bluuebunny/crossref_metadata_2025 huggingface dataset:
Mabthera (Rituximab) in the Treatment of 34 Adult Patients with Refractory Autoimmune Hemolytic Anemia (AIHA).
Authors: Cabrera JRafael , Penalver F. Javier , Millan Isabel , Alvarez-Larran Alberto , Diez-Martin Jose L. , Gallur Laura , Jarque Isidro ,   other 13 participants in the multicentric retrospective Spanish Study on use of Rituximab in refractory AIHA
Published: October 2019

Objectives: Rituximab (RB) is an anti-CD20 murine/human chimeric antibody that has been used in the treatment of auto-immune diseases. RB has shown to be effective in childhood AIHA but data on adults are lacking. The effectiveness of RB in the treatment of adult patients with AIHA has been evaluated.

Patients and methods: A multicentric retrospective study of patients with AIHA treated with RB has been carried out. A questionnaire about clinical characteristics and response to RB was sent to the participating centers. These questionnaires were filled out by the physician in charge of each patient. We received questionnaires from 20 centers (34 patients). Response criteria were defined as follows: Complete Response (CR): Hb >10 g/dl or Hb increase >1.5 g/dl; Partial Response (PR): Hb > 9 g/dl or Hb increase of 1–1.5 g/dl. Median age was 64 years (20–86), 62% were women. 13 cases were idiopathic and the remaining 20 were associated with chronic lymphoproliferative syndromes (CLS) (14), connective tissue diseases (2), allogeneic hematopoietic stem cell transplantation (2), liver transplant (1), amiloidosis (1) and MGUS (1). In 6 cases the immune hemolysis was due to cold-reactive antibodies. Before RB they have received several treatment schemes, 12 patients were splenectomized and only 6 (17.6%) had not been transfused. Median Hb before the treatment with RB was 6.9 g/dl (3.3–12.7). 27 patients received 4 doses of RB (1–6), 24 received it in association with other treatments, mainly steroids.

Results: 19 patients obtained CR (63%) and 4 patients obtained PR (13%). In 50 % of the cases the Coombs antiglobulin test became negative. The majority of the responses took place in 3 weeks (1–8) and maximum responses in 10 weeks (1–35). 15 out of the 19 patients that obtained CR, reached it from the beginning of the response. 82% (9/11) of the patients with AIHA associated to CLS obtained CR, 58% (7/12) of the patients with idiopathic AIHA obtained CR and 17% (2/12) PR. 50% (3/6) of the patients with immuno-hemolytic anemia due to cold-reactive antibodies obtained CR. There were no differences in the response between splenectomized and not splenectomized patients. Median increase in Hb level was 5.9 g/dl (1.2–9.8) with a monitoring median of 8.7 months (1–48). There were no significant toxicities.

Conclusions: These results suggest that RB can be effective in the treatment of adult patients with AIHA, both idiopathic and associated with CLS, with a very low toxicity profile.
Manipulation and Visualization of the Erythroid Lineage - In Vivo Models for Erythroid Disorders.
Authors: Heinrich Achim C. , Pelanda Roberta , Klingmueller Ursula
Published: October 2019

Red blood cells have a short half-life and are continuously renewed in a tightly controlled growth process. Dysregulation of erythropoiesis results in erythroleukemias or more frequently in anemias. To elucidate molecular mechanisms regulating red cell production in an animal model, we generated a mouse line (ErGFPcre) that simultaneously facilitates erythroid specific gene manipulation via the CreloxP recombination system and visualization of erythroid progenitor cells. To avoid site of integration effects frequently observed for randomly integrated transgenes, we used a knock-in strategy targeting the genomic EpoR locus that ensures a reliable EpoR promoter controlled transgene expression of our GFPcre fusion protein.

The flow cytometric analysis of GFP fluorescence in different hematopoietic subpopulations of adult and embryonic ErGFPcre mice revealed a strictly erythroid-specific expression pattern for GFPcre. Further studies on GFP positive erythroid progenitor cells indicated a developmental switch in lineage progression from the hematopoietic stem cell compartment to early erythroid progenitor cells that are Sca-1 negative and c-kit high. To monitor previous and persistent GFPcre expression during development and to determine the efficiency of Cre-mediated recombination in our mouse model, we crossed the ErGFPcre mouse line with the LacZ reporter strain R26R. The spatial and temporal analysis of GFPcre-mediated LacZ expression confirmed that within the hematopoietic system GFPcre expression is limited to the erythroid lineage. Surprisingly, non-hematopoietic expression of GFPcre is restricted to the vascular system. It is possible that the observed differential transcriptional activity of the knock-in and the wild-type allele is linked to the absence of intron 2 to 7 in the knock-in locus. The quantitative analysis of the recombination frequency confirmed that GFPcre-mediated recombination is limited to erythroid progenitor cells and showed that it occurs in the adult bone marrow at a frequency of up to 80% and in the fetal liver with an efficiency close to 100%.

Thus, our ErGFPcre mouse model offers the possibility to study regulatory mechanisms in erythroid progenitor cells and facilitates the establishment of mouse models for erythroid disorders.
Comparison of Conventional Versus Lineage-Specific Chimerism Analysis for Detection of Minimal Residual Disease or Relapse in Myeloid Malignancies after Myeloablative Allogeneic Hematopoietic Cell Transplantation with Respect to Clinical Follow-Up.
Authors: Zeiser Robert , Spyridonidis Alexandros , Waesch Ralph , Ihorst Gabriele , Grullich Carsten , Hartmut Bertz , Jurgen Finke
Published: October 2019

In order to detect minimal residual disease (MRD) or relapse after allogeneic hematopoietic cell transplantation (aHCT) the immunophenotype of the original leukemic clone can be used for separation of distinct cell populations, expected to harbor malignant cells.

