test animal-control animal pairs. Normal donor blood, collected into 4U/ml low MW heparin and 10 μM quinacrine dihydrochoride, was drawn at shear rates of 270–650 sec-1 through a parallel-plate flow chamber for which one surface was one of the above slides. Such shear rates give rise to shear stresses which approach the 20–25 dynes/cm2 estimated to exist in glomerular arterioles. Platelets depositing on the sections were imaged in real time using epifluorescence digital videomicroscopy, with quantitation of adherent platelets via image processing. For 16 pairs of test and control sections, the percent of image pixels occupied by adherent platelets after 6–9 min of blood flow was 17.7 ± 3.06 (mean ± SD) for test sections vs 5.71 ± 5.18 for controls (P<0.001). This 3.1-fold increase for sections from Stx-treated rabbits is all the more striking considering that individual platelet aggregates on these sections were also larger and thicker, as judged from greater local pixel intensities. In 4 additional experiments, preincubation of sections with 300 nM of a monoclonal antibody (courtesy of Y. Nemerson, Mt. Sinai Medical Center, NY, NY) directed against human tissue factor (TF) with known cross-reactivity to rabbit TF markedly reduced platelet adhesion/aggregation on both test and control sections. Residual platelet deposition, which only slightly favored test sections, may be due to either a non-TF-dependent mechanism or incomplete neutralization of TF activity by the amount of antibody used. Studies of parallel fibrin deposition are in progress. Thus, renal tissue from the Dutch belted HUS model is reactive to platelets in large part via expression of TF, an expression augmented on test sections. These observations support the hypothesis that the TF pathway of coagulation plays a major role in childhood HUS.
Does the Platelet Factor 4 (PF4) ELISA Correlate with the Clinical Diagnosis of Type II Heparin-Induced Thrombocytopenia?.
Authors: Streiff Michael B. , Kickler Thomas S. , Weir Edward G.
Published: October 2019

Type II heparin-induced thrombocytopenia (HIT) is an antibody-mediated complication of heparin therapy that is associated with a consumptive thrombocytopenia and a high risk of thromboembolism. It is now known that antibodies associated with type II HIT recognize sites on PF4 when complexed with heparin. Due to technical challenges associated with the serotonin release assay, most clinicians rely on the PF4 ELISA to establish a laboratory diagnosis of HIT. However, it is unclear whether the ELISA has predictive value in identifying those patients who have clinically apparent disease and whether ELISA optical density (OD) measurements correlate with the development of thrombosis. We retrospectively reviewed the medical records of 103 sequential patients who tested positive for HIT by a commercial PF4 ELISA from 2000–2002. While blinded to ELISA OD values, we recorded patient age and gender, admission diagnosis, hospital course, type of heparin administered, route of administration, baseline and nadir platelet counts, timing of thrombocytopenia, HIT therapy, clinical thrombotic events within 30 days, and other potential causes of thrombocytopenia to determine the clinical likelihood of HIT. The association between OD values and both the clinical likelihood of HIT and objectively confirmed thrombotic events was analyzed using the Pearson Chi-square test. Median patient age was 62 yrs and 52% of patients were female. Those with cardiac disease, cancer, and venous thromboembolism constituted 44%, 19% and 9% of the study population, respectively. Median platelet count nadir was 40K/mL (range, 2K–90K), and average onset of thrombocytopenia was 6.5 days. Median ELISA OD value was 0.77 (0.40–3.81). The clinical likelihood of HIT was noted in 24/103(23%) patients, and 22(21%) suffered a thrombotic event. An ELISA OD >1.0 was significantly associated with the clinical likelihood of HIT (p=0.03), though OD values ranged from 0.41 to 3.81 among these patients. Neither an OD threshold of 1.0 or 1.5 was associated with thromboembolism (p=0.2 and p=0.1, respectively). In contrast, the clinical likelihood of HIT was significantly associated with subsequent thrombosis (p=0.002). ELISA results were not significantly associated with the extent or onset of thrombocytopenia, type of heparin or route administered, patient age or gender, admission diagnosis, or hospital course. In conclusion, PF4 ELISA OD values are significantly associated with the clinical likelihood of HIT but are not predictive of HIT on an individual patient basis. Clinical judgment but not ELISA OD values is associated with subsequent thromboembolism. In summary, the clinical likelihood of HIT, and not ELISA results, remains the best parameter by which to modify anticoagulation therapy in patients being assessed for HIT.

