bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential of Angeli’s Salt To Reduce Nitric Oxide Scavenging in Hemolysis.
Authors: Kim-Shapiro Daniel B. , He Xiaojun , Azarov Ivan , Richardson Jodi , King S. Bruce , Gladwin Mark T.
Published: April 2020

Since cell-free hemoglobin (Hb) scavenges nitric oxide (NO) more effectively than that encapsulated in the red blood cell (RBC), hemolysis reduces NO bioavailability with pathological consequences in sickle cell disease and other hemolytic anemias, paroxysmal nocturnal hemoglobinuria, thalassemia intermedia, malaria, and cardiopulomonary bypass. The ability of the cell-free Hb to scavenge NO is reduced when the Hb is converted from its oxygenated form (HbO2) to its oxidized form, methemoglobin (MetHb). We show that Angeli’s salt (AS) can convert two OxyHb to two MetHb molecules through its release of nitroxyl (HNO). AS also further converts the MetHb to less potentially oxidative forms: nitrite bound MetHb and iron-nitrosyl hemoglobin. We also show that, due to the fast reactivity of HNO with Hb (like NO), AS preferentially reacts with cell-free rather than RBC encapsulated Hb. In conditions simulating sickle cell crisis, within six minutes, AS converted 45 ± 12% of cell-free Hb to non-NO scavenging forms (MetHb and iron-nitrosyl Hb) and only converted 0.4 ± 0.3% of RBC encapsulated Hb (n=3). Similar preferential reactivity was observed under other physiologically relevant conditions. We conclude that, as AS preferentially reacts with cell-free Hb compared to RBC encapsulated Hb, converting it to species that do not effectively scavenge NO, it is has promise as a treatment for increasing NO biavailability in conditions associated with hemolysis.
Phlebotomy and Bolus Subcutaneous Deferoxamine in Algerian Patients with Thalassemia Major or Intermedia or Sickle Cell Disease Treated with Hydroxyurea.
Authors: Bradai Mohamed , Bachir Dora , De Montalembert Mariane
Published: April 2020

Background: Iron chelation with continuous subcutaneous deferoxamine injections is painful and expensive, in particular due to the cost of infusion pumps. Oral chelators are not widely available in Algeria. This prospective study was designed to evaluate the safety and efficacy of bolus subcutaneous deferoxamine (DFO) and phlebotomy in patients with beta-thalassemia major (TM) or intermedia (TI), or sickle cell disease (SCD), treated with hydroxyurea.

Methods: 31 patients were enrolled (22 TM, 5 TI, 4 SCD). Mean hydroxyurea dosage was 17 mg/kg/d [15–24]. Phlebotomy was performed when hemoglobin was >8 g/dL and ferritin >1000 ng/ml; 13 patients met these criteria (5 TM, 4 TI, 4 SCD). One TI patient was not compliant; the 12 remaining patients underwent phlebotomy removing 3 to 7 ml/kg, every 2 to 4 weeks as tolerated. Three mild hypotension episodes were noted. Bolus subcutaneous deferoxamine was given to the 25 patients with adequate health insurance. Mean deferoxamine dosage was 30±6 mg/kg/d, in two daily boluses. Skin nodules developed in 6 patients and a subcutaneous abscess in 1 patient. Blood transfusions were given when hemoglobin was <6g/dL.

Results:

table 1 phlebotomy only DFO only Phlebotomy+DFO N patients 3 TM, 3 TI 17 TM, 2 TI 2 TM, 4 SCD Median age (yrs) 16.5 [7–50] 15 [6–21] 20.5 [15–24] Median F.U. (mos) 27 [12–38] 20 [12–28] 18 [13–23] Median N erythrocyte concentrates transfused during F.U. 0 [0–1] 6 [0–19] 0 Median baseline ferritin (ng/ml) 2800 4130 3760 Median last ferritin (ng/ml) 884 1510 1665

Conclusions: Combination of phlebotomy and bolus subcutaneous deferoxamine very effectively reduced iron overload in patients with TM, TI and SCD. Phlebotomies could be performed in 12 patients thanks to hydroxyurea treatment which had increased their Hb level above 8 g/dL. Such combined therapy can be proposed in countries which can not afford infusion pumps or oral chelators.
3D Nuclear Organization of Telomeres in Hodgkin and Reed-Sternberg Cells.
Authors: Knecht Hans , Sawan Bassem , Lichtensztejn Daniel , Lemieux Bruno , Wellinger Raymund , Mai Sabine
Published: April 2020

