bluuebunny/crossref_metadata_2025 huggingface dataset:
Humanized Anti CD-40 Antibody SGN-40 Effectively Induces Cytotoxicity Against Chronic Lymphocytic Leukemia (CLL) Cells through Antibody Mediated Cytotoxicity and Demonstrates Modest Biologic Evidence of CD40 Activation.
Authors: Gowda Aruna C. , Zhao Xiaobin B. , Cheney Carolyn , Mehter Najma , Lozanski Gerard , Lin Thomas S. , Guster Sara , Drachman J. G. , Muthusamy Natarajan , Byrd John C.
Published: October 2019

The CD40 antigen is involved in cell survival and differentiation of B-cells and is uniformly expressed on chronic lymphocytic leukemia (CLL) cells. The CD40/CD40L interaction stimulates B-cells, dendritic cells and monocytes to proliferate, differentiate, up regulate co-stimulatory molecules and increase antigen presentation. While activation of CD40 can protect CLL cells against early fludarabine-induced apoptosis, these cells become sensitive to delayed death by extrinsic pathway apoptosis. (Blood, 105: 3193–8, 2005). SGN-40 is a humanized anti-CD40 antibody entering clinical trials and has been reported to have weak agonistic properties following CD40 ligation. To pursue rational clinical development of SGN-40, we studied the effects of this antibody in fresh, non-cryopreserved primary CLL cells. These studies included classic antibody mediated killing mechanisms and evidence of both CLL cell activation and protection against early fludarabine-mediated apoptosis. CLL cells treated with SGN-40 (10 mcg/ml) for 2 hours (hrs) in the presence of human serum promoted no complement mediated cytoxicity (CDC) in 8 pts tested. Direct SGN-40 induced apoptosis of human CLL cells with or without anti-Fc IgG cross-linking at 24, 48 and 72 hrs was not increased over that observed with the isotype control antibody trastuzumab in 8 pts studied. In contrast, SGN-40 induced antibody dependent cellular cytotoxicity (ADCC) against CLL cells an average of 12% (±11.39 SD, range 2–32%) killing at 4 hrs (effector to target cell ratio 25:1) in 6 pts tested. The SGN-40 induced ADCC against CLL cells were similar to that observed with alemtuzumab (average 19%, SD 6.9, range10–30%) or rituximab (average 18%, SD 12.48, range 8–42.5%). SGN-40 also mediated death in Raji and 697 lymphoblastic lymphoma cell lines via ADCC. Similar to reports by others with CD40 ligand, SGN-40 mediated activation was noted with modest up-regulation of CD80 and HLA-DR at 48hrs. When administered prior to fludarabine, SGN-40 also protected against death in 5 consecutive samples, although this was less than observed with CD40 ligand transfected HeLa cells, consistent with incomplete CD40 activation. Concurrent administration of SGN-40 and fludarabine did not protect from drug-mediated apoptosis. In conclusion, these findings suggest that SGN-40 has dual property of mediating cytotoxic effect by ADCC and partial CD40 activation. Development of SGN-40 as a therapeutic agent in CLL is justified and future studies by our group are focusing on enhancing SGN-40 mediated ADCC against CLL cells and potentially designing combination studies with SGN-40 to exploit this agent’s ability to engage the CD40/CD40L network.
SPC2996 - A New Bcl-2 Inhibitor for the Treatment of CLL.
Authors: Kearney Phil , Westergaard Majken , Bo Hansen Jens , Hansen Henrik F. , Thrue Charlotte A. , Frieden Miriam , Koch Troels , Ørum Henrik
Published: October 2019

The proapoptotic oligonucleotide agent - SPC2996, has been developed by Santaris Pharma A/S, as a specific Bcl-2 mRNA antagonist. SPC2996 was selected from our inhouse discovery research in a library of short antisense oligonucleotides with Locked Nucleic Acid (LNA). LNA brings a significant increase in the affinity towards complementary RNA and in addition remarkably increases the bio-stability.

Stability was evaluated in human and rodent plasma with SPC2996 capable of 20day half life in human serum. mRNA and protein analyses were used for showing specific downregulation of Bcl-2 in cancer cell lines and this reduction was associated with induction of apoptosis. Cellular Bcl-2 responses on mRNA and protein are seen from 1nM of SPC2996. These effects lead to apoptosis induction. Anti- tumor activity was studied in xenotransplanted athymic mice models of human cancer. SPC2996 was equally effective as Genasense in these models.

That SPC2996 specifically dowregulates Bcl-2 was demonstrated in primate studies. Side by side comparison with Oblimersen sodium in these studies showed SPC2996 effectively downregulated Bcl-2 at 3mg/kg while Oblimersen sodium showed little or no target downregulation. Microarray analyses were used to demonstrate specificity of target downregulation.

With SPC2996 we bring the first member of a new generation of safe, potent and specific mRNA antagonist drugs, exploiting the benefits of LNA. SPC2996 provides the cancer patient with the possibility of lower and less frequent dosing, which is unprecedented by any other oligonucleotide inhibitor of Bcl-2 used in clinical trials.
Liposomal Amphotericin B (L-AMB) as Initial Therapy for Invasive Filamentous Fungal Infections (IFFI): A Randomized, Prospective Trial of a High Loading Regimen vs. Standard Dosing (AmBiLoad Trial).
Authors: Cornely Oliver A. , Maertens Johan , Bresnik Mark , Herbrecht Raoul
Published: October 2019

Background: L-AMB exhibits non-linear pharmacokinetics, with maximal values for Cmax and AUC achieved at a dose of 10 mg/kg/d. Maximal tolerated dose is >15 mg/kg/d. In animal models improved efficacy has been demonstrated with higher doses of L-AMB, but comparative clinical data for efficacy of higher than standard doses of L-AMB are lacking. A randomized, double blind study was performed to compare the efficacy and safety of a high loading dose regimen (HD) to standard dosing (SD) as initial therapy of IFFI.