Figure Figure
Restoration of the CD4+CD25bright Regulatory Cell Subset in Patients with Crohn’s Disease Following Autologous Non-Myeloablative Stem Cell Transplantation (NST).
Authors: Verda Larissa , Oyama Yu , Craig Robert , Statkute Laisvyde , Krosnjar Nela , Quigley Kathleen , Burt Richard K.
Published: October 2019

Regulatory CD4+CD25 bright cells are a distinct population of T cells which modulate Th1 and Th2 mediated immune responses and have a preventive role in the development of autoimmune diseases including Crohn’s disease. Using flow cytometry we analyzed the percentage and absolute number of CD4+CD25bright cells in the peripheral blood of Crohn’s disease (CD) patients before and following non-myeloablative stem cell transplantation. All patients (N=14) survived the NST procedure and showed marked clinical and histological improvements in disease severity. Prior to NST, both the frequency and absolute number of CD4+CD25 bright cells in CD patients were reduced compared to healthy volunteers (p<0.01). Following NST, a robust increase in the absolute number of CD4+CD25+ cells was observed: pre-NST 10.3 ± 3.5 (N=14) vs. 6 mo post-NST 28.1 ± 13.4 (N=13) vs. 1 yr post-NST 24.7 ± 12.3 (N=12) versus 2 yr post-NST 20.2 ± 10.2 (N=9); p < 0.01. Two patients who relapsed after NST had less prominent increases in CD4+CD25 bright cell subset. When these patients were excluded from analysis, the difference between pre and post NST measurements increased (p < 0.0001). We conclude that autologous non-myeloablative stem cell transplantation induces the recovery of CD4+CD25 bright regulatory T cells which may contribute to the restoration of immunological homeostasis and remission of disease.

Absolute number of CD4+CD25+ cells in PB of CD patients pre and post NST Absolute number of CD4+CD25+ cells in PB of CD patients pre and post NST
Rituximab Mediated Depletion of B Cells Following Transplant of Human CD20 Transgenic Mouse Bone Marrow Results in Prolonged B Cell Depletion and Augmented Anti-Tumor Immune Response.
Authors: Zhang Yu D. , Nechushtan Hovav , Jones Monica , Chan Andrew , Shin Seung-uon , Pham Dien , Cho Hyung-Mee , Yi Kyung-Hee , Levy Robert , Rosenblatt Joseph D.
Published: October 2019

We previously demonstrated rejection of murine tumors due to enhanced anti-tumor cytolytic and Th1 cytokine responses in mice lacking B cells (BCDM) compared to immunocompetent mice. We wished to compare immune and anti-tumor responses in BCDM to mice functionally depleted of B cells using an anti-human CD20 antibody (Rituximab). We evaluated Rituximab effects on the outgrowth of human CD20 transgenic mouse (huCD20 Tg) B cells following the transplant of huCD20Tg CD45.2+ bone marrow (BM) into sublethally irradiated congenic CD45.1+ mice. CD45.1 mice were irradiated with 9.5Gy at day -1, followed by infusion of 3x106 huCD20Tg BM cells on day 0, and by treatment with or without Rituximab at 400ug/mouse given once weekly i.v. for 4 weeks. Without Rituximab treatment, B cell percentages in the peripheral blood (PB) were approximately 46.4%±1.3; spleen, 77.8%±1.5; and peripheral lymph nodes (pLNs), 43%±1.5 at day 47. With Rituximab treatment, CD19+ cells in the PB, spleen and pLNs were 1.8%±0.4, 4.3%±1.3, and 0.8%±0.2 respectively at 47days post transplant. Higher percentages of CD3+ T cells in PB, spleen and pLNs (66.1%±0.5, 51.7%±1.1 and 43.0%±3.8) were noted in the Rituximab treated group compared to controls (19%±0.9, 10.3%±1.0 and 32.3%±1.7 respectively). The percentage of remaining B cells in PB declined progressively from 5.6% at day 13 to 1.8% at day 47 post transplant indicating that Rituximab depletion of huCD20+ transgenic B cells was progressive, durable and very effective. In contrast, CD3+ cells in PB increased from 11.2% (day 13) to 66.1 % (day 47) following Rituximab treatment. Interestingly reconstitution with endogenous murine B cells in huCD20 transplanted mice was minimal. The huCD20 Tg donor-derived T cells in the Rituximab treated group maintained a CD44low naive phenotype.

