bluuebunny/crossref_metadata_2025 huggingface dataset:
Telomerase Activity and Telomere Length of CD4+CD25+ Regulatory T-Cells under Conditions of In Vitro and In Vivo Expansion.
Authors: Wolf Dominik G.F. , Wolf Anna M. , Koppelstaetter Christian , Rumpold Holger F. , Mayer Gert , Gastl Guenther A. , Gunsilius Eberhard , Tilg Herbert
Published: October 2019

The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation.

In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.
Downregulation of TGF-β Signaling by Thrombin in Endothelial Cells.
Authors: Tang Hua , Low Brad , Rutherford Stacey A. , Hao Qin
Published: October 2019

Thrombin activates protease-activated receptor 1 (PAR1) on endothelial cells (ECs) and regulates many aspects of EC biology. A body of evidence has demonstrated that the thrombin-PAR1 pathway is critical for angiogenesis and vascular development. However, the mechanism underlying the proangiogenic effect of thrombin has not been elucidated yet. Here, we report the discovery of a novel functional link between thrombin-PAR1 and transforming growth factor-β (TGF-β) signaling pathways. Using mass spectrometry, confocal immunofluorescence microscopy, cell surface ELISA and a phospho-specific antibody against the serine-phosphorylated endogin, we demonstrated for the first time that thrombin via PAR1 induced the internalization of endoglin and type-II TGF-β receptor (TβRII) but not the type-I receptors in primary human ECs. This effect was mediated by protein kinase C-ζ (PKC-ζ) that was rapidly activated by thrombin in ECs since specific inhibition of PKC-ζ caused aggregation of endoglin and TβRII on cell surface and blocked their internalization in response to thrombin. Furthermore, acute (30 min) and long-term (24 h) pretreatment of ECs with thrombin or PAR1 peptide agonist suppressed the TGF-β-induced serine phosphorylation of Smad2/3, the critical mediators of TGF-β signaling. Moreover, activation of PAR1 led to a profound clustering formation of Smad2/3 and a more spread cytosolic localization, which sequestered Smad2/3 from nucleus and prevented their nuclear translocation evoked by TGF-β. Since TGF-β and its receptors play a crucial role in the resolution phase of angiogenesis, the downregulation of TGF-β signaling by thrombin-PAR1 pathway may provide a new insight into the mechanism of the proangiogenic effect of thrombin.
Long Term Outcome of Patients Undergoing Splenectomy for Immune Thrombocytopenic Purpura.
Authors: Novitzky Nicolas , Seth Yunus , Mothupi Rosalia
Published: October 2019

Splenectomy is very effective in patients with idiopathic thrombocytopenic purpura (ITP) unresponsive to steroids; however it is invasive and has been associated with longstanding immune suppression. We retrospectively reviewed the clinical outcome and laboratory parameters in 52 patients who had undergone splenectomy and had been followed up for a minimum of 5 years. We also studied the long term effectiveness of anti pneumococcal vaccination by testing antibody titres against 5 bacterial antigens. Hospital records of 52 individuals with ITP who required splenectomy were accessed. Of these 52, 47 patients were available for clinical review and consented to provide blood samples to have their laboratory parameters repeated. None of the patients had been treated with prophylactic antibiotics. Except for one, all had received anti pneumococcal vaccination before surgery. The serum anti pneumococcal IgG antibody titres were tested by enzyme linked immunosorbent assay and the results were related to a control group. The complication rate of surgery was 15%, but there was no mortality. Immediately post splenectomy all patients normalised their platelet counts but thrombocytopenia recurred in 6, who required further immunosuppression. Following splenectomy the median time to platelet recovery was 10 days (range 1–90). At 5 years from surgery, 92% remained in response and only 1 / 47 surviving subjects required continuing immunosuppression. In the intervening period subsequent to surgery 5 individuals died. Of these deaths, 4 were unrelated to ITP while 1 patient who had not received anti pneumococcal vaccination, developed rapidly fatal sepsis. At review, patients had significantly higher MCV (p= 0.001), lymphocyte count (p= 0.001) and lower IgM (p= 0.0001). Compared to control samples, of the 5 anti pneumococcal serotypes tested, only the 19F antibody titres were significantly reduced (15.07 ± 17.47 vs. 65.26 ± 79.35 pg/L; p= 0.03) in splenectomised subjects. Splenectomy is highly effective in ITP while the rate of short term complications and long term sequelae appear acceptable. In patients receiving vaccination, anti pneumococcal titres were well maintained and despite no antibiotic prophylaxis no patient in this group developed life threatening infections.
In Normal Controls, a Lower Hb Concentration and Female Sexe Increases Coagulability on the Thrombelastograph (TEG).
Authors: Roeloffzen W.W.F. , Kluin-Nelemans J.C , de Wolf Joost
Published: October 2019