Methods: Patients with proven or probable IFFI by modified EORTC/MSG criteria were randomized to receive L-AMB 3 or 10 mg/kg/d x14d, then 3 mg/kg/d until investigator-defined end of study drug treatment (EOT). Study drug was blinded for the first 14 days of treatment. The primary endpoint was favorable overall response (FOR) assessed at EOT. FOR=complete responses + partial responses. Survival was followed up to 12 wks. An independent Data Review Board (DRB) confirmed all IFFI diagnoses and response assessments.

Results: 201 patients with DRB confirmed IFFI diagnoses comprised the MITT population. 107 received SD, 94 HD. Groups were well matched in terms of risk factors. 93% of patients in each group had underlying hematological malignancies. 16% of SD and 19% of HD patients had allogeneic SCT. Neutropenia was present at baseline in 73% of patients overall, and persisted through EOT in 42%. Invasive aspergillosis (IA) accounted for 97% of cases. Median duration of study drug treatment was SD 15d (range 1–60d) and HD 14d (range 1–57d). FOR at EOT was 50% for SD vs. 46% for HD (p= NS). No significant differences in FOR by treatment group were seen for the subsets of IA, allo-SCT, neutropenia subsets, or site of IFFI. Survival at 12 wks was 72% SD vs. 59% HD (p= NS). Nephrotoxicity (serum creatinine ≥ 2x baseline) occurred in 14% SD vs. 31% HD (p<.01). Grade 3 or greater hypokalemia (K+<3.0 mmol/L) developed in 16% SD vs. 30% HD (p<.02), but no difference was found in grade 4 hypokalemia (K+ ≤ 2.5 mmol/L) 3% SD vs. 4% HD (p= NS).

Conclusions: In a population of highly immunocompromised patients (93% with underlying hematological malignancies, 73% with neutropenia at study entry), L-AMB as initial treatment of invasive aspergillosis and other filamentous fungal infections at a standard dose of 3 mg/kg/d had an overall favorable response rate of 50% and a 12 wk survival rate of 72%. L-AMB given as a high loading regimen of 10 mg/kg/d x14d did not demonstrate any benefit in overall response or survival, and was associated with higher rates of nephrotoxicity and hypokalemia.
CD117 Expression Is a Poor Prognostic Factor (PF) for Progression Free Survival (PFS) and Freedom from Progression (FFP) in Patients with Newly Diagnosed Acute Myelogenous Leukemia (AML).
Authors: Saber Wael , Abou-Jawde Rony , Hsi Eric , Jin Tao , Kalaycio Matt , Sobecks Ronald , Sekeres Mikkael , Pohlman Brad , Tripp Barbara L. , Advani Anjali
Published: October 2019

Background: There are conflicting reports on the prognosis of patients with newly diagnosed c-KIT positive (CD117) AML. In addition there is controversy on the best way to define CD117 positivity by flow cytometry (FC). We retrospectively reviewed data on 95 patients with newly diagnosed AML during the years 1998–2002 to determine the prognostic value of CD117 expression. Methods: CD117 expression was determined by staining cells with antibodies to CD45 and CD117 (BD Biosciences San Jose, CA). CD45-stained cells without CD117 antibody were used as a negative control. FC was performed on FACSCalibur instruments and data acquired using CellQuest software (BD Biosciences). Using a CD45/orthogonal light scatter gate to isolate blasts, the mean fluorescence index (MFI) was calculated as the CD117 mean channel number (MCN) of the blasts/MCN auto fluorescence. Statistical analysis was performed using Cox proportional hazards (CPH) and log rank (LR) testing; Recursive Partitioning analysis was used to Identify CD117 MFI cut-point.

Results: The median age of patients was 59 years. 30% had a history of an antecedent hematological disorder. Good, normal, unfavorable, and other cytogenetics were seen in 10%, 49%, 30%, and 11% of patients, respectively. Patients were treated with anthracycline based induction therapy, followed by consolidation therapy (45% chemotherapy, 23% stem cell transplant). The complete remission (CR) rate was 69.5% (58.7% for age ≥60, 79.6% for age <60). Median Follow up was 16.5 months (m). Median PFS and median overall survival (OS) was 11.2 m (8.5 m for age ≥60, 12.7m for age <60) and 21.3m (19.9m for age ≥ 60, 37.1m for age <60), respectively. CD117 expression (MFI) did not correlate with age, cytogenetics, CR rates or OS. However, patients with CD117 MFI > 10.7 had a median PFS of 9.5m vs. 14.0m for MFI ≤ 10.7 (p=0.033, LR). Median FFP for MFI > 10.7 was 10.0m vs. 33.6m for MFI ≤ 10.7 (p=0.019, LR). On univariate CPH analysis, CD117 (MFI >10.7) was associated with worse PFS (HR: 1.86, 1.04–3.32, p=0.036) and FFP (HR: 2.38, 1.13–5.03, p=0.023). Multivariate CPH analysis showed increasing CD117 expression (per 5 unit MFI increase) was associated with worse FFP (HR: 1.19, 1.01–1.4, p=0.042).