Telomerase activity is high in Hodgkin (H) and Reed-Sternberg (RS) cells and abundant telomerase RNA template (hTR) is identified in both by in situ hybridization. However, nothing is known about the three-dimensional (3D) structure of the telomeres in H and RS and the molecular events associated with the transition from the mononuclear H to the multinuclear diagnostic RS are still unclear. We analyzed the 3D structure of telomeres in interphase nuclei in the Hodgkin cell lines HDLM-2, L-428 and L-1236, where about 90 to 99% are mononuclear H and only 1 to 10% are multinuclear RS. We also analyzed the 3D structure of telomeres within 5 mm thin sections of three lymph node biopsies diagnostic for Hodgkin’s disease (HD). The stereometric (3D) organization of telomeres was investigated in 30 H and 30 RS of each cell line and in 30 H and 30 RS of each lymph node biopsy specimen as previously described (Proc Natl Acad Sci USA, 102: 9613). Cellular localization of key-proteins of the telomere localized shelterin-complex (TRF1 and TRF2), of the mitotic spindle (centrin and gamma-tubulin), and of double-stranded DNA breaks (gamma-H2AX), was also analyzed. In all three cell lines the multinuclear RS showed overall significantly shorter and also significantly less telomeres in relation to the total nuclear mass when compared to their mononuclear H precursors. Visualization of their 3D telomeric structure revealed that this difference was due to partial or nearly total loss of telomeres within single nuclei of multinuclear RS; in particular, one or two nearly telomere free nuclei were often adjacent to one or two nuclei displaying several huge telomeric aggregates. TRF1 and TRF2 were mainly cytoplasmic in H and RS whereas gamma-H2AX accumulated in the nuclei of RS but not H. Multiple pairs of centrosomes not correlating with the number of nuclei, as well as high numbers of multi-polar spindles and incomplete spindles were identified in RS. Importantly, results analogous to those described in the HD cell lines were found in the HD lymph node biopsy specimen. In conclusion, our results suggest that multinuclear RS represent end stage tumour cells, where further nuclear division gets impossible due to sustained 3D telomere aggregation, shortening or loss. In particular, the number of nuclei within RS correlates closely with the 3D organization of telomeres, thus allowing for the first time a mechanistic understanding of the transition from H to RS cells. This process is initiated in H and advances to end stage telomere free “ghost” nuclei as observed in many RS. The shelterin complex appears to be disrupted and the mitotic cycle is profoundly disturbed.
CD4+CD26 - T Cell Population in Classical Hodgkin Lymphoma Displays a Distinctive Regulatory T Cell Population.
Authors: Ma Yue , Visser Lydia , Blokzijl Tjasso , Harms Geert , Atayar Cigdem , Poppema Sibrand , van den Berg Anke
Published: April 2020

Introduction: Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) are surrounded by a majority of infiltrating reactive cells, which mainly consists of CD4+ T cells. These T cells express several activation associated surface markers but lack expression of the T cell co-stimulatory molecule CD26. Little is known about the significance of these rosetting CD4+CD26- T cells.

Methods: To characterize these T cells, CD4+CD26- and CD4+CD26+ T cells were sorted from lymph node cell suspensions from 7 cHL and 5 reactive lymph nodes (LN). Of 5 HL cases and 3 lymph nodes, parts of the cells were stimulated with PMA/ionomycin to get activated T cell subsets. mRNA profiles of activated and non-activated T cell populations were evaluated with quantitative RT-PCR for 46 selected genes.

Results: We observed a higher percentage of CD4+CD26- T cells in cHL compared to reactive LN. For the non-activated T cell subsets, CD4+CD26- T cells in cHL showed higher mRNA levels of IL2RA, CTLA4, TNFRSF4 and CCR4 compared to LN. Moreover, these cells displayed low or no expression of the Th1 or Th2 related cytokines IL2, IFNγ, IL13, IL12B, IL4, IL5 and the chemoattractant receptor GPR44. Overall, the profiling results support a regulatory T (Treg) cell type for the CD4+CD26- T cells in cHL. Besides Tregs, Th17 cells may exist in cHL based on the significantly higher IL17 mRNA level for both the CD26- and CD26+ T cells in cHL than in LN. Upon activation, the lack of up-regulation of mRNA levels of most cytokine genes (IFNγ, IL2, IL8, IL21, IL17, IL13, IL12A and IL4) indicated an anergic character for the CD4+CD26− subset in cHL.

Conclusion: A high proportion of CD4+CD26− T cells is characteristic for cHL. No evidence for a Th1 or Th2 cell type is found for these cells but they display a regulatory T cell phenotype. Anergy fits with the regulatory T cell profile of these cells, probably explaining the immunosuppressive mechanism involved in cHL.
Effects of c-Src Overexpression in CD40-Activated Human B Lymphocytes.
Authors: Jung Daniel , Cayer Marie-Pierre , Proulx Maryse , Ma Xue-Zhong , Sakac Darinka , Giguère Jean-Francois , Drouin Mathieu , Neron Sonia , Branch Donald R.
Published: April 2020

The 60-kDa c-Src is the normal human cellular protein counterpart of the highly transforming v-src gene. Recently, c-Src activity was reported to increase in CD40-activated human B cells in the presence of IL-4, suggesting its involvement in proliferation. We report here that c-Src expression is detectable concomitant with the detection of Stat5b and, therefore, Stat5b may serve as a substrate for tyrosine phosphorylation by c-Src, inducing the activation of Stat5b and initiating a transcriptional pathway important for B cell expansion. To elucidate the exact role of c-Src in the proliferation of normal B cells, we undertook c-Src over-expression experiments. Recombinant adenoviruses Ad5/F35 vectors, which we previously reported as highly efficient for B cell transduction, encoding wild-type c-Src(c-Src/WT), constitutively active c-Src(c-Src/CA), dominant negative c-Src(c-Src/DN) or EYFP were constructed. B lymphocytes purified from human peripheral blood were activated with soluble CD154 in the presence or absence of IL-2, IL-4 and IL-10, and infected with the viruses. Real-time PCR and Western blot analysis revealed that vector-transferred c-Src were strongly expressed in infected B cells as early as 48 hours post infection. Kinase assays confirmed that vector-transferred c-Src/WT and c-Src/CA display a strong kinase activity whereas negligible kinase activity was detected with Ad5/F35-c-Src/DN. No significant variation of B cell expansion could be observed between uninfected cells, Ad/F35-EYFP and Ad5/F35-c-Src/CA or Ad5/F35-c-Src/WT infected cells, suggesting that B cell proliferation induced by endogenous c-Src already attains a maximum rate of expansion, which cannot be further enhanced by supplemental exogenous c-Src. In contrast, overexpression of c-Src/DN results in a 40% inhibition of B cell expansion. These results suggest that transgenic dominant negative c-Src may compete with endogenous c-Src resulting in a partial inhibition of a transcriptional pathway involved in B cell proliferation. In conclusion, our results confirm an important role for c-Src in the expansion of normal human B cells in vitro.
KIR Positive Subsets of Human CD56+CD3+ NKT Cells Are Different in Frequency from Equivalant KIR Positive CD56+CD3- NK Cell Subsets.
Authors: Bondzio Ilka , Arendt Andreas , Schmitz Jurgen , Huppert Volker
Published: April 2020