Since conventional (CCA) and lineage-specific (LCA) chimerism analysis as diagnostic means have potential pitfalls, the present study assesses the results of both methods, to determine how predictive the evaluations prior to a hematological relapse were with respect to the risk that the patient would develop a relapse during the course of his disease.

118 individuals with acute myeloid leukemia (n=98) and myelodysplastic syndrome (n=20), investigated with CCA and LCA, were evaluated with a median follow-up of 26.6 months (range: 8–47).

Subset chimerism samples were cell-separated by using immunomagnetic beads with respect to the patient’s leukemia phenotype, expressed at diagnosis. Mean purity of the positive-selected CD34 fractions were 97% (93–99%). PCR products were analyzed and quantified by capillary electrophoresis. 33 patients experienced a clinical relapse after aHCT. CCA and LCA detected a mixed chimerism (MC) in 27/118 and 25/118 patients, respectively. MC was detected at a median of 27 (12–54) and 34 (10–71) days before clinical relapse by CCA and LCA, respectively. LCA reached a significantly higher sensitivitiy than CCA with 91% (CI:54–98%) as compared to 83% (CI: 69–91%) and was also more sensitive as compared to bone marrow morphology examination (BME) (61%; CI:44–75%). Specificity was also higher with LCA (98%, CI: 89–100%) as compared to CCA (92%; CI: 75–99%) and BME (91%; CI: 82–96%).

Our data suggest a higher sensitivity and specificity of LCA as compared to CCA and BME with respect to the clinical follow-up. We conclude from these data that due to the high predictive value subset chimerism monitoring may be employed for clinical decision making concerning adoptive immunotherapy after myeloablative aHCT for patients with AML and MDS, especially when lacking other molecular markers for detection of MRD.

Differential accuracy of Lineage-specific versus Conventional Chimerism analysis

Lineage-specific Chimerism analysis (LCA) Conventional Chimerism analysis (CCA) Bone marrow examination (BME) Sensitivity and specificity of lineage specific (LCA), conventional (CCA) chimerism analysis and bone marrow examination (BME) with respect to the event that the patient would develop a relapse in the clinical follow-up. Sensitivity in % (Confidence interval) 91 (54–98) 83 (69–91) 61 (44–75) Specificity in %(Confidence interval) 98 (89–100) 92 (75–99) 91 (82–96)
Improved Prediction of Acute Leukemia Relapse after Allogeneic SCT by Chimerism Analysis of Null Alleles and Indel Polymorphisms with Real-Time Quantitative PCR.
Authors: Jimenez-Velasco Antonio , Barrios Manuel , Roman-Gomez Jose , Navarro German , Buño Ismael , Diez-Martin Jose L. , Torres Antonio , Heiniger Ana I.
Published: October 2019

Increasing mixed chimerism (MC) predicts morphological relapse after stem cell transplantation (SCT) in patients with acute leukemia. Early treatment of increasing MC reduces the onset of relapse and improves survival. However, conventional methods for chimerism analysis fail to detect MC prior to a considerable number of relapses. We have tested a new approach for chimerism follow-up, based on amplification by real-time quantitative PCR (rtq-PCR) of null alleles and insertion/deletion (indel) polymorphisms, and compared it with conventional variable number of tandem repeats (VNTR)-PCR. Four null alleles: GSTM1, GSTT1, RhD and SRY, and 10 indels: Xq28, rs4399, DCP1, R271, FVII, THYR, MID-1039, MID-1335, MID-1385, MID-2062, were quantified by rtq-PCR using allele-specific primers and probes (LyghtCycler technology). Sensitivity studies (by mixture of positive and negative DNA), and intra- or inter-assay variability experiments were performed. All markers showed high sensitivity (at least 0.01%) and reproducibility. Accuracy of the method was also confirmed by dilution of positive in negative cells with subsequent DNA extraction and rtq-PCR quantification. Donor/host informativity (presence of the sequence in the recipient and absence in the donor) was tested retrospectively in a series of 68 acute leukemia patients, and at least one informative marker was obtained in 85.3% of donor/host pairs. Chimerism follow-up was performed on peripheral blood (PB) and bone marrow (BM) samples obtained after SCT. In non-relapsed patients, PB host chimerism values progressively descended from transplantation until reaching donor complete chimerism (CC); conversely, BM chimerism decreased but did not reach CC more than 3 years after SCT. From 18 relapses evaluable with rtq-PCR, 16 (89%) showed increasing chimerism in PB samples prior to relapse; by contrast, only 73% of such relapses were preceded by increasing MC in BM. When compared to VNTR, rtq-PCR predicted a significantly higher number of relapses (89% vs 42%) and anticipated them earlier (median of 56 vs 38 days). In summary, rtq-PCR determination of null alleles and indels constitutes a new validated simple procedure to monitor hematopoietic chimerism after SCT. It improves the predictive results obtained with conventional VNTR-PCR, and is specially useful when PB samples are analyzed.
Results of Bortezomib (BTZ) Therapy for Myeloma (MM) Patients Relapsing after an Allogeneic Transplant. Preliminary Results Show Efficacy without Induction of GVHD.
Authors: Giralt Sergio , Aleman Ana , Lei Xin , Davis Marilyn , Mickler Kerry , Weber Donna , Wang Michael , Champlin Richard
Published: October 2019

Background: The proteosome inhibitor bortezomib (BTZ) has demonstrated significant activity against multiple myeloma (MM). In vitro data suggests that protesome inhibition has immune-modulatory activity that may be relevant in the setting of allogeneic progenitor cell transplantation (allo SCT). However, there is limited experience with the use of BTZ in patients with MM relapsing after an allo SCT.