The growth of MC38 tumors implanted 15 days following transplant was significantly slowed in the Rituximab treated group. Increased IFN-γ production and higher expression of the cytolytic marker CD107a by T cells was seen in the Rituximab treated group compared to controls. The combination of myeloablative irradiation, huCD20 transgenic bone marrow transfer and Rituximab depletion, was able to generate mice devoid of CD20+ B cells. Rituximab treated huCD20+ reconstituted B cell depleted mice have substantially higher percentages of T cells than control non-Rituximab treated mice, show sustained depression of B cell numbers and manifest an increased anti-tumor Th1 response. The huCD20 transplant model will be useful in studying effects of post-transplant B cell depletion on immune reconstitution and in the design of future clinical transplant and immunization strategies.
A Novel C-Type Lectin Receptor DCL-1 (CD302) Is a Potential Endocytic Receptor for Tumor Antigen Loading into Antigen Presenting Cells.
Authors: Kato Masato , Khan Seema , McDonald Kylie J. , Hart Derek N.J.
Published: October 2019

DCL-1 (CD302) is a novel C-type lectin receptor, discovered as a genetic fusion partner of DEC-205 (CD205), a C-type lectin that delivers antigen from the surface into DC antigen processing pathway (J. Biol. Chem.2003. 278:34035). We have now examined the molecular nature and expression of DCL-1. DCL-1 contains an external single C-type lectin-like domain followed by a transmembrane and cytoplasmic domain with putative signaling and endocytic motifs. The DCL-1 gene, consisting of 6 exons, was mapped to chromosome 2q24, approximately 5 kb downstream of the DEC-205 gene. Human DCL-1 has 76% protein identity to the mouse ortholog, suggesting its highly conserved function. Northern blot analysis showed that the 4.2 kb DCL-1 mRNA was present in myeloid cell lines, but not in T and B cell lines. Specialized phagocytic cells (i.e. monocytes, macrophages, granulocytes), monocyte-derived DC (MoDC) and blood DC (BDC) expressed DCL-1 mRNA, detected by RT-PCR. Immunoprecipitation/ Western blot (IP/WB) analysis using FLAG-tagged DCL-1 transfectants detected a broad band (modal size 35 kDa), which became a single 26 kDa band after N-glycosidase F digestion, indicating that DCL-1 is N-glycosylated. Flow cytometry studies with DCL-1 mAb (MMRI-20, developed in house) detected moderate levels of DCL-1 expression on granulocytes, monocytes, macrophages, MoDC and BDC, but not on lymphoid cells. Concomitant WB/IP analysis using the DCL-1 mAb detected 26 and 33 kDa doublets in monocytes, macrophages and MoDC. DCL-1 transfectants, macrophages, MoDC and BDC endocytosed cell surface DCL-1 after crosslinking with the DCL-1 mAb. DCL-1-Ig fusion protein bound to GlcNAc- and Man-conjugated beads, but not to GalNAc-, Fuc- and mannan-conjugated beads in a calcium-dependent manner. Thus, DCL-1 is expressed by phagocytes and DC, behaves as a C-type lectin, and is likely to be involved in endocytosis/phagocytosis of microbes. We are exploiting DCL-1 as a potential antigen-loading receptor for DC tumor immunotherapy.
Cytokine Gene Polymorphisms in Turkish Patients with Childhood Chronic Myeloid Leukemia.
Authors: Ozdilli Kursat , Oguz Fatma , Duvarci Yeliz , Bilgen Hulya , Anak Sema , Carin Mahmut , Gedikoglu Gunduz
Published: October 2019

Cytokines are necessary for normal hematopoiesis in the bone marrow and provide a means of fine-tuning bone marrow function in response to stimulation. Several of cytokines generated during both innate and adaptive immune responses stimulate the growth and differantiation of bone marrow progenitor cells. The vast majority of polymorphism found in cytokine genes and their reseptors are located in non coding regions. Cytokine gene polymorphism may be implicated in the pathogenesis of infections, autoimmun disease and malignancies via their effect on cytokine production and regulation. It is known that leukemic cells proliferate under the influence of cytokines.