Conclusion: CD117 expression does not correlate with age, cytogenetics, or OS. However, CD117 (MFI>10.7) is a poor PF for PFS and FFP in AML patients. Higher CD117 ratio is also an independent poor PF for FFP. Whether or not agents that inhibit CD117, such as Imatinib Mesylate, can improve prognosis is the basis of ongoing trials.
T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN- γ Expression.
Authors: Long Meixiao , Slaiby Aaron M. , Hagymasi Adam T. , Mihalyo Marianne A. , Lichtler Alexander C. , Reiner Steven L. , Adler Adam J.
Published: October 2019

When Th1 effector CD4 cells encounter tolerizing antigen in vivo their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than non-effector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with antigen vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggests that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN γ expression potential through alterations in chromatin structure.
Development of Novel CD138 Antigen-Specific Peptide Capable of Eliciting Myeloma-Specific Cytotoxic T Lymphocytes Response.
Authors: Bae Jooeun , Martinson Jeff , Yu-Tzu Tai , Neri Paola , Tassone Pierfrancesco , Li Xianfeng , Coffey Rory , Xia Zhinan , Prabhala Rao , Anderson Kenneth , Munshi Nikhil
Published: October 2019

Multiple myeloma (MM) is characterized by the accumulation of mature plasma cells in the bone marrow. Despite progress in treatment, the disease still remains incurable, therefore novel therapeutic approaches are required to achieve better treatment. Development of peptide-based immunotherapies against specific tumor-associated antigens offers an attractive approach that may lead to tumor-targeted cellular therapy in MM patients. CD138 (Syndecan-1) is an unique target expressed on the surface of MM cells and normal plasma cells with very limited other distribution or expression. CD138 has been reported to play an important role in myeloma cell survival and, infact, is used to purify MM cells in laboratory experiments. The purpose of this study was to develop immunogenic peptides derived from CD138 antigen for the induction of antigen-specific cytotoxic T lymphocytes (CTLs) against MM. We have identified an immunogeneic HLA-A2-specific CD138 peptide that is capable of inducing MM specific-CTLs. To induce the specific CTLs, HLA-A2-positive normal human T lymphocytes were stimulated with autologous dendritic cells or T2 cells pulsed with the CD138 peptide. The generated CTLs showed a distinct phenotype consisting of 67% of CD69+/CD45RO+ and 1% of CD45RA+/CCR7+ T cells, characteristic of effector memory cell population. A significant (p < 0.05) increase in T cell proliferation and IFN-g secretion were observed by the CTLs following restimulation with HLA-A2+/CD138+ MM cells. The CTLs displayed HLA-A2-restricted CD138-specific cytotoxic activity against MM cell, demonstrated as 76% and 86% cytotoxicity at Effector:Target ratios of 20:1 or 60:1, respectively. The MM cell-specific cytotoxicity was confirmed in “cold” target inhibition assays using HLA-A2.1+/CD138+ MM cells as “cold” inhibitor, demonstrated 25% and 39% reduction in cytotoxicity when incubated with cold inhibitors at E:T ratios of 20:1 or 60:1, respectively. A distinct CD138 peptide-specific tetramer+/CD8+ population was also confirmed. Taken together, these data demonstrate the efficacy of the immunogenic CD138 peptide in inducing a cellular immune response in MM for future clinical application.
Two Functional Mutations in cis of the Ferrochelatase Gene (FECH) Cause Erythropoietic Protoporphyria (EPP).
Authors: Di Pierro Elena , Brancaleoni Valentina , Moriondo Valeria , Cappellini Maria D.
Published: October 2019

Erythropoietic protoporphyria is an autosomal dominant disease with incomplete penetrance, due to reduced activity of ferrochelatase (FECH), a mitochondrial enzyme that catalyzes the final step of the heme biosynthetic pathway. The clinical phenotype of EPP results from coinheritance of a mutated allele and a wild-type low expressed allele of the FECH gene. To date, more than 100 different mutations have been identified in the FECH gene of EPP patients. Different evidence suggests that an entire haplotype (−251G, IVS1-23T, and IVS3-48C) reduces allele expression. In two Italian EPP patients carring the −250G>C mutation in the promoter region we recently demonstrated the loss of an SP1 binding site causing a strong impairment of promoter activity. In both patients we searched for the GTC haplotype on the other allele. Family segregation studies established that the GTC haplotype was in trans to the −250C mutation. Indeed, asymptomatic parents carried either the three polymorphisms or the point mutation. In both patients only the IVS3-48C polymorphism was in apparent homozygosity since family studies established the absence of Mendelian segregation. To reduce the possibility of interfering polymorphisms in the primer sites, a second set of sequencing primers was designed. Apparent homozygosity was confirmed. We assumed the presence of an intronic deletion on the same allele carrying the −250G>C mutation. In order to establish the size of the deletion we performed a semiquantitative RT-PCR analysis on fresh RNA and XL-PCR analysis on DNA. In both cases the primers were situated in regions outside the heterozygotic polymorphisms. As expected RNA analysis revealed the presence of a normal fragment of 612bp and a shorter fragment of 343bp that was less abundant compared to controls. Sequence analysis on the isolated abnormal fragment showed loss of exons 3 and 4. DNA analysis revealed the presence of two fragments of 21440bp and 15864bp respectively. We performed an internal long PCR to obtain two shorter fragments 9717bp and 4141bp respectively. Sequence analysis on the isolated abnormal fragment showed a 5576bp deletion defined by two short direct repeats of about 40bp. The deleted region includes both a first repeat and interposed sequence. These results showed the presence of two functional mutations on the same allele. Deletion contributed to create a null allele preventing formation of the correct mRNA that is already strongly impaired by the −250G>C mutation. The deletion is caused by slipped strand mispairing or more probably by unequal intragenic recombination. These two mutations in the promoter region and intron 3 respectively, are located in the same regions as the −251G and ivs3-48C polymorphisms, strongly suggesting a functional relationship between these two regions.
Role of Lipid Rafts in GPVI Agonist-Induced Platelet Signaling.
Authors: Quinter Patricia G. , Quinton Todd M. , Dangelmaier Carol A. , Kunapuli Satya P. , Daniel James L.
Published: October 2019