Killer cell immunoglobulin-like receptors (KIRs) are known to modulate the cytotoxic ability of human Natural Killer (NK) cells, as well as a subset of T cells. To date, only a very small number of publications have discussed the role of KIRs on T cells, e.g. CMV-specific CD4+CD28-KIR+ cytotoxic T cells (van Bergen, J., J Immunol. 2004), so we investigated whether CD56+CD3+ NKT cells might also have KIR-positive subsets. Whole human blood as well as magnetically sorted human CD56+CD3+ NKT cells were analyzed for their expression of various KIR molecules using a novel panel of fluorochrome-conjugated, anti-KIR monoclonal antibodies (CD158a/h (KIR2DL1/DS1), CD158b (KIR2DL2), CD158e (KIR3DL1), CD158i (KIR2DS4), KIR2D; Miltenyi Biotec). KIR-positive CD56+CD3+ NKT cells were identified in every donor tested. Donors possessing NK cells of a specific KIR phenotype also possessed CD56+CD3+ NKT cells with the same KIR phenotype. KIRs were also expressed in a clonal fashion on CD56+CD3+ NKT cells, similarly to NK cells. The investigated KIRs were also shown to be expressed on unseparated NK and CD56+CD3+ NKT cells from whole blood. In addition, the ratio between KIR expression on NK and CD56+CD3+ NKT cells was calculated for each donor analyzed. The results show that there is no correlation between the frequencies of KIR expression on NK cells with that of CD56+CD3+ NKT cells. For example, the expression of CD158a/h in one donor was found to be the highest of all CD56+CD3+ NKT cells analyzed, but the lowest of all NK cells by comparison to the other donors tested. For all KIR phenotypes analyzed, the frequency of KIR+ NK cells was higher than the frequency of KIR+ CD56+CD3+ NKT cells in all samples (range: 1.1 to 25.3-fold higher). Interestingly, the frequency of KIR+ NK cells versus KIR+ CD56+CD3+ NKT cells differs significantly between donors: in one donor the frequency of KIR expression is between 7.3 to 25.3-fold higher in NK cells for multiple KIR phenotypes, while this range is more narrow in other donors (2.0–5.4-fold higher). The frequencies of CD56+CD3+ NKT cell subsets staining positive for particular KIRs differ significantly between donors, e.g. for CD158b, the number of positive CD56+CD3+ NKT cells fall within a range of 4.8% to 43.3%. For CD56+CD3+ NKT cells sorted with MACS® Technology, a similarly wide-ranging distribution of CD158b (KIR2DL2) expression was found (0.85%–5.82%), though at a lower level. Further research will be required to explore these differences as they may point to different mechanisms of KIR regulation. The identification of KIR-positive CD56+CD3+ NKT cells may also provide an opportunity for their use for functional KIR studies instead of NK cell clones, as the cloning of CD56+CD3+ NKT cells may prove easier (i.e. using standard T cell cloning methods) than that of NK cells.
Irreversible Platelet Activation Requires PAR1 Regulation of Phosphatidylinositol Phosphates (PIPns) Activation of Rap1.
Authors: Holinstat Michael , Hudson W. James , Preininger Anita M. , Hamm Heidi E.
Published: April 2020

Thrombin is the most potent activator of platelets, through reversible and then irreversible aggregation and mediates a diverse range of vascular effects which may result in acute coronary syndrome and stroke. We demonstrate that PAR1 signals human platelet activation through generation of a phosphatidylinositol phosphate signaling pathway; inhibition of this pathway blocked the formation of a stable platelet aggregate. Early Rap1 activation and platelet aggregation are insensitive to perturbations along the PI-kinase pathway, while stable aggregate formation requires this pathway. Computational lipidomics identified a single phosphatidic acid species required for PAR1-, but not PAR4-mediated Rap1 activation and platelet aggregation. Both PI5K and PI3Kg were required for stable PAR1-mediated platelet aggregation, and we show that these enzymes are present in a complex with activated Rap1. This novel lipid signaling pathway is a potential target for anti-platelet therapy.
Phase II Study of R-CHOP Followed by 90Y-Ibritumomab Tiuxetan in Untreated Mantle Cell Lymphoma: Eastern Cooperative Oncology Group Study E1499.
Authors: Smith Mitchell R. , Zhang Lijun , Gordon Leo I. , Foran James , Kahl Brad , Gascoyne Randy D. , Advani Ranjana , Paietta Elisabeth , Weller Edie , Horning Sandra J.
Published: April 2020

Background: As MCL has a continuous relapse pattern with current treatments, we designed a study to determine the safety and efficacy of the anti-CD20 radio-immunoconjugate,90Y-ibritumomab tiuxetan (90Y-RIT), following 4 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) induction.

Methods: Patients (pt) with untreated stage II-IV MCL (CD20+, cyclin D1+) ≥18 yr with measurable/evaluable disease and adequate organ function were eligible. At 4–8 weeks after 4 cycles of R-CHOP, responding (CR/PR) and stable pt received 0.4 mCi/kg 90Y-RIT. The primary endpoint was failure-free survival (FFS) and secondary objectives were evaluation of response and toxicity after R-CHOP and after 90Y-RIT. The study design required 52 eligible pt to demonstrate a prolongation of FFS by 50% compared with R-CHOP alone (median FFS 16 mo, Howard et al., JCO20:1288, 2002).