Our aim is to analyze cytokine gene profiles in patients with chronic myeloid leukemia (CML)in order to clarify the pathogenesis of CML.

Genomic DNA from 26 CML patients were analysed. Genotyping was performed by polymerase chain reaction with sequence specific primers (PCR-SSP) using “ One lamda” kit in 26 childhood CML patients 60 unrelated healthy individuals. Genotype frequencies between CML patients and controls were compared using Chi-Square Yates, Fisher’s Exact Tests. In this study; 26 CML patients with a mean age of 15,6±5,3 and 60 healthy controls with a mean age of 18,2±5,4 were investigated.

In CML patients the frequencies of TGF-b(TC/GG) genotype (in chi-square p=0,01,odds ratio(OR)=3,46, 95% confidence interval (CI) 1,3–9,04) and IL-10 (GCC/ATA) genotype (in chi-square p=0,04,OR=3,3, 95% CI 0,9–11,1 were found higher in patients with CML compared to the control group. On the other hand IL-6 (CC) genotype (in chi-square p=0,012,OR=0,17, 95% CI 0,4–0,76 frequency was found higher in the control group compared to the patients with CML.

As a conclusion higher frequency in TGF-b(TC/GG) and IL-10(GCC/ATA) genotype polymorphism were significantly higher in the patients with CML so it may predispose to CML. On the other hand IL-6 (CC) genotype might be the preventive factor for CML.

The present study is rather significant that it is the study which assessed the relation of the cytokines in patients with CML compared to the control group taken from the genetic pool of The Turkish population, we believe studies like this will eventually help to understand the pathogenesis of CML and the role cytokines play in CML,but larger groups of studies must be done in future.
Short Nucleotide Insertions in the MCL-1 Promoter Affect Its Gene Expression.
Authors: Moshynska Oksana , Moshynskyy Igor , Bosch Mark , Qureshi Tania , Saxena Anurag
Published: October 2019

We have recently reported a short nucleotide (six and eighteen) insertion (6- and 18-nt) in the MCL-1 promoter (−190bp upstream of the major transcription start site, GenBank, AN: AF198614) in B-cell chronic lymphocytic leukemia patients. These insertions were found to be polymorphisms. CD38-negative CLL patients with these promoter insertions had higher mRNA levels, poor response to standard chemotherapy and shorter survival. The aim of the present study was to test the hypothesis that these insertions directly effect MCL-1 gene expression. K562 and HeLa cells were transfected with pGL3-Basic, pGL3-Control and four reporter plasmids were constructed with a +0/0, +6/6, +18/18, and −23/23 MCL-1 promoter linked to Luciferase. Firefly activity in each transfection was normalized to Renilla Luciferase activity. At 24-hours post-transfection cells were induced with phorbol 12-myristate 13-acetate (PMA), granulocyte-macrophage colony stimulating factor (GM-CSF), both PMA and GM-CSF, or with fresh media only. At 48-hours post-transfection cells were harvested and Dual-Luciferase Assay was performed. Compared to transfection with pGL3-(+0/0) plasmid, the Luciferase PMA-stimulated activity for K562 cells transfected with pGL3-(+6/6) and pGL3-(+18/18) was 1.7 (P=0.0277) and 1.5 times higher (P=0.0067) respectively, and for pGL3-(−23/23) was 3.2 times lower (P=0.0021). The Luciferase PMA-stimulated activity for HeLa cells transfected with pGL3-(+6/6) was 3.3 times higher (P=0.0038), pGL-3-(+18/18) was 2.3 times higher (P=0.0245), and did not differ significantly for pGL3-(−23/23), compared to pGL3-(+0/0). Similar results were observed for GM-CSF-stimulated activity for HeLa cells, where activity was 3.9 times (P=0.0068) for pGL3-(+6/6) and 2.5 times (P=0.0258) higher for pGL3-(+18/18). Unstimulated Luciferase activity was also 2.5 times higher (P=0.0115) for pGL3-(+18/18) compared to pGL3-(+0/0) plasmid. These results provide experimental evidence for the positive effect of these nucleotide insertions (6- and 18-nt) on MCL-1 gene expression in both hemopoietic (K562) and epithelial (HeLa) cells.
Gene Expression Studies Reveal a Potential Mechanism of Glivec Resistance in Patients with Ph+ ALL Treated.
Authors: Loi To Ha , Ma David D.F.
Published: October 2019