Purpose: Determine safety, efficacy and effects on GVHD of BTZ in MM patients relapsing after an allo SCT.

Methods: Retrospective analysis of outcomes on 9 MM patients who received BTZ therapy for MM relapse after allo SCT

Results: Nine patients were identified. Median age was 51 (range, 38–64); all patients underwent an allo SCT because of failure to respond or relapse after primary therapy including autograft in 8 patients. At the time of allo SCT; 3 patients had refractory disease and 6 had chemo-sensitive relapses. The median time from allo SCT to relapse was 8.5 months (range, 5–117); the median time from relapse to initiation of BTZ therapy was 3.4 months (range, 0.1–20). BTZ was the 1st line of therapy for relapse in 5 patients. No patient had active GVHD at the time of BTZ therapy. BTZ was given at a dose of 1.3 mg/m2 on days 1,4,8 and 11 every 21–28 days for a median of 3 cycles (range, 2–8). 4 patients received BTZ alone, 3 in combination with DEXA, and 2 in combination with DEXA/THAL. 6 of 9 patients had either a 50% reduction or greater of monoclonal peak, of which 2 were very good PR (>90% reduction) to BTZ. BTZ was well tolerated and no flares of GVHD were seen. Only 2 patients developed severe thrombocytopenia and fatigue, which required discontinuation of therapy (one in the context of relapsing disease). 2 of the responding patients have relapsed at 4 and 7 months after initiation of BTZ therapy. 4 of the 6 responses are ongoing 3 to 9 months after initiation of BTZ therapy. Individual patient outcomes are summarized in Table 1.

Conclusions: BTZ therapy is effective as treatment of MM relapse post allo SCT, it is well tolerated and does not promote GVHD reactions. The response rates would seem to be higher than the responses reported to donor lymphocyte infusions in this setting. BTZ therapy should be studied as front-line therapy for patients relapsing after allo SCT and as maintenance therapy post high risk allograft.

Individual Patient Outcomes

Pt# #Cycles BTZ BTZ plus Response Duration of Response NCI Toxicity 3-4-5 Outcome 1 3 Dexa PR Ongoing None Alive PR 3+m 2 2 - PR 4m Fatigue 3/Heme 4 Died PD 5m 3 2 Dexa PR Ongoing Fatigue 3/Heme 4 Alive PR 9+m 4 2 - PD 0 Diarrhea Alive PD 2+m 5 8 - VGPR Ongoing Alive VGPR 5+m 6 8 Dexa VGPR Ongoing Alive VGPR 6+m 7 3 - PD 0 Hypotension 3 Died PD 5m 8 4 Dexa/Thal PR 7m Alive PD 7+m 9 3 Dexa/Thal PD 0 Alive PD 4+m
Arginase Activity Is a Determinant of Nitric Oxide Levels in Adult Vasoocclusive Sickle Cell Crisis.
Authors: Lopez Bernard L. , Telem Dana , Flenders Pamela , Ma Xin L. , Ballas Samir K.
Published: October 2019

A number of studies have demonstrated both low nitric oxide (NO) and l-arginine (l-arg, the substrate for NO synthesis) levels during adult sickle cell vasoocclusive crisis (VOC). L-arg levels, while diminished, are 10-fold higher than the km (3 mM) of endothelial NO synthase (eNOS) and more than sufficient to saturate this enzyme (a state known as the arginine paradox). Diminished l-arg thus cannot directly explain lower NO levels. Two explanations, enhanced arginase activity (catalyzing the conversion of l-arg to ornithine), and inhibition of NOS by increased levels of asymmetric dimethylarginine (ADMA) have been proposed to explain this arginine paradox in VOC. We sought to investigate these mechanisms during adult VOC in the emergency department (ED). L-arg, ornithine, and citrulline (the end product of ADMA metabolism) were measured via ion exchange chromatography from blood obtained from adult ED VOC patients. Citrulline levels were used as an indirect indicator of ADMA and ornithine levels were used as an indicator of arginase activity. Samples from sickle patients in steady-state and from normal healthy volunteers were obtained for comparison. As expected, VOC subjects had significantly lower median l-arg levels (23.16 μM/l, 18.74–29.81 IQR, p<0.001 vs steady state, 38.56 μM/l, 5.88–20.26 IQR, and control, 73.01 μM/l, 27.42–37.2 IQR). There was no significant difference in citrulline levels. Ornithine, the end product of l-arg metabolism by arginase, was lower in VOC (median: 22.61 μM/l, 17.22–27.3 IQR, p<0.001 vs steady state median of 36.78 μM/l, 30.28–42.26 IQR). It has been suggested that the l-arg:ornithine ratio may be a better reflection of total arginase activity - in this study, the l-arg:ornithine ratio was significantly lower in adult VOC (1.02 vs 2.71) when compared to healthy controls, indicating enhanced total arginase activity. We conclude that the l-arg-arginase-ornithine pathway may play a significant role in the diminished NO seen in adult VOC patients via enhanced arginase activity. This enhanced arginase activity may alter available l-arg, resulting in decreased NO. ADMA does not appear to be an important contributor to the arginine paradox found in adult VOC.
Is Screening for Microalbuminuria Warranted in Children with Sickle Cell Hemoglobinopathies?.
Authors: Alvarez Ofelia , Montane Brenda
Published: October 2019