Results: The characteristics of 56 eligible pt are: 73% male, median age 61 (33–83) yrs, 91% stage III/IV, 68% >1 extranodal site, 78% marrow-positive. IPI was 0–2 in 51%, 3–5 in 49%. Fifty-one (91%) pt received all treatment and best response (n=50) was 42% CR/CRu, 32% PR, 12% stable and 4% unevaluable, with an improvement in response in 16 pt after 90Y-RIT. After 90Y-RIT, 55% had grade 3/4 neutropenia with no febrile neutropenia and 45% had grade 3/4 thrombocytopenia with recovery at 12 weeks in 22/23 pt. Median follow-up (all pt) is 24.4 months. Median FFS for all 56 pt is 27 months with an estimated 71% FFS and 93% overall survival at 18 months. Among pt who completed all treatment and have been followed ≥1.5 yr (n=45), 33 remain failure-free and 12 have progressed (4 dead).

Conclusions: 90Y RIT after 4 cycles of R-CHOP in untreated MCL is safe and improves the number and quality of responses. These data suggest prolongation of FFS over that expected with R-CHOP alone. Consolidation of remission in MCL with 90Y-RIT shows potential as a strategy to prolong remission duration and is applicable to most pt with MCL. However, longer follow-up in needed to evaluate the durability of remissions achieved.
Platelet Septins: Interaction of the Human Septins SEPT5, SEPT7 and SEPT11.
Authors: Zieger Barbara , Bartsch Ingrid , Bläser Susanne , Anja Busse , Roeseler Sabrina , Bauer Heike , Lieber Mareike , Brendle Cornelia
Published: April 2020

Septins are GTP-binding proteins which form heteropolymeric complexes. In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis. The SEPT4, SEPT5 and SEPT8 are expressed in platelets. Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets. Transmission electron microscopy of platelets revealed that SEPT4, SEPT5 and SEPT8 are localized surrounding alpha-granules suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology. Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in granular secretion. Previously, we identified the interaction of SEPT5 with SEPT8 and showed that SEPT5 partners with SEPT11. The aim of this study was to identify other interaction partners of SEPT11. Using the yeast two-hybrid system we demonstrate that SEPT11 partners with SEPT7. Western analysis revealed that SEPT7 is also expressed in platelets. The molecular interaction of SEPT11 with SEPT7 was verified by precipitation of the SEPT7/SEPT11 complex from lysates of human platelets. Since SEPT5, SEPT7 and SEPT11 are members of the same septin complex and since SEPT5, SEPT7 and SEPT11 are expressed in platelets, SEPT5, SEPT7 and SEPT11 may play an important role in exocytosis in platelets.
Clinical Characteristics and Laboratory Analysis of Patients with Thrombocytopenia and Thrombocytosis.
Authors: Do Young-Rok , Kim Jin Young , Park Woo Young , Lee Sang Min , Kwon Ki Young
Published: April 2020

The normal range of platelet counts of peripheral blood is 150–450 × 109/L. Decreased platelet count results in the increased risk of bleeding. On the other hand, thrombosis formation is more prevalent in the patient of thrombocytosis. The mean platelet volume(MPV) is the mode of the measured platelet volume. There is an inverse relationship between platelet size and number. Therefore, the total platelet mass, the product of the MPV and platelet count is closely regulated. When platelets decrease in number, bone marrow megakaryocytes are stimulated by thrombopoietin, and their nucleus becomes hyperlobulated, with much greater ploidy. These stimulated megakaryocytes produce larger platelets. The platelet distribution width(PDW), is an indicator of the degree of variation in platelet size. PDW is defined as the coefficient of variation of the distribution of individual platelet volumes. So PDW is rising if the platelet destruction is increased while MPV is decreased. 48,340 patients were performed CBC test at Dong-san medical center through out the year of 2004. The platelet count below 150 × 109/L were 5,501(11.4%). Below 100 × 109/L of Platelet count is 2,303(41.8%). Platelet count Below than 20 × 109/L is 73(1.3%), 20–50 × 109/L is 323(5.9%), 50–100 × 109/L is 1,907(34.6%). Hemoglobin is 11g/dL in 50–100 × 109/L group and the hemoglobin is also rose in proportion to platelet count. WBC count is 10,540/L in below 20 × 109/L group and WBC count is decreased in proportion to platelet count. MPV is 9.5fL in 20–50 × 109/L group. PDW is 48.54% in below 20 × 109/L and the percentage is rose in proportion to platelet. Total bilirubin is 3.08g/dL in 20–50 × 109/L. Cause of thrombocytopenia is chronic liver disease, malignancy, infection and hematologic disease and secondary origin is more frequent than primary origin. Patients with thrombocytosis are 8,027(16.6%). The platelet count more than 600 × 109/L is 1,199(14.9%), 600–1,000 × 109/L is 1,051(13.1%), above 1,000 × 109/L is 148(1.8%). The PDW is 49.2% in 600–1,000 × 109/L group, 53% in above 1,000 × 109/L group.
New Compound Heterozygous Mutations of GPIIb in Patient with Glanzmann Thrombasthenia.
Authors: Yu Ziqiang , Su Jian , Bai Xia , Wang Zhaoyue , Ruan Changgeng
Published: April 2020

Glanzmann thrombasthenia (GT) is a homozygous or compound heterozygous autosomal recessive bleeding disorder caused by the qualitative or quantitative deficiency of integrin GPIIb-IIIa, which acts as the receptor of platelet fibrinogen. Here we report a case of GT with a compound heterozygous mutation in GPIIb according to the results of flow cytometry and genetic investigation.The flow cytometry was used to measure the average amounts of integrin GPIIb-IIIa on the patient’s platelets, and all 30 exons of GPIIb were amplified and sequenced with the corresponding primers.The average fluorescence intensity of integrin GPIIb-IIIa were 3.07 and 12.5, respectively, compared with 23.7 and 254, respectively, in the normal healthy individuals. And sequencing analysis of all exons of GPIIb demonstrated that there existed following compound heterozygous mutations in GPIIb gene: one heterozygote mutation (68 C→A) in the 1st exon, which resulted in Pro 23 His substitution in signal peptide domain; one nonsense heterozygous mutation (1750 C→T) in the 17th exon, which result in premature termination; one heterozygote mutation (2159 T→C) in the 21stexon, which resulted in Leu 720 Pro substitution. According to Glanzmann thrombasthenia database of ISTH (http://sinaicentral.mssm.edu/intranet/research/glanzmann/listmutations?mut=GPIIb), 68 C→A mutation and 2159 T→C mutation are novel mutations in the GPIIb heavy chain. These compound heterozygous mutations in GPIIb gene might be a novel pathogenetic mechanism of GT, which impaired the protein translation and co-expression with GPIIIa on the membrane of platelet.
Hemorrhagic Fever in Schoenlein Henoch Purpura.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