Background. The TEG is used in situations were point of care testing of hemostasis is desired, although its value is still controversially because of insufficient test validation. The main parameters of the TEG are (a) the reaction time (R), the time until the initial fibrin formation and comparable with the coagulation times PT and APTT; (b) clotting time (K), the time until a fixed level of clot firmness is reached; (c) the angle (alpha) is closely related to K and measures the rapidity of fibrin build up and gives information about the clot strength; R, K and alpha are prolonged by anticoagulants and factor deficiencies; (d) maximum amplitude (MA) is a measurement of maximum strength or stiffness of the developed clot; it is especially influenced by platelets and fibrin.

Methods. We performed a multivariate analysis using the Cox multiple-regression model to study the effects of sexe, age, Hb, platelet count, PT, APTT and fibrinogen on the TEG parameters.

Results. Ninety nine normal controls (54 men) age 51 ± 17 years (range 19–87) were studied; no anticoagulants, antithrombotics or oral contraceptives were used. Native whole blood samples were used. The R is significantly influenced by sexe (p<0.001; r=0.4) and APTT (p<0.001; r=0.4); R is in women 19 ± 4, versus 24 ± 5 minutes in men. K is significantly influenced by Hb (p<0.001; r=0.5), fibrinogen (p<0.001; r=−0.4), platelet count (p=0.01; r=−0.3), sexe (p=0.018; r=−0.5) and APTT (p=0.009; r=0.4). The alpha is significantly influenced by the Hb (p<0.001; r=−0.5), fibrinogen (p=0.001; r=0.4), sexe (p=0.008; r=0.5) and APTT (p=0.03; r=−0.4). In normal controls with a Hb of resp.< 8, 8–9 or >=9 mmol/l the K was resp. 6.8 ± 1.7, 8.5 ± 2.5 and 10.5 ± 2.8 minutes; the alpha was resp. 31.4 ± 8.1, 26.1 ± 7 and 21.4 ± 5.8 degrees. The MA was significantly influenced by fibrinogen (p<0.001; r=0.5), age (p<0.001; r=0.5), sexe (p<0.001; r=0.4), platelet count (p=0.02; r=0.2) and PT (p0.03; r=0.1).

Conclusion.. Coagulability measured in the TEG is increased in women compared to men (shortened R and K, larger alpha, increased MA) and coagulability increases with a decrease of the Hb concentration (shortened K and larger alpha).
A Novel Assay for Tissue Factor Pathway Inhibitor (TFPI) Anticoagulant Activity in Plasma.
Authors: Dahm Anders E.A. , Andersen Trine O. , Rosendaal Frits , Sandset Per Morten
Published: October 2019

Introduction. Several studies have shown that full-length free TFPI is a much stronger anticoagulant in diluted prothrombin time (dPT) assays than truncated and lipoprotein associated TFPI. Although it has been suggested that TFPI anticoagulant activity is of importance for the effect of TFPI in vivo, no assays have so far been established and validated to assay TFPI anticoagulant activity in patient samples.

Aims. To develop and validate an assay of TFPI anticoagulant activity and study the relationship between TFPI anticoagulant activity and TFPI free and total antigen and TFPI chromogenic substrate activity.