Renal impairment occurs in adults and children with sickle cell disease. Microalbuminuria (MA) defined as albumin excretion >2.9 mg/dl in random urine sample or 30–300 mg in 24 hour urine collection is an early, sensitive indicator for glomerulopathy in both diabetes mellitus and sickle cell disease. Therefore, we conducted a universal screening in our patients (pts) with sickle cell hemoglobinopathies in order to identify the prevalence of MA and other indicators of early renal impairment. Children and young adults with hemoglobin SS, SC, Sβ+thalassemia and Sβ0thalassemia, ages 4–21 years (yrs), had urinalyses and random urine measurements for protein, microalbumin, and creatinine. All were in good state of health, free of pain, and without prior diagnosis of renal dysfunction when the samples were obtained. Serum electrolytes, creatinine, osmolality, and 24-hour urine collection for protein, microalbumin, β2-microglobulin, creatinine, and electrolytes were done in pts with abnormal urine samples. There were 101screened pts (53 males; 76 SS, 20 SC, 3Sβ0, and 2 Sβ +). Nineteen pts had MA detected by random urine. MA was confirmed in 24-hour samples in 46% of studied pts. In addition to MA, tubular proteinuria (increased β2-microglobulin) was detected in 1 pt, isolated hematuria in 3 and increased urine protein/creatinine (>0.2) in 4. From the MA+ pts, 15 had SS (19.7% of SS group) and 4 had SC (20% SC group). 25% of children 10 yrs of age or older had MA (mean± standard deviation MA 2.83±3.94), as compared to 6% of younger children (mean 1.21±0.80), (p=0.02). African Americans had lower incidence of MA (11.4%), compared to other children of Haitian (21.3%), Hispanic Caribbean (25%), and other Caribbean (28.5%) descent (p=not significant [NS]). There was no correlation between MA and history of stroke, acute chest syndrome/asthma, brief use of non-steroidal anti-inflammatory medications, hemoglobin or white cell values. From the subgroup of 16 pts on hydroxyurea, 11 were 10 yrs of age or older; of these, 5 (45.5%) had MA, compared to 21% of the same age non-treated group (p=NS). We analyzed the effect of age at onset of chronic transfusions and presence of MA. Six transfused pts had MA (age at start of transfusion 8.6–13.5 yrs, mean 11 yrs; mean MA 5.3±3.4), while there were 15 MA free pts (age 0.8–12.4 yrs, mean 7 yrs; mean MA 1.4±0.6), suggesting that early age at start of chronic transfusions may be protective for MA (p=0.0004). We conclude that: (1) urine screening of children with sickle cell hemoglobinopathies 10 yrs or older is recommended, (2) chronic transfusions starting at an early age may protect against MA, and (3) validity of random versus 24-hour urine collections in determining MA should be further investigated. Studies to further define the risk factors, prognosis, and intervention in children with sickle cell hemoglobinopathies are warranted.
Cerebral Oxygen Saturation in Sickle Cell Patients Treated with Hydroxyurea:.
Authors: Nahavandi Masoud , Tavakkoli Fatemeh , Wyche Melville Q. , Hasan Syed P. , Castro Oswaldo
Published: October 2019

Recently we reported the use of a non-invasive near-infrared optical spectroscopy technique to measure cerebral oxygenation (cerebral oximetry, rSO2%) in agroup of 27 adult patients with sickle cell disease (Eur J Clin Invest, 34:143,2004). The sickle cell patients’ rSO2 values were significantly lower (mean 47.7%) than those in normal subjects (mean 61.3%) even though none of the patients had clinical evidence of stroke or cerebral ischemia. We included patients with all Hb phenotypes, and regardless of hydroxyurea (HU) treatment. Transfusions improved cerebral oxygen saturation but the post-transfusion values still did not reach normal levels. Our findings were corroborated independently by Raj et al. who studied 25 children with sickle cell disease (J Pediat Hematol Oncol 26:279,2004). In order to determine if long-term HU treatment affects rSO2, we analyzed cerebral oximetry results in a subset of 31 patients with sickle cell anemia (Hb SS). Eleven of them were on long-term (more than 6 months) HU treatment at stable doses (1000–1500 mg/day). The table shows that the mean rSO2, Hb, Hct, and MCV in HU-treated patients were significantly higher than those in sickle cell anemia (SCA) patients not on HU. The rSO2 in HU-treated patients was 12.5% higher than in SS patients not on this drug. By comparison, we previously reported a 24% increment in rSO2 following transfusions.

A group of 8 patients who were on long-term HU treatment were given also single 1000 mg oral doses of HU and their rSO2 was measured for 12 hours without noticeable change in cerebral oxygenation. Nor did rSO2 change after oxygen inhalation (3L/min). The cause of the low rSO2 in sickle cell patients is unknown and still under investigation. It is probably not related exclusively to the anemia, since, as previously reported, anemic subjects without sickle cell disease appear to have normal rSO2. These preliminary results indicate that chronic HU treatment is associated with higher rSO2 values in SCA. If validated in a larger number of patients, our findings suggest that cerebral oximetry could be a useful, non-invasive method for assessing a new in vivo effect of HU and red cell transfusion in sickle cell disease: increased blood oxygen saturation in the cerebral vasculature.