The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.
Validation of T2* Technique in Ex-Vivo Myocardial Tissue.
Authors: Kirk Paul , Firmin David N. , Sampson Barry , Porter John B. , Walker Malcolm J. , Pennell Dudley J.
Published: October 2019

Background

The T2* technique has been validated as a non-invasive measurement of myocardial iron load. However, calibration of the technique against myocardial tissue remains to be determined. Since such calibration is easiest to perform using post-mortem tissue, it is useful to know the effects of storage of the tissue in formalin, and in particular whether iron leaks from the heart during storage, and whether fixation alters the myocardial T2* significantly.

Method

A heart was donated after death of a patient with sideroblastic anemia who had been poorly compliant with deferoxamine treatment and died in heart failure. The pre-mortem myocardial T2* was 5.2 ms indicating severe myocardial iron loading. The majority of the heart was assigned to a calibration study of myocardial iron, but 2 samples from the heart were available for this study.

Sample 1 was an apical short-axis slice of the left ventricle. This was preserved in formalin for 3 days prior to measurement of the initial post-mortem myocardial T2* which was 5.2 ms (Siemens Sonata 1.5T MR scanner). The T2* of the slice was then measured weekly for 4 weeks; then 2 weekly for 8 weeks; and finally 4 weekly.

Sample 2 was a 7.35 g piece of apical myocardium which was stored in 280ml formaldehyde. A 1ml sample of formaldehyde solution was taken for iron analysis from sample 2 initially and at 2 days, and then weekly for 4 weeks; two-weekly for 6 weeks; and then 4-weekly for 12 weeks. The iron concentration in the formalin was measured using Inductive Coupled Plasma with Mass Spectometry (ICPMS).

Results

The results from sample 1 are shown as myocardial T2* against days elapsed. The T2* rose from a pre-mortem and post-mortem (equivalent values) of 5.2ms to a mean of 6ms over the first 20 days, and after this was stable (figure 1).

The results from sample 2, show an increase from 16.6 microgram to 74.9 microgram over the initial 3–4 weeks after which little significant additional accumulation occurred (figure 2). Estimation of the total iron content of the myocardial sample (prior to destructive iron measurement at the end of the study) suggested that the total iron leaked into the formalin was 0.44% of the iron in the myocardial sample.

Conclusion

There was a small increase in myocardial T2* over the first 20 days of formalin fixation, but little significant change after that. This was associated with a loss of approximately 75 micrograms of iron into the formalin fixation solution, which was estimated as only 0.44% of the iron in the myocardial sample. This suggests that formalin fixation has a small but measurable effect on myocardial T2* over time, and this is not explained by loss of iron into the fixation fluid.

Figure 1 Figure 1. Figure 2 Figure 2.
Is Primary Biliary Cirrhosis a Rare Cause of Eosinophilia? A Case Report.
Authors: Tsesmeli Niki E. , Savopoulos Christos G. , Kaiafa Georgia D. , Hatzitolios Apostolos I. , Vretou Eleni E. , Papadopoulos Athanasios , Koliouskas Dimitrios P.
Published: October 2019

INTRODUCTION: Evaluation of eosinophilia involves various medical sybspecialties and the list of diseases, syndromes and inflammatory processes associated with peripheral blood (>300 eosinophils/mm3) and/or tissue eosinophilia, is quite extensive. Little attention, however, has been given to its possible association with primary biliary cirrhosis (PBC).

AIMS & METHODS: To identify PBC as a rare cause of eosinophilia

RESULTS: A 59 female patient, complaining of fatigue, referred to us for abnormal liver function tests. She had no prior history of liver disease, alcohol abuse or medication use. The haematological parameters were normal except for eosinophilia: WBC: 7.5 x 109 /lit, eosinophils 1125/mm3 -15% (normal range: 3–5%) and the biochemical abnormalities included an alkaline phosphatase (ALP) of 314 U/L, γ-gt 146 U/L, total bilirubin 2.2 mg/dl (direct 1.6 mg/dl), AST 62 U/L, ALT 88 U/L and cholesterol 302 mg/dl. Eosinophilia was first detected during an initial evaluation for fatigue 20 months before. She had shown a normal serum biochemistry and negative stool examinations for intestinal parasites and ova at that time. Other causes of eosinophilia, such as skin, allergic, pulmonary, collagen, vascular or immunodeficiency disorders and malignancies, had been excluded by history, physical examination, imaging procedures (chest X-ray and abdominal CT scan) and laboratory investigation, including serum protein electrophoresis, quantitative immunoglobulins, serum immunoelectrophoresis, tumor markers and bone marrow aspirate. On admission, both abdominal and ultrasonography examination revealed a mild hepatomegaly. AMA were detected and liver biopsy was performed. After the serological and histological identification of PBC, ursodeoxycholic acid (15 mg/kg daily) was administered. Three months later, her haematological and biochemical profiles were improved (eosinophils 6%, ALP 234 U/L, γ-gt 80 U/L, AST 59 U/L, ALT 72 U/L)