Materials and Methods. TFPI anticoagulant assay: 32 μL of plasma was first incubated for 5 min with either 8 μL TBS or anti-TFPI immunoglobulin, which completely blocked TFPI activity. Then, 30 μL recombinant, relipidated tissue factor (Innovin®) at 1/200 fold dilution was added and incubated for 3 min. Clotting was initiated by the addition of 30 μL CaCl2 (35 mM). Results were expressed as a ratio, with clotting times in the presence of anti-TFPI as the denominator. This ratio was normalized against the ratio obtained with a reference plasma within each run to minimize day to day variation in clotting times. After normalization, high ratios reflect high functional TFPI anticoagulant activity, whereas low ratios reflect low activity.

TFPI antigen assays: TFPI free and total antigen were assayed with commercial kits from Stago, France. TFPI free antigen is specific for circulating full-length TFPI, whereas TFPI total antigen detects both full-length and truncated forms of TFPI.

Study population: Control subjects (n=436) of the Leiden Thrombophila Study (LETS).

Results. Mean TFPI anticoagulant ratio was 1.03 (SD 0.0066, range 0.87–1.22). The assay coefficient of variation was 2.8% between days and from 0.6% to 1.5% within days. Linear regression showed that the n-TFPIac ratio was strongly associated to the levels of TFPI free antigen, but weakly associated to bound TFPI (table 1).

Conclusions. We have established a novel and reproducible assay for detection of TFPI anticoagulant activity in plasma. TFPI anticoagulant ratio was strongly associated to free antigen, and may be considered an activity assay of full-length TFPI in plasma.

Table 1 - The association of normalized TFPI anticoagulant ratio to other TFPI assays

TFPI assay Normalized TFPI anticoagulant ratio- b (95% CI) Correlation coefficient- r Standardized regression coefficient (b), 95% confidence interval (CI) and correlation coefficient (r). (b) shows the change in n-TFPIac ratio per increase of one standard deviation in the TFPI assay studied. TFPI free antigen 0.049 (0.044 to 0.053) 0.73 Total TFPI 0.033 (0.027 to 0.038) 0.48 TFPI chrom. subst. activity 0.031 (0.025 to 0.037) 0.46 Bound TFPI 0.020 (0.014 to 0.026) 0.30
Muscle Cells in Fibrin Matrix Actively Collapse the Network Leading to Rapid Allignment and Formation of Muscle Bundles.
Authors: Carr Marcus E. , Lam Phillipe , Simpson David G. , Wnek Gary E. , Bowlin Gary L.
Published: October 2019

The goal of in vitro organogenesis has been hampered by cell culture techniques that promote two dimensional cell proliferation. Since cell growth stops once confluence is achieved, three-dimensional organization does not occur. Possibly due to a lack of normal spatial orientation and cell-to-cell interactions, cells grown in such systems often assume morphologies significantly different from those assumed in normal biological tissues. We have recently developed a simple technique for growing cells in fibrin gels. This is accomplished by suspending cells in fibrinogen solutions and subsequently mixing the fibrinogen-cell suspension with buffered thrombin. The fibrinogen solution can be prepared from purified protein or from plasma diluted with growth media. The mixture rapidly forms a fibrin gel to which the cells are attached. When muscle cells are cultured in fibrin matrices, they initially form a latticework of proliferating cells. Over the next forty-eight hours, the cells begin to collapse the edge of the structure in a process similar to platelet induced clot retraction. As the fibrin network collapses, muscle cells are brought into linear alignment to form structures closely resembling muscle bundles. This process is relatively rapid. Within forty-eight to seventy-two hours, three-dimensional structures morphologically similar to muscle tissue are formed. By manipulating the gel structure through the imposition of outside forces, the alignment of muscle cells can be caused to occur even more rapidly. This system is simple to use and rapidly results in a muscle culture structure that resembles muscle tissue. This technique holds promise to significantly advance the goal of true in vitro “tissue” formation.
The Care of Immigrant Children with Haemophilia.
Authors: Barnes Chris , Hilliard Pam , Stain Ann Marie , Floros Georgina , Blanchette Victor , Carcao Manuel
Published: October 2019