HYDROXYUREA AND CEREBRAL OXYGEN SATURATION IN PATIENTS WITH SICKLE CELL DISEASE

NO HYDROXYUREA (N=20) HYDROXYUREA (N=11) P value* rSO2 = cerebral oxygen saturation. *= t-test. Plus/minus figures represent SD Mean rSO2 (%) 41 ± 6.6 46 ± 7.6 0.025 Mean Hb (g/dl) 8.4 ± 1.4 9.68 ± 1.2 0.029 Mean Hct (%) 24± 3.4 28± 4.4 0.027 Mean MCV (fl) 89± 8 102± 7 0.028
The Genetic and Phenotypic Diversity of Dyskeratosis Congenita.
Authors: Vulliamy Thomas J. , Marrone Anna , Walne Amanda , Stevens David , Mason Philip J. , Dokal Inderjeet S.
Published: October 2019

Two of the genes responsible for the bone marrow failure syndrome dyskeratosis congenita (DC) have been identified. These have evoked considerable interest as the products of both of these genes are involved in the telomerase complex. Telomerase is responsible for maintaining telomere length at the ends of each chromosome and hence the replicative potential of stem cells and cells in the germ line. In order to define the heterogeneity of DC, we have reviewed the clinical features and genetic basis of 213 families that have been referred to the DC registry at the Hammersmith Hospital. In 67 families we have identified 34 different mutations in the gene that encodes the protein dyskerin (DKC1), 9 of which are unpublished (P10L, T67I, H68Q, K314R, D359N, A386T, T408I, S420Y and IVS14nt473A->G). While most of these mutations are unique, the remarkably frequent A353V substitution is seen in 28 different families. Presentation of cases with this amino acid substitution ranges from a very severe disease, causing death at the age of 4 years, to a man who is alive into his 40s. In 10 families we have found mutations in the gene encoding the RNA component of telomerase (TERC), all of which are unique. Presentation of these heterozygous TERC mutations is even more variable, ranging from an asymptomatic 80 year old woman, to a girl presenting with hypoplastic MDS at the age of 3. In a substantial proportion of families (40/213) the index case presents with very severe disease, previously defined as the Hoyeraal-Hreidersson (HH) syndrome (characterized by growth retardation, microcephaly, cerebellar hypoplasia, and the development of bone marrow failure/immunodeficiency usually below the age of 5 yrs). In 28 of these families there is affected male(s) only, 13 of which have DKC1 mutations. Of these, 4 have the same amino acid substitution (T49M), which is only seen in patients with HH. In the 12 HH families with affected females, none have DKC1 mutations, 6 are sporadic and 6 appear to have an autosomal recessive form of inheritance. In a large number of families we have been unable to find either a DKC1 or TERC mutation. This includes not only the majority (51/60) of the families in which there is an affected female, but also nearly two thirds (77/113) of the sporadic male cases. Other important gene(s), possibly related to the telomerase complex, are therefore yet to be identified.
Prothrombin Residues 473 to 487 Contribute To Factor Va Binding in the Prothrombinase Complex.
Authors: Yegneswaran Subramanian , Mesters Rolf M. , Fernandez Jose A. , Griffin John H.
Published: October 2019

To identify sequences in prothrombin (fII) involved in assembly of the prothrombinase complex (fXa:factor Va:fII:phospholipids), synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. The fII peptide comprising residues 473–487 (designated PT473–487 that are homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity with 50% inhibition observed at 12 microM and 10 microM peptide, respectively. Prothrombinase inhibition by PT473–487 was fVa-dependent and sequence-specific since the peptide did not inhibit fII activation in the absence of fVa and a scrambled sequence peptide, PT473–487SCR, was not inhibitory. Peptide PT473–487 also inhibited cleavage at Arg271 in meizo-thrombin, showing that it similarly inhibited fXa cleavage at both Arg320 and Arg271. Peptide PT473–487 did not inhibit the amidolytic activities of fXa or thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine if PT473–487 interacted directly with fVa, fluorescein labeled-fVa (Fl-fVa) was prepared. When PT473–487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 microM peptide) whereas the control peptide PT473–487SCR peptide did not alter the anisotropy, suggesting a direct binding interaction between PT473–487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473–487 provide fVa binding sites and mediate interactions between fVa and fII in the prothrombinase complex that contribute to cleavages at both Arg271 and Arg320 by fXa.
The Regulation of Factor IXa by Phosphatidylserine.
Authors: Majumder Rinku , Monroe Dougald M. , Cole Daud , Lentz Barry R.
Published: October 2019

A number of clinical studies have demonstrated correlation between elevated levels of factor IX and the risk of coronary heart disease. Studies by different groups have shown that blocking procoagulant activity of factor IXa could reduce the risk of thrombosis. We propose to study the regulation of factor IXa by phosphatidylserine (PS) which further could open up possibilities to design drugs that might inhibit the up regulation of the procoagulant protein factor IXa. The activated form of factor IX, IXa, forms the Xase complex together with factor VIIIa and Ca2+ on an activated platelet membrane surface. We hypothesize that PS regulates factor IXa by binding to specific sites on factor IXa and thereby enhancing its catalytic activity. We used a soluble form of phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS) as a tool in order to address the role of PS in regulating factor IXa. Intrinsic fluorescence measurements demonstrate that C6PS binds tightly (Kd ~1.3 μM) to and induces changes in conformation of factor IXa. We also monitored the amidolytic activity of 300 nM factor IXa in the presence of C6PS using synthetic substrate Leu-PHG-Arg-pNA. There is a reduction in the amidolytic activity of factor IXa with increasing addition of C6PS and the hyperbolic fit gives an apparent Kd of 130 μM. Recent results show that calcium is required for both amidolytic and proteolytic activity of factor IXa in the presence of C6PS. Our data shows that amidolytic activity of factor IXa in the presence of 600 mM C6PS is saturated at a concentration of calcium near 3 mM. We have also demonstrated that both C6PS and calcium enhance proteolytic activation of factor X by factor IXa. The data shows that as the concentration of Ca2+ increases, the proteolytic activation of factor X by factor IXa increases as well. Enhancement of factor X activation appears to saturate at a calcium concentration of 3 mM. Based on these results, we conclude, 1) C6PS induces a conformational change in factor IXa, 2) C6PS regulates the amidolytic and proteolytic activity of factor IXa, 3) Ca2+ is needed for PS mediated regulation of factor IXa.
Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X.
Authors: Gilbert Gary E. , Bhimavarapu Anu , Price Patricia , Jacquemin Marc
Published: October 2019