Schoenlein Henoch Purpura (SHP) may show a cohort of symptoms such as: a palpable purpuric rash where lesions may be prominent mostly on lower extremities, abdominal pain, arthritis and nephritis. It is an inflammatory disorder characterized by an IgA-mediated vasculitis with immune complexes deposition in smaller veins, capillaries and arterioles which often occur as a response to infections (mycoplasma, streptococcus group A, Campylobacter enteritis, Helycobacter, Herpes virus, Parvovirus B19, Epstein-Barr virus), or to an exposure to allergens, drugs, some particular food, cold, or even insect bites. Symptoms are often dramatic at the onset, and the exitus occurs sadly between 30 – 50% of the patients, despite the therapeutic support, for this reason the diagnosis and the therapy should be precocious. Clinically we can observe multi organ failure, hypotension, flushing, capillary leak haemorrhage secondary to thrombocytopenia, platelet dysfunction, disseminated intravascular coagulation, shock. We have studied a young boy (19 years old) with an urticarial wheals, erythematous maculo-papules, purple spots, and larger petechiae, migrant arthritis and arthralgia, renal disease (low grade hematuria with proteinuria), weakness, necrotic and hemorrhagic plaques, with haematemesis, diarrhoea, abdominal pain, fever (38.5°/39.5° C), and increased blood level of human Parvovirus-B19 NS1-gene component. We performed a skin biopsy of SHP that showed: leukocytoclastic vasculitis, with angiocentric neutrophilic infiltrate, fibrin deposition and focal fibrinoid necrosis. We also evaluated a kidney biopsy which showed: mild increase of mesangial cellularity, with segmental capillary luminal leukocytes and scattered capillary wall fuchsinophilic deposit, while the diagnostic finding is constituted by granular mesangial IgA with C3. The management is based on support therapy, with plenty of haemotrasfusions, and immunosuppressive therapy (prednisone 40 mg os/die, and cyclophosphamide100–200 mg/daily/os for 30–60 days (may decrease inflammation, capillary permeability and suppressing granulocytes activity and migration. The prognosis is usually excellent when the syndrome is self-limited and resolves spontaneously often 4/8 weeks without recurrence. Sometimes the diagnostic investigation may show an elevated level of vascular IgA deposition (direct immunofluorescence), elevated plasma levels of C3 and C4, elevated levels of IgA, ANCA (antineutrophil cytoplasmic antibodies: pANCA anti-MPO, cANCA anti-PR3), CRP, fibrinogen, ICAM-1, and increase of TNF, IL-1, IL-6, TGF-beta that reflect endothelial cell damage and dysfunction.
A High Purity Factor VIII/vWF Complex Concentrate Is Effective in the Management of Mild Hemophillia A with Inhibitors: A Case Report.
Authors: Dukka Srivasavi , Kaye Susan , Ashcroft John , Hill Anita , Parapia Liakat A.
Published: April 2020

Introduction: Inhibitor development in Mild Haemophilia A (MHA) is an uncommon complication, occurring in approximately 15% of MHA patients. Risk factors associated with the development of inhibitors in MHA include change in recombinant factor VIII (rFVIII), intensive replacement therapy, immunological challenges and missense mutations in the FVIII gene, including Tyr2105Cys mutation on exon 22. Only few reports have addressed the therapeutic options to eradicate these inhibitors, either by immune tolerance or immunosuppressive protocols.

Patient: We present a case report of a 36-year-old MHA patient (original factor VIII activity of 17.5% (10–20%) with a Tyr2105Cys mutation. He developed a low titre inhibitor after intensive exposure to rFVIII to cover for spinal epidural steroid injection, switch of recombinant product (full length rFVIII to B-domain deleted rFVIII) and intensive antiviral treatment (pegylated interferon and ribavarin) for Hepatitis C. His quality of life was very poor due to recurrent spontaneous bleeds - ecchymoses, haematomas and haemarthroses. His baseline factor VIII activity dropped to 1% with an initial inhibitor titre of 2.6 BU and factor VIII recovery of less than 25%.

Results: The patient responded positively when started on an immune tolerance protocol with a low dose of high purity FVIII/vWF complex concentrate. All clinical parameters improved two weeks after commencing 50 IU/kg on alternate days of the high purity FVIII/vWF product. FVIII recovery levels improved to 50% and he was asymptomatic in terms of bleeding manifestations for the following 17 weeks. Inhibitor titres stabilised at 3.5 BU. Recently, the recovery levels dropped to 26% and the frequency of bleeds increased, necessitating treatment with additional doses. The immune tolerance dose of the high purity FVIII/vWF was increased to 100IU/kg/day. Haemostatic and clinical parameters again recovered (factor VIII recovery levels of 43%) with no further spontaneous bleeds.