Canada, like many industrialised countries, has a history of accepting immigrants and a significant proportion of the population of major cities like Toronto is comprised of visible minority groups. The provision of health care to recently arrived immigrant families presents a number of challenges including difficulties in communication, economic inequalities experienced by migrating families and complications of underlying conditions not often seen in countries with well-resourced health care systems. Over the last decade the Hospital for Sick Children (HSC) has been referred a number of children with haemophilia who have recently immigrated. We reviewed the presenting features and subsequent management of recently immigrated children with haemophilia at HSC. We compared objective joint assessment scores of these patients at presentation to follow-up joint assessment scores. Joint assessment scores were performed according to published guidelines with a normal joint score being graded as zero and the maximum joint score (total scores of both elbows, knees and ankles) being 162. Seventeen patients with haemophilia (haemophilia A N=15, haemophilia B N=2) were identified representing 9.2% of all patients with haemophilia at HSC and is comparable to the proportion of people who have recently immigrated to the Toronto area from other countries over the years 1995 to 2001 according to census data. The patients were seen initially at HSC at an average age of 7.3 years (range 0.6 years to 12.2 years) and have been managed at HSC for an average of 3.1 years (range 0.5 to 9.3 years). Fifteen patients have severe haemophilia and of these patients, 7 had no definite previous treatment with clotting factor concentrate. Two patients had been exposed to infection with the hepatitis C virus. None of the patients were HIV virus positive. Joint assessment scores at initial presentation to HSC were available for 13 patients with a mean score of 9.4 (range 0 to 24). Two of the patients were not weight-bearing at presentation. The mean repeat joint assessment scores for ten of the recently immigrated patients was 7.8 (range 0 to 27). With specific treatment and rehabilitation the joints scores improved in the majority of patients. Addressing the challenges of providing health care to recently immigrated children with haemophilia in a culturally sensitive manner can lead to effective medical management and greatly improve joint function in these children.
Short-Term Use of Phenobarbital in the Treatment of “Superwarfarin” Poisoning.
Authors: Oliveri Cecilia V. , Stubbs James R. , Chernova Tatyana , Carnahan Gary Ellis , Evans Tara N. , Gammill Andrew , Siegel Jonathan
Published: October 2019

Background: “Superwarfarin” was developed in the 1970s as an anticoagulant rodenticide. The half-life of the “superwarfarin”, brodifacoum, is four times longer than warfarin. Patients with significant brodifacoum ingestions have often required long-term treatment with vitamin K1 (e.g., 6 months), occasionally with adjunctive phenobarbital therapy.

Case: A 52-year-old male was admitted to the hospital after being found unconscious at home. He was pale and tachycardic at presentation with an oozing laceration on his right leg and bright red blood per rectum. Two liters of maroon fluid were recovered via nasogastric lavage. Admission laboratory values included: PT > 300 seconds, aPTT > 300 seconds, hemoglobin 2.8 g/dL, hematocrit 8.8%, and platelet count 400,000 per μL. Metabolic acidosis with an increased anion gap was present. A drug screen (Toxi-Lab®) was negative. Alcohol, salicylate, and acetaminophen assays yielded negative results. The following day, the patient admitted ingesting with a total of 170 grams of brodifacoum with suicidal intent. Subcutaneous vitamin K1 (10 mg QID), octreotide acetate (50 mg PO), and pantoprazole sodium (40 mg PO) were administered. He improved clinically, but his PT (range: 22.4–47.4 seconds), INR (range: 2.1–5.5), and aPTT (range: 40–70 seconds) remained abnormal. On hospital day 5 his vitamin K1 dose was increased to 25 mg QID and oral phenobarbital (30 mg TID) was started. A total of thirty-eight units of plasma and 8 red cell units were transfused during the admission. He was transferred to an inpatient psychiatric facility on day 8, and he was discharged from that facility after 5 days. Vitamin K1 and phenobarbital were discontinued at this time. Three and one-half months later, the patient was asymptomatic. His laboratory values included: PT 13.2 seconds, INR 1.1, aPTT 25 seconds, hemoglobin 14.3 g/dL, and hematocrit 42%. A serum brodifacoum level yielded negative results.