The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.
Molecular Follow-Up of Patients Treated with HSV-TK/ΔLNGFR Engineered Donor Lymphocytes after Allogeneic Stem Cell Transplantation.
Authors: Bonini Chiara , Magnani Zulma , Recchia Alessandra , Urbinati Fabrizia , Muraro Sara , Tech Serenella Sartori , Ciceri Fabio , Bernardi Massimo , Pescarollo Alessandra , Gallo-Stampino Corrado , Bregni Marco , Bordignon Claudio , Mavilio Fulvio
Published: October 2019

Donor lymphocyte infusions (DLI) play a crucial role in promoting immune reconstitution and anti-tumor activity in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). The efficacy of DLI is, however, limited by the occurrence of graft-versus-host disease (GvHD). In three different clinical trials, we showed that the infusion of lymphocytes transduced by a retroviral vector expressing a suicide gene (HSV-TK) and a surface marker (ΔLNGFR) allows to control GvHD in 100% of cases, while preserving anti-tumor and antiviral activities. In >40 patients treated with TK-cells in the context of HLA identical and haplo-identical HSCT, we observed consistent expansion (up to 40% of circulating cells) and long-term persistence (>10 years) of transduced cells. No acute or chronic adverse or toxic effect due to the gene transfer procedure was observed in these patients, who were treated with a total of >1011 cells generated by >60 independent transductions. Analysis of vector integration sites was preformed by LM-PCR on lymphocytes obtained up to 10 years after treatment from 4 patients, and selected for ΔLNGFR expression. 60% of the sequences met our validity criteria and were unambiguously mapped onto the human genome by Ensembl BLAST analysis. Transduced T-cells were highly polyclonal, with vector integrations occurring preferentially within genes (52%), and particularly within the first intron (25%). Quantitative PCR analysis of selected integrations was performed to follow the dynamics of individual T-cell clones during time. Microchip analysis of the gene expression profile showed that <200 out of >22,000 genes (0.9%) were differentially expressed in ΔLNGFR+ vs. ΔLNGFR- T-cells from two different patients, suggesting that no significant perturbation was induced by retroviral transduction or HSV-TK/ΔLNGFR expression in human lymphocyte populations. Interestingly, these data also show the substantial biological identity of T-cell population generated by HSCT and those administered as DLI. Finally, the influence of proviral integration on the expression of targeted genes was studied in individual T-cell clones transduced in vitro or obtained ex vivo from treated patients by quantitative gene expression analysis.
The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE): Venous Thromboembolism Prophylaxis Practices in Acutely Ill Medical Patients.
Authors: Tapson Victor F. , Decousus Herve , Bergmann Jean-Fran[ccedi]ois , Chong Beng H. , Froehlich James B. , Kakkar Ajay K. , Merli Geno J. , Monreal Manuel , Nakamura Mashio , Pavanello Riccardo , Pini Mario , Piovella Franco , Spyropoulos Alex C. , Turpie Alexander G.G. , Zotz Rainer B. , FitzGerald Gordon , Anderson Frederick A. ,   for the IMPROVE investigators
Published: October 2019

Background Despite consensus group recommendations indicating that medical patients should receive appropriate venous thromboembolism (VTE) prophylaxis, prophylaxis practices remain poorly characterized. This analysis of IMPROVE, a prospective study of acutely ill medical patients, describes in-hospital prophylaxis practices prior to the publication of updated VTE prevention guidelines by the American College of Chest Physicians.

Methods Patient recruitment began in July 2002. Patients ≥18 years old, and hospitalized for ≥3 days with an acute medical illness are enrolled consecutively. Exclusion criteria are: therapeutic antithrombotics/thrombolytics at admission; major surgery or trauma during 3 months prior to admission; and VTE treatment begun within 24 hours of admission.

Results Data were from 4315 patients (32% from USA) enrolled up to 30 June 2004 in 37 hospitals in 11 countries (76% with 3-month follow-up data). Patients are 50% female, median (IQR) age 69 (50–80) years, median length of hospital stay 8 (5–14) days, median weight 68 (58–80) kg, and 40% were immobile for ≥3 days (median length of immobility 7 [4–14] days, including immobility immediately prior to admission). In-hospital VTE prophylaxis was received by 41% of patients (Table 1). Of patients with no risk factors (44%), one risk factor (40%), or ≥2 risk factors (16%), 25%, 49%, and 67% received prophylaxis, respectively. 12% of IMPROVE patients would have been eligible for inclusion in the MEDENOX study. Of these, only 52% received prophylaxis in hospital. Prophylaxis was provided to 6% of patients during the 3-month follow-up period, and continued in 11% of patients after discharge.

Conclusions Only 41% of IMPROVE patients received VTE prophylaxis, with considerable variation in types and regimens of prophylaxis used. While MEDENOX showed the benefits of VTE prophylaxis (enoxaparin 40 mg) in acutely ill medical patients, only half of MEDENOX-eligible patients received prophylaxis.