Conclusions: The use of recombinant factor VIII, especially high intensity replacement may pose a risk of inhibitor development and bleeding complications during the management of MHA patients with Tyr2105Cys mutation. An immune tolerance protocol with FVIII/vWF concentrate is effective in the management of MHA patients with inhibitors and can improve the quality of life soon after the start of the treatment. There is no anamnestic response observed. Though low dose immune-tolerance protocol may be effective, the benefits may be short-lasting, necessitating treatment with high daily dose protocol.
Identification of Trigger Factors Initiating an Acute TTP Episode in 68 Patients.
Authors: von Auer Charis , Wilken Andreas , Scharrer Inge
Published: April 2020

Recent research efforts have provided significant insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). However, the low incidence of the disease hampers the identification of trigger factors initiating an acute TTP episode. In the present work data from 68 TTP patients were analyzed. The patients were asked by a questionnaire to provide data concerning age, gender, medical history, underlying and concomitant diseases and potential triggers. Additional laboratory and clinical data from onset of disease and remission phase were obtained by a second questionnaire completed by the physicians. Our study showed that long term abuse of nicotine is a predisposing factor with an OR of 3.19. The OR for obesity and development of TTP was 2.01. The influence of nicotine abuse has not yet been described whereas the association between obesity and development of TTP is already known. The intake of oral contraceptives (13 of 46 women/28%) of Tamoxifen, of Fluvastatin, of Ticlopidine and of Quinine was identified to be potential triggers. 55% (34 patients) of the patients reported infections and inflammations as triggers (mostly influenza (n=16) and gastroenteritis (n=14)). The relation of female and male patients was 3:1. The mean age, in which the initial TTP episode occurred, was significantly lower in female patients compared with the male patients (33.8 versus 43.8 years). Severe ADAMTS-13 deficiency was found in 91% in all investigated episodes of the patients. Inhibitory activity was associated with severe ADAMTS-13 deficiency in 81% of the cases. The comparison of patients during remission with and without relapses of TTP revealed a significant lower ADAMTS-13 activity in patients with relapsing TTP compared to patients without a relapse of TTP. This result suggests that ADAMTS-13 activity has prognostic value with respect to the occurrence of relapse.
Multiple Complete Responses in a Phase 1 Dose-Escalation Study of the Antibody-Drug Conjugate SGN-35 in Patients with Relapsed or Refractory CD30-Positive Lymphomas.
Authors: Younes Anas , Forero-Torres Andres , Bartlett Nancy L. , Leonard John P. , Lynch Carmel , Kennedy Dana A. , Sievers Eric
Published: April 2020

Figure Figure

Background: The antibody-drug conjugate SGN-35 is being developed as a targeted therapy for Hodgkin lymphoma (HL) and other CD30-expressing hematologic malignancies. SGN-35 binds to CD30 and is internalized into the tumor cell, with subsequent release of the antitubulin agent monomethyl auristatin E (MMAE). Binding of released MMAE to tubulin disrupts the microtubule network, prompting cell cycle arrest and apoptosis.

Methods: A multicenter phase 1 dose-escalation study was conducted in 45 patients with refractory or recurrent CD30-positive hematologic malignancies, including HL (n=42), systemic anaplastic large cell lymphoma (sALCL; n=2), and angioimmunoblastic T cell lymphoma (n=1). Median age was 36 (range 20–87) and most patients (93%) had an ECOG performance status of 0/1. Patients had a median of 3 prior chemotherapy regimens (range 1–7); 73% of patients previously received an autologous stem cell transplant. SGN- 35 dose levels ranged from 0.1 to 3.6 mg/kg (2-hr outpatient IV infusion, premedications not required) every 3 weeks. Patients with stable disease or better after 2 doses were eligible to receive additional doses of SGN-35. The study has completed enrollment and a weekly SGN-35 dosing regimen is currently being investigated.

Results: The most common adverse events (occurring in ≥20% of patients) were fatigue, pyrexia, nausea, and diarrhea. Dose-related neutropenia was observed. The maximum tolerated dose was 1.8 mg/kg every 3 weeks. One patient treated at 3.6 mg/kg developed febrile neutropenia and presumed sepsis and died 14 days after the 1st dose of SGN-35. Exposure to SGN-35 increased relative to dose level, and the terminal elimination half-life was 5.0 ± 1.8 days at 1.8 mg/kg. Approximately 75% of patients reporting B symptoms at baseline experienced symptom resolution on study. Most patients (86%) had reductions in target lesion size. Among 28 evaluable patients treated at doses ≥1.2 mg/kg, the objective response rate (CR+PR) was 46% (n=13) and the complete remission rate was 25% (n=7). Two additional PRs were observed in the 0.6 mg/kg cohort. Median response duration to date is 22 weeks (range, 0.1+ to 38+ weeks). Thirteen patients continue on study.

Conclusions: The antibody-drug conjugate SGN-35 induced durable objective responses in heavily pretreated Hodgkin lymphoma and systemic ALCL patients at well-tolerated doses, providing evidence of selective tumor targeting. Pivotal studies with SGN-35 are planned.
Enhanced Fibrinolysis of Coarse Fibrin Networks Reflects Cleavage of a B Knob Containing Fragment(s)
Authors: Galanakis Dennis K. , Neerman-Arbez Marguerite , Henschen Agnes , Kudryk Bodan
Published: April 2020