CONCLUSIONS: Our case suggests that primary biliary cirrhosis should be contemplated in the differential diagnosis of eosinophilia, when all common causes are excluded. In particular, clinicians should evaluate AMA in cases of otherwise unexplained eosinophilia, even in the absence of symptoms or laboratory findings compatible to PBC.
Differential Expression of Granzymes A and K in Subsets of Human T-Cells and NK-Cells.
Authors: Zeberg Lennart , Olofsson Tor
Published: October 2019

Granzymes are highly specific proteases stored in secretory granule in T-cells and NK-cells. Five different granzymes are known in human (A, B, H, K and M). A and B are the best studied and both can induce apotosis when delivered to target cells. The role for granzyme H, K and M are principally unknown. We have studied the expression of granzymes in subsets of T-cells and NK-cells both in fresh circulating cells and cultured cells stimulated with IL-2, IL-15 and OKT3/antiCD28. Protein expression (biosynthesis/immunoprecipitation) and mRNA level (RT-PCR) was studied in parallel. This report focus on Granzyme A and K which are close relatives, both tryptases located 70 kb apart on chromosome 5.

Granzyme A have the highest expression in NK-cells were granzyme K are barely detected. Granzyme K on the other hand have a particular high expression in CD8+ T cells. Stimulation with IL-2 or IL-15 give a strong upregulation of both enzymes in NK-cells. Stimulation of CD8+ T cells gives mainly upregulation of granzyme A. Fresh CD4+ T cells show very weak synthesis of granzymes but treatment with IL-2 or IL-15 give a clear upregulation of both granzyme A and K. T-cells (both CD4+ and CD8+) are highly responsive to stimulation with OKT3/anti CD28 producing granzyme B but not A and K. This rather discordant pattern of expression for granzyme A and K talks in favour of different modes of regulation and most likely different functions.

A striking difference between granzyme A and K concern the propensity for granzyme A to be secreted into the culture medium after stimulation while this is not the case for granzyme K. The secreted form of granzyme A has a molecular weight slightly higher than the intracellular form indicating the presence of a proform. Secretion of granzymes to the extracellular medium is probably not only an in vitro phenomena since both granzyme A and B can be detected in serum from healthy individuals in picomolar concentrations. Moreover, significant elevation of granzyme levels in serum can bee seen in different diseases with activation of cellular immunity and in T- and NK-cells malignancies. We have previously reported that proforms of certain serine proteases including the five human granzymes have a downregulating activity on myeloid proliferation, a mechanism probably implicated in certain forms of neutropenia. We propose that secreted granzymes (especially A, B and H) constitute a part of T regulatory function working both locally at the site of inflammation and distantly via the circulation.
The Effects of Glycoprotein IIb/IIIa Antagonists and Chloride Channel Blockers on Platelet Cytoplasmic Free Calcium.
Authors: Yin Songmei , Li Yiqing , Xie Shuangfeng , Nie Danian , Li Haiming , Ma Liping , Wang Xiuju , Wu Yudan , Feng Jianghong
Published: October 2019

Objective

To explore the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on the platelet cytoplasmic free calcium ([Ca2+]i ).

Methods

We washed and suspended fresh platelets with Hepes buffer containing 0.1% bovine serum albumin (BSA), then loaded platelets with 5μmol/L Fura-3/AM. Then RGDS, the GPIIb/IIIa antagonists, and the chloride channel blockers 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene(DIDS) or niflumic acid(NFA) were added to the platelet suspension. After 2 minutes incubating, we observed the effects and interactions of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i by measuring the Fura-3 fluorescence intensity.

Results

1. Effects of GPIIb/IIIa antagonists and chloride channel blockers on platelet [Ca2+]i.

The fluorescence intensity of resting platelet [Ca2+]i were 369.6±62.2, 381.9±72.4, 392.8 ±69.9 after adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L) respectively. every agent had no significant effect on resting [Ca2+]i (p>0.05). After thrombin(0.03 U/ml) stimulating and adding RGDS(250 μmol/L), DIDS(100 μmol/L) or NFA( 100 μmol/L), the platelet [Ca2+]i were 883.9±107.0, 789.8±99.8, 564.1±79.4. Compare with the control(977.9±108.8), the three agents could inhibit the elevation of [Ca2+]i stimulated by thrombin (p<0.05). The inhibiting rates were (9.37±7.5)%, (18.7±10.4)% and (41.8±10.1)% respectively.

2. Combined effects of GPIIb/IIIa antagonists and chloride channel blockers

The fluorescence intensity of resting platelet [Ca2+]i was 383.9±67.9 after incubated with RGDS and DIDS. That had no significant effects. When platelets were stimulated by thrombin (0.03 U/ml), the combined inhibition rate was (24.4±10.8)%, RGDS and DIDS couldn’t weaken or enhance each other on thrombin-induced elevation of [Ca2+]i (p>0.05). Neither RGDS nor NFA had significant combined effects on resting [Ca2+]i(p>0.05). The combined inhibition rate was (46.0±7.3)%, they had no interactions too(p>0.05).