In Ph+ ALL, patients respond to Glivec but nearly always relapse due to acquired resistance. The biological processes associated with Glivec resistance in Ph+ ALL are yet to be fully elucidated.

The aim of this study is to use microarray and Q-PCR technology to dissect changes in signaling pathways of blast cells in Ph+ ALL patients treated with Glivec. Peripheral blood (PB) samples were taken before treatment and on consecutive days after administration of Glivec from two Ph+, c-ALLA+ and CD19+ ALL patients. Blast cells were isolated and their gene expression assayed using 19K cDNA microarrays.

Over 400 differentially expressed genes were identified with at least a 1.5-fold up- or down-regulation in treated cells compared to cells collected pre-treatment. Based on these gene expression results, three main gene ontology groups were further evaluated: Apoptosis, Proliferation and B cell differentiation. The up-regulation of Bim and Bcl-6, and down regulation of Cyclin D2, confirms the induction of apoptosis via the FOXO3a pathway in cells treated with Glivec in vivo. Interestingly, both the proliferation genes, Tcl1-A and PKCe, and B cell differentiation associated genes, including CD79a, ETS1 and a cohort of IGH and Igl and k genes, were up-regulated during therapy.

These gene expression changes observed in vivo were confirmed by Q-PCR in the Ph+ cell lines K562 (derived from CML blast crisis) and SUP-B15 (derived from ALL) treated with Glivec. The unexpected finding of increased expression of pro-proliferative genes and B cell differentiation genes by microarray revealed potential links with early B cell development and B cell receptor (BCR) signaling.

Evidence for apoptosis and proliferation of Ph+ cell lines treated with Glivec were then examined by FACs. After 5 days of treatment with Glivec, 90% of K562 and 50% SUP-B15 cells underwent apoptosis. Furthermore, cell cycle analysis revealed the existence of a population of cells in G2 phase even after 6 days of Glivec treatment in SUP-B15 but not K562 cells, thus providing evidence of a population of cells undergoing proliferation during Glivec treatment in vitro.

In summary, our in vivo observations supported by in vitro experiments suggest that Glivec induces the majority of Ph+ ALL blasts to undergo apoptosis. However, as treatment is prolonged, a population of ALL cells escapes death and undergoes proliferation and differentiation. We hypothesise that Glivec induced differentiation and proliferation of Ph+ cells may result in the clonal enrichment of cells resistant to Glivec.
Antitumor Genotoxic Agents, Such as Fludarabine*, Doxorubincin or Cis-Platine, Induce Activation of STAT1 in a p53 Protein Dependent Manner.
Authors: Youlyouz-Marfak Ibtissam , Le Clorennec Christophe , Najjar Imen , Baran-Marszak Fanny , Gachard Nathalie , Remenieras Liliane , Jaccard Arnaud , Bornkamm Georg W. , Fagard Remi , Feuillard Jean
Published: October 2019

Introduction:

Chemotherapeutic drug such as Fludarabine*; doxorubicin or cis-platine induce cell cycle arrest and apoptosis via activation of p53. Convergent studies suggest that p53 and STAT1 cooperate in the induction of apoptosis, and that STAT1 favors p53 activation. However, to our knowledge, the role of p53 in the activation of STAT1 is not documented. We present our results suggesting that (i) genotoxic agents are STAT1 inducers, (ii) STAT1 activation depends on the presence of p53 protein, and (iii) this phenomenon is modulated by the tyrosine kinase inhibitor STI571.