Coarse fibrin network clots display faster fibrinolysis than their fine network counterparts (Collet et al, Arterioscler Thromb Vasc Biol2000, 20: 1354–61), and this is ostensibly of pathophysiologic relevance. Since relative to fine networks, coarse networks can be predicted to possess a relatively higher content of “B knob” (-”b” and/or -”a hole”) contacts, we investigated whether or not such contacts are more susceptible to plasmin than their A knob counterparts. Owing to much slower FpA than FpB release from Aα G16H fibrinogen, its clots acquire a high content of B knob contacts throughout polymerization (Galanakis et al, Bld Coag Fibrinol, 2007, 18: 731–737) and were therefore used for this purpose. Plasminogen-free fibrinogen was obtained by Lys-Sepharose chromatography, and clots of isolates from one homophenotypic (HM) and one (unrelated) heterophenotypic (HT) Aα G16H proband were compared with normal controls. Plasmin digests of fibrinogen, monitored by PAGE-SDS, disclosed no differences from controls. Thrombin was added to fibrinogen (1–5 μM), pH 7.4, 8 mM CaCl2, with or without 20% afibrinogenemic plasma so as to achieve > 96% clottability. For lysis times, clots were suspended in 2 volumes of a lysis solution. For turbidity (340 nm) measurements the lysis solution was carefully layered on the cuvette clot top. To obtain clots of similar turbidities 0.8 to 1.2 thrombin U/ml were used. The lysis solution contained either 2 μM plasmin in buffer (37º) or 80 μM rtPA with or without 50 μM plasminogen. Selected clots were prepared in solutions containing either anti-β 15–42 IgG (Mab, 3 mols/mol fibrin) or the GlyHisProArgProOH peptide (50 mols/mol fibrin). Using fibrin (70 nM)-enhanced glu-plasminogen (250 nM) activation by rtPA (10 μM) and S2251 substrate (40 mM), no differences (405 nm) among control, HM, and HT fibrin could be shown in two separate sets of experiments. Clot lysis times (n=3) were 31% (HM) and 54% (HT) of controls, respectively. Turbidity measurements (n=3) also disclosed HM and HT clot lysis onset ~2 and 1.5 fold faster than controls, but similar lysis rates. Duplicate clots lacking >90% of intact αC (i.e. fraction I-9) yielded similar differences. Also assessed by turbidity, clots formed in Mab containing buffer lacked lysis differences from non-immune IgG controls. Using turbidity measurements of normal fibrin, lysis differences between coarse and fine clots were abolished when clots had been obtained from fibrinogen/GlyHisProArgProOH solutions (n=2). We conclude that B knob contacts are constitutively more susceptible to plasmin than their A knob counterparts. This property is attributable to faster release by plasmin of at least one of the two β 15–53 (B knob containing) fragments relative to release of either or both α 17–19 (A Knob) fragments from the DDE complex (Olexa et al, Biochemistry1981, 20:6139–45).
Invariant NKT Cells Regulate Osteoclast Development and Function Under Homeostatic and during Conditions of Immune Activation
Authors: Hu Ming , Basssett J.H. Duncan , Danks Lynette , Spanoudakis Emmanouil , Xu Ke , Howell Peter GT , Boyde Alan , Horwood Nikki , Williams Graham , Roberts Irene AG , Karadimitris Anastasios
Published: April 2020

Invariant NKT cells, a small subset of immunoregulatory T cells restricted by the glycolipid-presenting non-polymorphic CD1d molecule, are able to modulate a variety of innate and adaptive immune responses. Osteoclasts (OC) are bone resorbing polykaryons of hematopoietic lineage, that have the capacity to regulate myeloid cell egress from bone marrow (BM) thus making them an integral part of the innate immune response. We and others previously showed that NKT cells regulate hematopoiesis in mice as well as humans. In this work, we investigate the role of NKT cells in OC development and function in homeostasis and after their specific activation by the model glycolipid alpha-galactosylceramide (aGC). Using quantitative back scattered electron scanning microscopy, we found that TCR Ja18 −/− mice which selectively lack development of NKT cells, exhibit a moderate osteopetrotic phenotype affecting trabecular as well as cortical bone. Histologically, these mice had the same number of TRAP+ OC as WT mice suggesting a maturation rather developmental defect in the TCR Ja18 −/−-derived OC. In vitro differentiation in the presence of RANKL and M-CSF showed that while TCR Ja18 −/− BM cells are capable of forming multinucleated OC, these, as assessed by confocal microscopy, fail to form F-actin rings and sealing zone and thus are unable to resorb bone. Further underscoring the effect of NKT cells in this process, CD45.1+ BM cells highly purified from CD45.2+ WT/CD45.1+ TCRJa18 −/− mixed BM chimeras displayed restoration of their OC F-actin rings. Next we investigated whether in vivo activated NKT cells regulate OC function. We found that a single injection of aGC dramatically increased the number of CD3-B220-CD11b-c-fmshighc- kithigh BM OC progenitors and accelerated the in vitro development of OC in WT but not TCR Ja18 −/− mice. Furthermore, this resulted in high serum levels of IFN-g and IL-4 but not IL-1 or IL-17. An aGC-mediated increase of OC progenitors was observed in IFN-g −/− but not IL-4 −/−mice suggesting that NKT cell-derived IL-4 is the main cytokine promoting osteoclastogenesis in this context. Taken together, our data demonstrate a novel role of NKT cells in homeostatic bone mass regulation and in the orchestration of innate immune responses through regulation of OC development and function.
A Potential Novel Therapeutic Approach for Fanconi Anemia
Authors: Lambert W. Clark , Brown Monique M
Published: April 2020

Lymphoblastoid cells from normal subjects and from patients with the bone marrow failure and cancer prone inherited disease, Fanconi anemia (FA) were treated in culture with psoralen plus ultraviolet A radiation (PUVA) in a scheme shown to produce interstrand crosslinks in cellular DNA. Hypersensitivity to DNA interstrand crosslinks, with associated increased clastogenicity, is considered to be a diagnostic hallmark of the disease. Following this cells were treated with hydroxyurea, 5 fluorouracil, or high dose thymidine for 24 hours. Clastogenicity and cytotoxicity, measured as trypan blue exclusion, were then found to be markedly increased in FA cells but not in FA cells subsequently treated with any of these other agents. Similar results were also found when all drugs were removed after these treatments and the cells cultured for 10 days without any drug in colony forming ability assays. We propose that the mechanism is related to decrease in the rate of DNA synthesis, which we have shown occurs in normal but not FA cells following PUVA, and which is also produced by these other drugs in the concentrations used here. Hydroxyurea has been used for many years as a safe and effective treatment for sickle cell anemia. It is now proposed as a possible treatment for Fanconi anemia to delay or even prevent development of bone marrow failure and/or other complications, including leukemogenesis and carcinogenesis, with or without prior bone marrow transplantation.
IL-18 Elicited Suppressor NK Cells with Immunoregulatory Functions
Authors: Ullrich Evelyn , Terme Magali , Aymeric Laetitia , Apetoh Lionel , Chaput Nathalie , Ryffel Bernhard , Mackensen Andreas , Schultze Joachim L , Zitvogel Laurence
Published: April 2020