Conclusion

The GPIIb/IIIa antagonists RGDS and the chloride channel blockers DIDS or NFA have no effect on resting platelet [Ca2+]i. All of them can inhibit the elevation of platelet [Ca2+]i induced by thrombin. There are no interactions between GPIIb/IIIa antagonists RGDS and chloride channel blockers (DIDS or NFA) in resting platelet [Ca2+]i and elevation of platelet [Ca2+]i induced by thrombin, and their effects were independent.
Circulating Platelet-Leukocyte Aggregates as a Marker of Microvascular Lesions in Diabetic Patients.
Authors: Elalamy Ismail , Chakroun Tahar , Robert Francoise , Lecrubier Chantal , Elgrably Fabienne , Samama Meyer M. , Gerotziafas Grigoris T. , Hatmi Mohamed T.
Published: October 2019

Introduction: Evolution of diabetes is associated with multiple disorders including metabolic, cellular and blood disturbances leading to various vascular complications (micro-and macro-angiopathies). Increased circulating levels of platelet-leukocyte aggregates (PLA) have been described in several thrombotic diseases.

Material and Methods: In this study, we have evaluated the circulating PLA in diabetic patients and we have investigated whether they may be linked to the vascular complications currently occurring during diabetes evolution. Using flow cytometry assay, we have quantified PLA percentages in 65 diabetics (37 males/28 females, 57 ± 11 years old) including 20 patients with type I and 45 with type II diabetes, and 25 healthy subjects (15 males/10 females, 44 ± 9 years old). Labelling approach using specific monoclonal antibodies permitted us to identify platelet-polymorphonuclear aggregates (PPA) and platelet-monocyte aggregates (PMA).

Results: We have observed a significant increase of PPA and PMA levels in diabetics (22 ± 12% and 45 ± 18%, respectively) compared to control subjects (7 ± 4% and 19 ± 10%, respectively). However, both PPA and PMA values were similar in the two types of diabetes and they were not correlated to the disease duration. Circulating PPA and PMA percentages were significantly enhanced in diabetics with vascular lesions (n=37; PPA: 24 ± 13%; PMA: 50 ± 18%) than in diabetics without vascular lesions (n=28; PPA: 18 ± 8%; PMA: 38 ± 15%). Patients with PPA > 18% and /or PMA > 38% had a more important vascular thrombotic damage (OR: 6; 95%; IC: 1.6; 23). The increased PMA circulating percentage seems to be more specific for micro-retinopathy occurrence (OR: 19, 95% IC: 2.3; 154). The rare patients with elevated PMA percentage and without vascular lesions have a shorter duration of diabetes (7 ± 5 years) than patients presenting retinopathy lesions (16 ± 11 years).

Conclusion: Together our findings established a relationship between increased circulating PLA rate, particularly that of PMA, and the incidence of microvascular complications in diabetic patients. In addition, they reinforce the concept that pro-inflammatory cells are involved in diabetic retinopathy pathogenesis.
Urinary 11-Dehydrothromboxane B2 Levels in Healthy Individuals Following a Single Dose Response to Two Concentrations of Aspirin.
Authors: McGlasson David L. , Chen Michael W. , Knight Zita A. , Dobbs Michael
Published: October 2019

Introduction: The purpose of this protocol was to determine the effect of aspirin on urinary 11-dehydrothromboxane B2 (11-DHT) levels in normal healthy adults who had ingested a single dose of aspirin (ASA).

Methods: We enrolled 50 normal healthy volunteers 18–58 years of age who had not ingested ASA or other NSAIDs for the previous 14 days. A baseline urine specimen was obtained followed by ingestion of a single 81 mg enteric-coated ASA. Twenty-four hours later a post-ASA urine specimen was obtained. Two weeks later another baseline urine specimen was obtained followed by a single 325 mg ASA enteric-coated tablet. Twenty-four hours later another post-ASA urine specimen was obtained. Urines were stored at -70°C until testing. Platelet response to aspirin was assessed using the 11-DHT immunoassay (Cayman Biochemical, Ann Arbor, MI). Aspirin Works® controls (Creative Clinical Concepts, Denver, CO) were utilized to control for lot to lot and run to run variability. Testing was completed by Hemostasis Reference Laboratories (Hamilton, ON). In 2002, HOPE study investigators computed quartiles by comparing clinical outcomes to urinary 11-DHT levels from subjects who were taking ASA to prevent a second adverse cardiovascular event. A level of 298 pg 11-DHT/mg urinary creatinine was the limit for determining platelet response to ASA and for projecting cardiovascular risk While 298 pg/mg was the lower limit of the fourth quartile, this limit may not apply to the 11-DHT effect of a single dose of ASA. Our limit for ASA response was established by determining the 50% difference between baseline and outcome. This approach was applied to both ASA concentration arms.

Results: Twenty-two of 48 subjects in the 81 mg ASA group showed an ASA response (46%). Forty of the 46 subjects (85%) in the 325 mg dosage group responded. The 325 mg ASA dose showed a significantly greater platelet response, based on mean 11-DHT levels.