Conclusion: Superwarfarin has been noted to be an increasingly common cause of poisoning in the past 20 years, especially in intentional ingestions. Many cases have been successfully treated with long-term vitamin K1 — with adjunctive phenobarbital given to some patients. The rationale for using phenobarbital in this setting is that it increases warfarin metabolism by inducing cytochrome P450 isozymes known to be major catabolic enzymes for warfarin (CYP2C9/19) in the liver. We report the successful treatment of significant brodifacoum toxicity with a short course of vitamin K1 (13 days) in combination with phenobarbital (6 days).

Figure Figure
Compare Two Arsenic Trioxide Administration Methods in APL Therapy.
Authors: Zhou Jin , Meng Ran , Yang Baofeng
Published: October 2019

Background As2O3, on the one hand is successful in the treatment of acute promyelocytic leukemia (APL), on the other hand many side effects may occur during the early arsenic treatment. We assessed the effectiveness and security of two As2O3 administration methods in acute promyelocytic leukaemia treatment.

Methods seventy-four cases of APL treated using As2O3 in two administration methods have been studied randomly. Thirty-two cases were administrated As2O3 with routine method as control, the other 42 cases were given As2O3 with a ‘continuous and slowing intravenous infusion’ method. Firstly, we calculated the total quantity of As2O3 needed every day of every patient according to the criteria of 0.16mg/kg, then gave half of the total amount of As2O3 in the morning and the other half in the afternoon, diluted each half of total amount of As2O3 with 5% glucose 250ml and infused at the speed of 8 drops/min, collected the blood serums of the patients treated with the two methods respectively at different time points, and finally we assayed arsenic concentration in blood serums.

Results Most of the APL treated by As2O3 gained complete remission at the time of 4th week with the two administrated methods. The total effective rate in ‘continuous and slowing intravenous infusion ‘ group was 93.75%, while that in control group was 83.3%. The effective arsenic level in blood circulation in ‘continuous and slowing intravenous infusion’ group was lasted at least 18.38±3.27 hours, while in control group was lasted 9.38 ±1.58 hours, the average hospitalization duration in ‘continuous and slowing intravenous infusion ‘ group was26.4±2.04 days, while that in control group was 35.7±4.78 days.

Conclusions The ‘continuous and slowing intravenous infusion’ method can relieve leukocytosis, increase complete remission (CR) rate of APL, and accelerate to CR.

Table1 arsenic concentration, its lasting time and the average time reaching CR (mean± SD)

Group (n) The peakconcentration in 24h μmol/L) ( The lasting time above μmol/L(h / 24h) 1 The average time reaching CR(d) Experiment 42 3.32±0.55 18.38±3.27 26.4±2.04 control 32 5.84±1.05 9.38 ±1.58 35.7±4.78 t-value 6.08 3.64 4.78 P -value < 0.01 < 0.01 < 0.01

Table2 The incidence of side effects in As2O3 therapy ( %)

peripheral WBC↑ DIC QTc prolongation nerve conductspeed ↓ cerebral vasospasm liver disfunction experiment (n=42) 31.3(15) 16.7(8) 8.1(4) 8.1(4) 14.6(7) 14.6(7) control (n=32) 71.2(34) 35.4(17) 25.0(12) 6.3(3) 33.3(16) 22.9(11) X2 - value 17.64 4.38 4.80 0.14 4.62 1.08 P - value <0.001 < 0.05 < 0.05 > 0.1 < 0.05 > 0.05
Peripheral Blood HPC Counts Are Augmented in CML Patients with Cytogenetic Findings of Disease as Compared to Patients in Cytogenetic Remission.
Authors: de Meis Ernesto , Morais Rita C.B. , Carvalho Luize O. , Moellmann-Coelho Arthur , Martinez Maria M.K. , Costa Ana J.U.B. , Barros Ailce S. , Castilho Ilda A.M.
Published: October 2019