Materials and Methods:

To analyse the role of p53 in STAT1 activation, we have used different cellular models with different p53 status: PRI (p53wt), BL2 (p53wt), BL41 (p53 mutated on Arg248, resulting in the loss of p53 DNA binding activity (p53mut)), Jurkat, HL60 and MEF (the 3 latter being p53 null). The following cDNAs were used for functional studies: p53wt, p53mut, MDM2 and MTBP (MDM2 transforming protein). These cDNAs were cloned either in a pcDNA3 vector or a pRT-1 inducible vector (in the latter, the gene of interest is expressed from a bidirectional doxycycline regulatable promoter allowing simultaneous expression of truncated NGF receptor, used as a surrogate marker of inducibility).

Results:

Treatment of the different cell lines with the 3 genotoxic drugs Fludarabine*, doxorubicin or cis-platine induced STAT1 activation in p53wt BL2 or PRI cells and in p53mut BL41 cells, but not in Jurkat cells neither in HL60 or MEF cells. Induction of STAT1 was also obtained in presence of the RNA synthesis inhibitor Actinomycin D or in presence of secretion inhibitor Brefeldine A. Over-expression of p53wt or p53mut markedly increased STAT1 activation in PRI cells. This effect was reversed by over-expression of MTBP. Complementation of both HL60 and MEF cells with both p53wt and p53mut cDNA induced constitutive STAT1 activation, an effect that was increased by treatment with doxorubine in transfected HL60 cells. This effect was reversed by over-expression of MDM2 in HL60 cells. Finally, we found that treatment of cells with the inhibitor STI 571 of c-Abl tyrosine kinase, a kinase known to be associated with ATM during p53 activation, decreased STAT1 activation by genotoxic drugs.

Conclusion:

Our results show that genotoxic agents are inducers of STAT1, that p53 protein but not p53 transcriptional activity is responsible for this STAT1 activation, and suggest a possible involvement the cABL tyrosine kinase.
The TCL1 Gene Is Involved, Other Than in Lymphoid Differentiation and Early Embryogenesis, Also in the Development of the Hair Follicle.
Authors: Ragone Gianluca , Bresin Antonella , Picchio Maria Cristina , Lazzeri Cristina , Cristofoletti Cristina , Kang Sang-Moo , Narducci Maria Grazia , Russo Giandomenico
Published: October 2019

The TCL1(T-cell leukaemia lymphoma1) oncogene plays a causative role in the onset of T and B cell leukaemia of mature phenotype, such as T-prolymphocytic leukema T-(PLL) in human and mice and B-cell-chroni lymphatic leukaemia (B-CLL) in mice due to its overexpression. Moreover, Tcl1 −/− mice show that this gene is also important during the early embryogenesis and lymphocyte differentiation. An undiscovered role was found for TCL1 gene in the skin. In this work, we show evidences that give Tcl1 a new, important role in mouse hair follicle (HF) and skin homeostasis. In fact, Tcl1 −/− adult mice develop premature hair loss leading to alopecia followed by extensive skin lesions, starting from the dorsal region. The expression pattern of Tcl1 in the skin throughout the different stages of hair and skin differentiation, by in vivo immunofluorescence and fluorescent in situ hybridization experiments, shows a high level of the mRNA and protein in HF mainly in the secondary hair germ cells and hair bulge but not in dermal papilla (DP), both in early anagen and late catagen phases, and to less extent in the epidermis in all the stages examined.

Loss of Tcl1 expression in the skin, although initially does not show any apparent morphological defects in the HF and epidermis, nonetheless leads to an increased rate of apoptosis and to a parallel reduction of proliferation through an unknown pathway, independent from the phosphorylation of akt proteins. Indeed, we show evidences that loss of Tcl1 affects CD34 high expression level in the bulge stem cells that is necessary, to these population, to maintain slow-cycling and stem characteristics along the time. Finally, the higher expression of the Tcl1 ortholog Tcl1B2 in epidermis and hair bulb can further explain the late onset, in adult mice, of alopecia and skin lesions. Our findings suggest that Tcl1 gene(s) have important roles in cycling, self-renewing stem cell populations in skin. This observation, coupled with other previous work showing that TCL1 gene is expressed in B-cell entering the germinal follicle in the lymph-node and in the preimplantation embryo until the 8-cell stage, points out to an interesting role for this gene for cells that at some point in their life must re-enter cell cycle.