Discussion: Previous studies have shown that non-responders to ASA have a greater risk of cardio-vascular problems using the quartile distribution. The current study may be used to extend these findings by applying them to single-dose ASA in normal healthy subjects. Further studies may apply these data to primary cardiovascular risk.
A Meta-Analysis on the Use of Intravenous Gamma Globulin and Corticosteroids in Children with Acute Immune Thrombocytopenic Purpura.
Authors: Chua Mary N. , Manuela Tintin , Sarol Jesus N. , Ligsay Antonio D.
Published: October 2019

To determine the difference in proportion of patients who achieved a rise in platelet count and to determine the risk difference of adverse events in patients with acute immune thrombocytopenic purpura treated with corticosteroids or gamma globulin (IVIG). Medline, EMBASE, Pediatric Hematology and Oncology, Pediatrics and Journal of Pediatrics (all up to 2005) and personal collections of the authors. Systematic search of articles utilizing a search strategy was performed to identify relevant articles according to prior inclusion and exclusion criteria. Fixed effects and random effects models were used to determine summary effect estimates. Difference in proportion of patients who achieved the desired platelet count and risk difference of adverse events between treatment groups. Analysis of 9 randomized controlled trials showed that IVIG produced an additional 12–14% increase in proportion of patients with platelet count >20 x 109/L and 15% increase in proportion of patients with platelet count >50 x 109/L on days 2 to 4. No significant differences were seen between the treatment groups on days 14 and 28 except for platelet count >50 x 109/L. More patients in oral steroid treated group had adverse events compared to IVIG treated group but no differences were noted in the parenteral treated group. IVIG is more effective compared to corticosteroids in raising the platelet count during the early part of treatment. IVIG and parenteral steroids shows less adverse events as compared to oral steroid.
Characterisation of the Novel, High Purity, Double Virus Inactivated Von Willebrand Factor and Factor VIII Concentrate Wilate®.
Authors: Stadler Monika , Gruber Gerhard , Kannicht Christoph , Biesert Lothar , Radomski Kai-Uwe , Suhartono Haryadi , Pock Katharina , Neisser-Svae Andrea , Weinberger Josef , Roemisch Juergen , Svae Tor-Einar
Published: October 2019

Purpose: This study summarizes the characteristics and virus safety profile of Wilate®, a new human plasma-derived, high purity, double virus inactivated VWF/FVIII concentrate. The manufacturing process comprises two chromatographic steps, ensuring high purity and preserving the integrity and functionality of the VWF/FVIII complex. The optimised solvent/detergent treatment (S/D) and terminal dry-heat treatment (PermaHeat) of the lyophilised product provide two effective and robust virus inactivation steps for enveloped (EV) and one step for non-enveloped viruses (NEV).

Methods: Functional and structural properties of the VWF/FVIII concentrate were studied utilising state-of-the-art assays. The reduction factors for a panel of model viruses were determined in down-scale experiments valid for the respective manufacturing steps.

Summary of results: The integrity of the VWF/FVIII concentrate was confirmed by all tests. The ratio between VWF:RCo and FVIII:C was close to that in plasma of blood donors. The VWF multimeric profile demonstrated >10 bands in all batches and the triplet structure was very well preserved. A high specific activity, on average 122 IU FVIII:C/mg total protein, was measured in 15 batches recently produced. SE–HPLC revealed a one-peak-product, representing the VWF/FVIII-complex. All viruses tested were rapidly inactivated by at least 4 log10. The PermaHeat treatment of the lyophilised product at +100°C for 2 hours, at a specified residual moisture of 0.7–1.6%, was efficient for the inactivation of both EV and NEV.

Conclusion: Wilate® is a high purity, double virus inactivated concentrate containing functional VWF and FVIII in a ratio close to plasma of healthy subjects. The product has an excellent viral safety profile. The safe and effective treatment of patients with von Willebrand disease and haemophilia A was demonstrated in multiple clinical trials.
Central Line-Associated Venous Late Effects in Children without Any Prior History of Thrombosis - A Cross-Sectional Study of Children Previously Treated for Malignant or Hematological Diseases.
Authors: Ruud Ellen , Holmstrøm Henrik , Hopp Einar , Wesenberg Finn
Published: October 2019

Background: The frequency of asymptomatic central line-associated thromboses is high and well recognised among children with cancer, while the long-term consequences are mainly unknown. In a cross-sectional study, we have evaluated clinical and radiological venous outcome in children with previous long-standing intravascular catheters.

Methods: The study enrolled 71 children previously treated for malignant or haematological diseases, 4–180 (median 37) months after removal of their central lines. The central lines had been placed in a jugular vein for a minimum of six months, and none had a previously identified event of thrombosis. The children were clinically examined for post-thrombotic syndrome (PTS) and had Doppler ultrasonography of their central neck veins. In a subgroup of twelve children, venous magnetic resonance imaging (MRI) of the same area of interest was performed.

Results: We observed mild PTS with increased superficial collaterals in four children (6 %), but no cases of more severe PTS. None complained of symptoms related to venous late effects. By ultrasonography, post-thrombotic venous alterations were detected in 17 children (24 %), and five of these had complete occlusion of veins. The sensitivity for pathologically increased collaterals to identify occlusive thrombosis was 0.6, while the specificity was 0.98. Occlusive thrombosis was associated with previous central line-associated infections (p = 0.001) and duration of central lines in place (p = 0.042). MRI did not give any extra information, and only occlusive thromboses were confirmed at MRI.

Conclusions: In spite of no prior history of thrombosis, children with previous long-term jugular lines had often local thrombotic sequelae, while clinical symptoms of PTS were rare.
Evaluation of Progenitor Cells (CFU-GM, CFU-GEMM, BFU-E, CD34+ Cells) and White Blood Cells in Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma Treated with Rituximab-CHOP-14 and Supported with Pegfilgrastim.
Authors: Bonfichi Maurizio , Brusamolino Ercole , Marseglia Carmela , Rusconi Chiara , Lazzarino Mario
Published: October 2019

Purpose: To investigate the behavior of CFU-GM, CFU-GEMM, BFU-E, CD34+ cells and white blood cells before and during six courses of dose-dense immune-chemotherapy (R-CHOP-14) supported by pegfilgrastim in eight patients affected with newly diagnosed diffuse large B cell lymphoma (3 men, 5 women, aged 18–51).