Chronic Myeloid Leukemia (CLM) is a stem cell disease characterized by inhibition of apoptosis and increased proliferation of bone marrow (BM) compounds. Evaluation of hematopoetic precursors cells on the peripheral blood (PB) of these patients show a high prevalence of leukemic CD34+ cells. Recently it was demonstrated that measurement of HPC by IMI channel on Sysmex showed an acceptable correlation between Hematopoetic Progenitors CD34+ cells counts. The aim of this work was to compare precursor cells (HPC) numbers in the PB of CML patients with disease and under remission. For this we studied 61 CML patients that had been treated with different protocols. From these 22 were in cytogenetic remission but 39 still presented more than 5% BM cells with Philadelphia chromosome (Ph+). Although the normal levels of CD34+ HPC in PB is considered to be very low, we considered less than 0.05% of leukocytes as normality. HPC in PB were counted using an automated peripheral blood counter (Sysmex XE-2100). We found that Ph+ CML patients had a significant increased number of PB HPC compared to CML patients under remission (with a confidence interval of 95%, and a corrected Yates analyses with X2 16.78 and p<0.0001). The increase in PB HPC was not related with the number of white cells counts at moment of the exam. These findings suggest that the presence of a high number of PB HPC in CML can be used as a non-invasive marker of the disease, more reliable than total and differential leukocyte count.
Constitutive JAK3 Activation as a Mouse Model for Human Peripheral T-Cell Lymphoma.
Authors: Cornejo Melanie G. , Kharas Michael G. , Lee Benjamin , Moore Sandra A. , Gilliland Gary , Mercher Thomas
Published: October 2019

Molecular pathogenesis of human peripheral T-cell malignancies remains poorly understood. We aimed to establish a model for these disorders by using the recently identified JAK3A572V allele. This mutation, which lies in the pseudokinase (JH2) domain of JAK3, transforms Ba/F3 cells to factor-independent growth and causes a striking lymphoproliferative phenotype in a murine bone marrow transplantation assay. Further characterization of JAK3A572V animals revealed that the aberrantly expanded cell population consisted of a mature effector/memory subtype of CD8+T-cells that infiltrated all major lymphoid and several non-lympoid organs and could be transplanted into secondary and tertiary recipients. These JAK3A572V T-cells had increased proliferative capacities and displayed enhanced phosphorylation of common JAK3 targets, such as STAT5 and S6-kinase. Proliferation of primary T-cells transformed by JAK3A572V was effectively inhibited with a small molecule JAK inhibitor that had no effect on the proliferative potential of control cells transduced with a wildtype JAK3 allele. Furthermore, the mutant cells showed increased production of cytotoxic cytokines, such as IFN-γ and TNF-α, compared to wildtype counterparts, which correlated with an increased cellular cytotoxicity towards allogeneic target cells. Of particular interest, JAK3A572V animals presented with skin lesions and histopathologic analysis showed aberrant skin-homing T-cells tagging along the epidermal/dermal junctions. Mice receiving Rag1-deficient donor cells transduced with the JAK3A572V allele also developed a lethal lymphoproliferative disease characterized by the expansion of immature CD3−TCRβ−CD4+/−CD8+ cells, suggesting that the JAK3A572V-dependent lymphoproliferation does not require proper TCR rearrangement. Altogether these results indicate that in this murine model, constitutive activation of JAK3 results in peripheral/cutaneous T-cell lymphoma (PTCL) that closely resembles the human disease. These findings suggest the possibility that the molecular basis of human PTCL could include aberrant JAK3 signaling and might provide a useful platform for deciphering the molecular and cellular mechanisms and requirements for peripheral lymphoid disease development and progression. Furthermore, it provides an opportunity to investigate the therapeutic potential of selective JAK3 inhibitors for this subset of lymphoid disorders, whose treatment remains a challenge.
PIR-B-Deficient Mice Are Susceptible to Salmonella Infection.
Authors: Oka Satoshi , Torii Ikuko , Hotomi Muneki , Benjamin William H. , Takai Toshiyuki , Kearney John F. , Briles David E. , Kubagawa Hiromi
Published: October 2019