Cancer-induced tolerance involves multiple immunosuppressive pathways, which subvert adaptive immune responses against tumor cells. Extensive phenotypic characterization of NK cells from tumor-bearing mice led us to the observation of a consistent expansion of c-kit-expressing NK cells (Kit+NK cells) compromising the NK cell arm of tumor immunosurveillance. In naïve animals, Kit+NK cells (CD3−NK1.1+CD117+) represent about 3.3±0.7% of blood, 4.8±1.4% of spleen and 13.2±2.4% of lymph node NK cells. In tumor-bearing animals, percentages and absolute numbers of Kit+NK cells increased to levels that mediate inhibitory effects on mature NK cells. Purified Kit+NK cells failed to produce IFNγ or GM-CSF in response to IL-2 and could not promote DC maturation in contrast to conventional Kit−NK cells. Moreover, adoptive transfer of Kit+ (but not Kit−) NK cells into mice injected with CT26 colon carcinomas or B16F10 melanomas promoted the establishment of lung metastases. Micro array comparison of CD3−NK1.1+Kit+ and CD3−NK1.1+Kit− cells revealed profound differences in their transcriptomes. Two major sets of genes involved in tolerance (B7-H1, CTLA4, Lag3, Hmox1) or tissue repair and bone marrow homeostasis (Cxcl2, CD81, CD63, Csf2) were markedly up-regulated in Kit+NK cells. Among these, B7-H1 appeared particularly intriguing as we found that Kit+NK cells killed Kit−NK cells in a B7-H1/PD-1-dependent manner. Moreover, IL-18 produced by CT26 or B16F10 tumors converts Kit− into Kit+NK cells endowed with immunoablative functions in lymph nodes. Upon tumor inoculation, Kit+ NK cells, which upregulate B7-H1, accumulate in lymphoid organs in an IL-18R/MyD88-dependent manner and directly kill conventional NK cells, thereby promoting the progression of NK-controlled cancers. Importantly, IL-18R−/− mice lost the development of Kit+NK cells during tumor progression. Neutralization of IL-18 by RNA interference in tumors or systemically by IL-18-binding protein (IL-18BP) reduced the expansion of Kit+NK cells, stimulated the NK cell-dependent immunosurveillance and significantly reduced tumor growth.

The inflammatory cytokine IL-18 is known to accumulate in cancer-bearing patients but its pathophysiological role still remains unclear. Here, we show that IL-18 is a major tumor-derived immunosuppressant affecting the innate arm of tumor immunosurveillance. In summary, these data suggest that IL-18 promotes the expansion of Kit+NK cells endowed with immunosuppressive functions that could act as negative regulator of general NK responses.
Molecular Characteristic of Variant and Classic Hairy Cell Leukemia.
Authors: Arons Evgeny , Suntum Tara , Sunshine Joel , Orthwein Anna , Margulies Inger , Stetler-Stevenson Maryalice , Kreitman Robert
Published: April 2020

10–20% of patients with hairy cell leukemia (HCL) have a variant (HCLv) and present with high tumor burden in spleen and peripheral blood, and are less responsive to purine analogs. HCLv cells lack CD25 and sometimes CD103. Differentiating HCLv from classic HCL (HCLc) is sometimes difficult and it is unclear whether HCLv and HCLc are different disorders. Detailed molecular distinctions between HCLv and HCLc have not been reported. Patients with HCL were studied by flow cytometry and PCR to sequence the monoclonal immunoglobulin heavy chain rearrangements. 50 and 21 VH-D-JH rearrangements were obtained from 49 HCLc and 19 HCLv patients, respectively. All rearrangements except 2 each for HCLc and HCLv were productive. The incidence of VH4 usage was higher in HCLv than in HCLc (57% vs 20%, p=0.0041). VH4-34 was the most common VH gene in HCLv and was more common in HCLv than in HCLc (33% vs 8%, p=0.012). The percentage of rearrangements which were unmutated (defined as < 2% somatic mutations) was higher in HCLv than in HCLc (43% vs 18%, p=0.038), but % homology was similar in both groups by rank order (94.9 vs 94.3% p=0.22). However, in comparing 11 VH4-34 with 60 other rearrangements, homology was higher with VH4-34 (median 99.2 vs 95.2%, p < 0.0001). In fact, the higher frequency of unmutated rearrangements in HCLv vs HCLc was due to VH4-34 cases, since the 7 HCLv VH4-34 rearrangements were all unmated and had higher homology than the other 14 HCLv rearrangements (median 99.6 vs 94.2%, p=0.006). Moreover, unmutated rearrangement incidence was similar between HCLv and HCLc for non-VH4-34 cases (14 vs 13%, p=1.0, median homology 94.2% vs 95.3%, p=0.5). Of the 4 VH4-34+ HCLc (CD25+) patients, 3 (75%) had unmutated rearrangements, and these 3 all had clinical features of HCLv, including presenting with lymphocytosis, large splenomegaly, absent cytopenias, and primary failure of purine analog treatment. Our data shown that homology of monoclonal immunoglobulin rearrangement to the germline sequence of HCL patients appears more related to VH4-34 status than to whether patients have HCLv or HCLc. In fact, no molecular distinctions between HCLv and HCLc were observed in VH4-34-negative patients. Our data suggest VH4-34-positive HCL is itself a variant of HCL affecting ~15% of our patients. Such patients have features of HCLv, but their HCL cells can be CD25+.