Table 1. Use of in-hospital VTE prophylaxis (N=4315)

VTE prophylaxis Patients receiving VTE prophylaxis, % ROW, rest of world; *Excluding elastic stockings and aspirin ≥1 type of VTE prophylaxis* 41 LMWH - USA (Q12h, Qd) 7 (5, 1) LMWH- ROW (Q12h, Qd) 31 (29, 2) UFH - USA (Q12h, Q8h) 28 (15, 11) UFH - ROW (Q12h, Q8h) 6 (5, 0) Intermittent pneumatic compression (USA, ROW) 6 (19, 0) Aspirin (USA, ROW) 4 (7, 3) Elastic stockings (USA, ROW) 6 (3, 8)
Safety and Efficacy of Alfimeprase for Restoring Function in Occluded Central Venous Catheters: Interim Results of a Phase 2, Multicenter, Randomized, Double-Blind Study (NuCath).
Authors: Deitcher Steven R. , Moll Stephan , Homesley Howard D. , Bertoli Luigi , Kenyon Peter , Swischuk Peter , Pena Luis
Published: October 2019

Alfimeprase, a recombinantly produced 22.6 kilodalton metalloproteinase, is a genetically modified variant of fibrolase. Alfimeprase, like fibrolase, proteolytically cleaves both the alpha and beta chains of fibrin(ogen) independent of plasminogen activation to plasmin and directly dissolves thrombi. Readily abundant alpha-2 macroglobulin acts as an endogenous circulating alfimeprase inactivator, forming a covalent, irreversible bond with alfimeprase which results in an inactive complex. Based on the direct thrombolytic effect of alfimeprase, rapid activity was hypothesized. We performed a Phase 2, randomized, double-blind, controlled, dose-ranging study to compare the safety and efficacy of one or two instillations of three intraluminal doses of alfimeprase (0.3 mg; 1.0 mg; and 3.0 mg) and Cathflo® Activase® (Genentech) 2.0 mg in reestablishing patency to occluded central venous access devices (CVADs). This report describes an interim analysis of 48 patients with CVAD withdrawal occlusion who were enrolled and randomized to treatment. Catheter patency was assessed at 5, 15, 30, and 120 minutes after each dose of study drug. Adverse events including bleeding events were assessed for a 30-day period after exposure to study drug. Cumulative patency rates are shown in the table. All study arms had similar patency rates at 120 minutes after the first dose. The alfimeprase 3.0 mg dose produced the highest patency rate at 120 minutes after the second dose. All three alfimeprase doses were more effective than Cathflo® Activase® during the first 30 minutes of treatment. The alfimeprase 1.0 mg and 3.0 mg doses resulted in ≥ 50% patency restoration rates at 15 minutes compared to 0% for Cathflo® Activase®. No intracranial hemorrhage, major hemorrhagic, or embolic events were reported in any treated patients at 5 days. Efficacy and safety results of this study support further evaluation of alfimeprase doses ranging from 1.0 mg to 3.0 mg for treatment of occluded CVADs.

Cumulative Patency Rate (%)

Alfimeprase 0.3 Alfimeprase 1 Alfimeprase 3 Cathflo Activase N=13 N=14 N=9 N=12 D=dose; min=minutes after dose Baseline 0 0 0 0 D 1; 5 min 15 14 44 0 D 1; 15 min 15 50 57 0 D 1; 30 min 30 50 57 25 D 1; 120 min 46 50 57 50 D 2; 5 min 46 50 57 58 D 2; 15 min 46 50 57 67 D 2; 30 min 46 50 57 67 D 2; 120 min 46 50 78 67
Prognostic Relevance of Lipoprotein Lipase (LPL) Expression in B-CLL.
Authors: Heintel Daniel , Kienle Dirk , Martin Bilban , Shehata Medhat , Kroeber Alexander , Mitteregger Dieter , Porpaczy Edith , Kainz Birgit , Auer Michael , Gleiss Andreas , Dohner Hartmut , Gaiger Alexander , Wagner Oswald , Stilgenbauer Stephan , Jaeger Ulrich
Published: October 2019

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic and potential functional relevance. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 55 CLL patients differed by 2 orders of magnitude between cases with mutated (N=28) or unmutated (N=27) IGVH (median: 1.62 vs. 100.43 compared to normal PBMNC). LPL expression correlated strongly with IGVH mutational status (R=0.78; P<0.0001). High LPL expression (≥ 10) predicted unmutated IGVH status with an odds ratio of 128.71 (P < 0.0001) and discriminated between mutated and unmutated cases in 51 of 55 patients (93%). In an extended cohort of 104 patients, a trend for LPL as a prognostic factor regarding overall survival was found: While the median overall survival (OS) in patients with LPL expression of more than 10 was 105 months, the median OS was not reached in the low LPL group. Importantly, high LPL expression (≥ 10) was significantly associated with a short treatment-free survival (median 40 vs. 96 months, P = 0.001). By indirect immunofluorescence, LPL was detected in the cytoplasm of leukemic B-CLL cells and showed intense, large granular and punctate distribution. Fine granular and diffuse staining was observed in monocytes but no immunoreactivity was detected in T- cells. In order to compare the LPL protein expression unmutated (LPL mRNA high) and mutated (LPL mRNA low) samples from 10 patients (5 mutated and 5 unmutated) were prepared and stained under identical conditions.The intensity of intracellular immunoreactivity to LPL antibody in B-CLL cells was higher in each unmutated compared to the mutated samples. In order to investigate the association of LPL with other genes we performed gene expression profiling on 12 patient samples selected for high or low LPL yielding 251 differentially expressed genes: 171 genes associated with LPL only (group 1), 58 genes with LPL and mutational status (group 2) and 22 genes with mutational status only (group 3). While group 2 and 3 contained a number of genes previously described by Rosenwald and Klein*, a considerable number of genes associated with lipid metabolism were clustered in group 1, indicating a functional role of LPL in B-CLL cells. Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

*J Exp Med 2001
PROphylaxis for ThromboEmbolism in Critical Care Trial (PROTECT): A Pilot Study.
Authors: McDonald Ellen , Poirier Germain , Hebert Paul , Pagliarello Joe , Rocker Graeme , Langevin Stephane , LeBlanc Francois , Mehta Geeta , Skrobik Yoanna , Fowler Robert , Granton John , Freitag Andreas , Jones Graham , Cooper Jamie , Meade Maureen , Guyatt Gordon , Anderson David , Geerts Bill , Crowther Mark , Zytaruk Nicole , Griffith Lauren , Cook Deborah ,   For the PROTECT Investigators and the Canadian Critical Care Trials Group
Published: October 2019

Rationale: No published randomized trials have compared low molecular weight heparin (LMWH) to unfractionated heparin (UFH) for thromboprophylaxis in medical-surgical ICU patients.