Patients and Methods: The standard-dose CHOP chemotherapy was delivered every 14 days, preceded on day 1 by rituximab (375 mg/m2) and followed on day 3 by pegfilgrastim (6 mg per cycle). In each course of therapy, the evaluation was performed before treatment (day 1) and on days +3, +6, +8, +10 and +13. Hemopoietic progenitors were assayed in a complete StemCell medium (MethoCultTM GF H4434 StemCell, Canada) and counted with an inverted microscope after 14-day incubation at 37°C, in 5% CO2. CD34+ cells were evaluated with flow cytometry (EPICS XL, Beckman Coulter); WBC were evaluated with an automatic counter (ADVIA 120 Bayer).

Results: The peak of CFU-GM, CFU-GEMM, BFU-E colonies and of CD34+ cells was invariably reached on day +13 and the highest absolute values were detected after the first cycle of therapy: CFU-GM/ml 3704±738; CFU-GEMM/ml 199±62; BFU-E/ml 8848±3499; CD34+cells/μL 33±3. In the subsequent courses, the number of both hematopoietic colonies and of CD34+ cells progressively lowered; however, the median value of CD34+ cells was still over 20/μL (range 20–35/μL) before the start of the fourth cycle of R-CHOP-14. A significant direct correlation was found between the number of CFU-GM, CFU-GEMM and BFU-E colonies and that of CD34+ cells (p<0.05). The WBC peak was constantly reached on day +6, with a mean value, all cycles considered, of 32±14 x 109/l.

Comments: Hemopoietic progenitor cells kynetic showed that colonies and CD34+ cells started to increase at the WBC nadir. Most important, the level of circulating CD34+ cells at day 13 of the first three cycles of therapy was above 20/μL which we consider permissive for a productive leukaferesis; this observation adds further evidence of the pegfilgrastim progenitor cells mobilizing capacity.
Potential for Mesenchymal Stem Cell Isolation from Human Placenta.
Authors: Atkinson Kerry , Chatterjee Konika , Barlow Sarah , Brooke Gary
Published: October 2019

Aims: To determine if mesenchymal stem cells (MSCs) can be isolated and cultured from human placenta.

Methods: Placentas were obtained at elective caesarean section from normal term pregnancies. Working from the foetal side, pieces of placenta were excised and washed in HBSS to remove excess blood. Tissue was then incubated in collagenase I and DNase I. After enzymatic digestion, a cell strainer was used to obtain a single cell suspension. The resulting cells were washed and centrifuged on a 1.073 g/ml Percoll density gradient and the interface collected. Cells were then cultured in DMEM (low glucose) with FCS. After 24 hours, non-adherent cells were removed and the remaining cells cultured until almost confluent before passage. Media was changed every 3–4 days. After two or more passages, the cells were analysed.

Results: Using this technique, placental plastic-adherent cells could be maintained in culture for long periods in a manner similar to bone marrow-derived MSC. The cells were large and exhibited an elongated, fibroblast-like morphology. In terms of phenotype, the cells were positive for CD29, CD44, CD73, CD90, CD105, CD166 and MHC class I expression, whilst negative for CD34, CD45 and MHC class II expression. The placental cells were shown to be capable of differentiating into cells of the mesenchymal lineage, namely osteocytes, chondrocytes and adipocytes and they could inhibit allogeneic T cell proliferation. However, some phenotypic differences, at both protein and mRNA level were noted between bone marrow and placental derived MSC, in particular only placental MSC showed expression of integrin VLA-4 and the chemokine receptor CXCR4.

Conclusions: The placental-derived cells exhibited characteristic MSC morphology and mesenchymal differentiation. Placental derived MSC therefore represent an alternative and more easily obtainable and abundant source of MSC than bone marrow.
Depletion of the Leukemic Fusion Protein MLL-AF4 with Short Interfering RNAs (siRNAs) Affects Post-Translational Modification of Aldolase A.
Authors: Greil Johann , Gessner Andreas , Thomas Maria , Heidenreich Olaf
Published: October 2019

The chromosomal translocation t(4;11) marks a therapy-resistant infant leukemia with very poor prognosis. It results in the expression of two fusion-proteins, MLL-AF4 and AF4-MLL. We addressed the role of MLL-AF4 in t(4;11) positive SEM cells by siRNA-mediated suppression. Depletion of MLL-AF4 results in induction of apoptosis, inhibition of proliferation, decrease in colony formation and diminished leukemic engraftment in vivo. Currently, we are analyzing global changes in protein expression. For that, we compare the proteome of MLL-AF4 depleted SEM cells with those of control cells. The analysis is performed by 2D-gelelectrophoresis followed by mass spectrometry identification and immunoblot validation of differentially expressed spots. One of these spots was identified as Aldolase A. Comparison of MLL-AF4 depleted SEM cells with control cells showed neither change in mRNA levels nor in absolute protein levels of Aldolase A. Two-dimensional western blotting, however, revealed differences in the protein pattern, suggesting changes in Aldolase A modifications upon MLL-AF4 depletion. These analyses will provide us with a better insight into the effects of siRNA-mediated MLL-AF4 knockdown on the proteome, and may enable us to identify new targets for molecular therapeutic approaches.