Paired Immunoglobulin-like receptors of activating (PIR-A) and inhibitory (PIR-B) isoforms are expressed by many blood cell types including B cells, dendritic cells, monocyte/macrophages, granulocytes, mast cells and megakaryocyte/platelets. To determine the role of PIR-B in bacterial infection, PIR-B deficient (PIR-B−/−) and control C57BL/6 mice of the same age and sex were infected intravenously with the attenuated strain of Salmonella typhimurium (WB335). When injected with high doses of bacteria (5 × 104 ∼ 5 × 106 CFU), both types of mice died within 3 to 14 days post infection. When given small doses (103 ∼ 104 CFU), however, PIR-B−/− mice exhibited progressively increasing morbidity beginning at 4-day post-infection over a 4-week period, whereas many control mice could survive (see Figure). PIR-B−/− mice were found to have higher bacterial loads in liver, spleen and lungs than the control mice and bacteremia at days 3 and 7 post-infection. In liver histology typhoid nodules were observed as early as day 1 and their incidence increased during the next six days in both groups of mice. However, quite distinct changes were observed at day 7: in control mice inflammatory cellular lesions were localized and exhibited nodular distribution, whereas in PIR-B−/− mice mononuclear cells infiltrated along the liver sinusoids and exhibited a spreading distribution. There were no significant differences in plasma levels of liver enzymes (ATL, AST and ALK), cytokines (TNFα, IL-10 and IFNγ) and antibodies (IgM, IgG and IgA) against Salmonella between PIR-B−/− and control mice. While splenic lymphoid and myeloid subsets were indistinguishable during the first week of infection, the increases of CD19+ B and CD8+ T cells in infected PIR-B−/− mice were not as marked as those in infected control mice at day 14 post-infection (see Table). For phagocytic cell functions ex vivo, there were no differences in phagocytosis and intracellular killing of bacteria and superoxide anion release between PIR-B−/− and control mice. However, PIR-B−/− macrophages were found to release more nitrite and TNFα upon bacteria stimulation than the control macrophages. These results suggest that PIR-B plays an important regulatory role in bacterial infection.

(Supported by NIH/NIAID grants AI42127 and AI52243).

Mac-1+ MΦ CD4 + T CD8+ T CD19+ B cell numbers (× 107), mean ± 1 S.D. Uninfected Control 1.3 ± 0.6 * 1.4 ± 0.4 0.7 ± 0.3 5.1 ± 1.8 Uninfected PIR-B−/− 1.2 ± 0.3 1.5 ± 0.2 1.2 ± 0.2 4.9 ± 0.3 Infected Control 19.4 ± 7.0 5.5 ± 1.5 2.6 ± 0.7 10.0 ± 2.6 Infected PIR-B−/− 16.5 ± 2.3 4.2 ± 2.1 1.3 ± 0.6 4.5 ± 1.3 Figure Figure
Macrocytosis in COPD and Course of the Disease
Authors: Kontoninas Zisis , Girtovitis Fotios , Kaiafa Georgia , Ntaios George , Saouli Zoi , Hatzitolios Apostolos , Charisopoulos George , Savopoulos Christos , Papadopoulos Athanasios
Published: October 2019

INTRODUCTION: Macrocytosis (MCV>94fl) is frequently observed at patients suffering from chronic obstructive pulmonary disease (COPD) with or without respiratory insufficiency. Recent studies have demonstrated the role of erythropoietin massive excretion subsequent to hypoxemia as a trigger factor in excessive marrow erythropoiesis. Thus, a large number of immature red cells are released in peripheral blood leading to macrocytosis. AIM: To determine possible correlation between macrocytosis and severity of he disease in patients with COPD.

PATIENTS AND METHODS: Thirty eight patients (mean age: 62,16±10,88 years, twenty nine male-Group A and nine female-Group B) with COPD were attended regarding their clinical status for one year, diagnosed according to spirometric classification of COPD based on post-bronchodilator FEV1 (Global Initiative for Chronic Obstructive Lung Disease, 2006). All patients were smokers suffered from mild or moderate COPD (FEV1/FVC <0,70, FEV1≥50% predicted, with PaO2 more than 60mmHg). MCV and hematocrit were measured separately for male and female additionally to the whole sample. Exacerbations considered to be happen when dyspnea or PaO2<60mmHg occurred. Patients with anemia, hematological malignancy, hypothyroidism, heart or renal failure and diabetes mellitus excluded.