Objectives: A) Our Feasibility Objectives were to assess: 1) timely enrollment and complete, blinded study drug administration, 2) LMWH bioaccumulation in renal insufficiency, 3) twice weekly leg ultrasounds, and 4) recruitment rates for a future trial. B) Our Clinical Objectives were to assess 1) the effectiveness of LMWH vs UFH by estimating DVT, and pulmonary embolism (PE) rates, and 2) the safety of LMWH vs UFH regarding bleeding, thrombocytopenia and HIT.

Methods: In this prospective randomized stratified concealed blinded multicenter trial, we included patients ≥18 with expected ICU stay ≥72h. We excluded trauma, orthopaedic, cardiac, or neurosurgery, severe hypertension, DVT, PE or hemorrhage within 3 mos, INR >2ULN, PTT >2ULN, platelets <100 x109/L, creatinine clearance <30ml/min, therapeutic anticoagulation, >2 doses of heparin, and palliative care patients. By centralized telephone randomization, we allocated patients to dalteparin 5,000 IU QD SC or UFH 5,000 IU BID SC. All clinicians caring for patients were blinded except the pharmacist. Bilateral leg compression ultrasounds were performed within 48h of ICU admission, twice weekly, on suspicion of DVT, and 7 days post ICU discharge.

Results: A) Feasibility Objectives: 1) Timely, complete study drug administration occurred for >99% of scheduled doses; every dose was blinded. 2) No LWMH bioaccumulation was observed as measured by anti-Xa levels when creatinine clearance decreased to <30 ml/min. 3) Ultrasounds were performed without exception. 4) Estimated recruitment for a future trial is 4–5 patients/month/center. B) Clinical Objectives: 1) Effectiveness: Among 128 patients, 11 (8.6 %) had DVT, 2 (1.6 %) had PE. 2) Safety: 6 (4.7 %) had clinically important bleeds, 23 (18 %) had minor bleeds, 4 (3.1%) developed a platelet count <50 x109/L, and 2 (0.8%) had HIT diagnosed by serotonin release assay.

Conclusions: DVT rates remain high in medical-surgical ICU patients despite universal heparin thromboprophylaxis. This pilot study suggests that a randomized clinical trial comparing LMWH vs UFH feasible. An adequately powered trial is needed to achieve the clinical objectives.
Argatroban Inhibits Human Factor Xa at Therapeutic Drug Levels: An Explanation for Its Prolongation of the INR.
Authors: Warkentin Theodore E. , Greinacher Andreas , Craven Sharon , Dewar Lori , Sheppard Jo-Ann I. , Ofosu Frederick A.
Published: October 2019

Three direct thrombin inhibitors (DTIs) differ in their ability to increase the International Normalized Ratio (INR) for a given prolongation of the activated partial thromboplastin time (APTT), as follows: argatroban > bivalirudin > lepirudin. This phenomenon is clinically-relevant, as it can complicate the transition to coumarin therapy (a risk factor for venous limb ischemia in patients with heparin-induced thrombocytopenia and deep-vein thrombosis, i.e., a clinical situation in which a DTI may be given). Since this observed order of INR prolongation by DTIs is the reverse of that expected based on their known relative affinities for human thrombin (see Table), we tested whether these DTIs (as well as the small-molecule thrombin inhibitor, D-Phe-Pro-ArgCH2Cl [PPACK]) also inhibit free and/or prothrombinase-bound factor Xa (FXa). Prothrombinase-bound FXa was assembled on rabbit brain phospholipids supplemented with human plasma factor X-depleted plasma, to which human FXa (0.5 nM, final) was added. We found that argatroban inhibited free human FXa, i.e., FXa in the fluid-phase (IC50 = 5 μM), whereas it inhibited bovine FXa much less effectively (IC50 = 110 μM). In contrast, only supratherapeutic concentrations of bivalirudin inhibited human and bovine FXa, whereas lepirudin had no effect (see Table). Neither therapeutic nor supratherapeutic concentrations of the three DTIs inhibited prothrombinase-bound FXa.

Comparison of DTIs on INR, APTT, IIa, and Inhibition of Human and Bovine Fxa

PPACK Argatroban Bivalirudin Lepirudin * published values Effect on INR N.D. Greatest Intermediate Least [DTI] doubling APTT N.D. 1 μM 0.25 μM 0.06 μM Ki for human thrombin* ~40 nM ~2 nM ~0.0001 nM IC50 human FXa 3 μM 5 μM 1,700 μM >10,000 μM IC50 bovine FXa 5 μM 110 μM 2,900 μM >10,000 μM

CONCLUSION: Compared with the other two DTIs, argatroban effectively inhibits human FXa at concentrations (IC50 = 5 μM) within the order of magnitude of the usual therapeutic drug levels (about 1 to 2 μM). Inhibition of free FXa is a plausible reason why argatroban prolongs the INR more effectively than the other two DTIs.