RESULTS: Mean value for MCV was 98,84±2,66 fl (range:95–107 fl). Average for Group A estimated at 99,52±2,92 (range: 95–107) and for Group B at 96,87±1,40 (range: 95–99). Mean value of exacerbations for both groups in one’s year period was 0,89±0,61 (range :0–2), that was not significantly different from those concerning Group A and Group B (0,79±0,60, 1,22±0,74 respectively). Inverse correlation between MCV and number of exacerbations was statistically significant (r = − 0,43, p<0,01). That correlation was more apparent for Group A (r = −0,45, p<0,01) than Group B (r = −0,35, p<0,05). Mean value of hematocrit was determined at 45,92 ± 3,77 (range: 40–54). Correlation between MCV and Ht was not significant (r=0,2).

CONCLUSIONS: An unexpected result revealed considering that elevated MCV in patients with mild to moderate COPD correlates with fewer exacerbations of the disease. A game of statistics or something more important for the pathophysiology of mild COPD?
Correlation of Red Blood Cells and Platelets Parameters.
Authors: Saouli Zoi , Kaiafa Georgia , Girtovitis Fotios , Kontoninas Zisis , Ntaios George , Charisopoulos George , Tsavdaridou Vasiliki , Aggouridaki Christina , Papadopoulos Athanasios
Published: October 2019

INTRODUCTION: Platelet along with red blood cell count is a part of complete blood cell count, one of the most frequent laboratory tests in medicine. Platelet distribution width, plateletcrit and mean platelet volume are three indices provided by hematological analyzers. There are few reports in literature regarding the correlation of these three parameters with red blood cell parameters.

AIM: Aim of this study is to investigate the correlation between these platelets parameters and red cell parameters: hematocrit, mean corpuscular volume and red blood cell distribution width.

METHODS: Three hundred and three healthy blood donor volunteers (176 men and 127 women, mean age 37,3 years) were included. None of them had any known hematological disease in the past. The parameters mentioned above were measured by the automated hematological analyzer Coulter®LH780.

RESULTS: The mean values for platelets were: PCT: 0,25±0,11%, MPV: 8,11±1,94 fL and PDW: 15,89±2,74%. The mean values for their parallel red blood cell parameters were: HCT: 40,55±2,63%), MCV: 91±4,17 fL, RDW: 13,3±1,35% Statistical and regression analysis including the correlation coefficient between platelet and red cell parameters as well as Student’s t-test was carried out.

CONCLUSIONS:

There seems to be no significant correlation between HCT and PCT. MCV and MPV were not correlated significantly as well, indicating that red blood cell and platelet sizes are independent. But there is a statistically significant correlation between RDW and PDW (r: 0,68, p<0,01) demostrating that anisocytosis of red blood cells and platelets might occur simultaneously. Based on these observations, further more studies should be carried out for the correlation between platelets and red blood cell indices in certain disorders.
Rabbit Antithymocyte Globulin in Treatment of Children with Severe Aplastic Anemia.
Authors: Pawelec Katarzyna , Matysiak Michal J. , Niewiadomska Edyta , Jerzy Kowalczyk , Stefaniak Jolanta , Balwierz Walentyna , Zaleska-Czepko Ewa , Chybicka Alicja , Szmyd Krzysztof , Sonta-Jakimczyk Danuta , Bubala Halina , Wysocki Mariusz , Kurylak Andrzej , Wachowiak Jacek , Kaczmarek-Karnold Malgorzata , Stolarska Malgorzata , Krawczuk-Rybak Maryna , Leszczynska Elzbieta , Urasinski Tomasz , Peregud-Pogorzelski Jaroslaw , Balcerska Anna , Wlazlowski Marek
Published: October 2019

In children with severe aplastic anemia (SAA) bone marrow transplantation is the chosen therapy. However, it is only possible in about 15–20% patients who have family donors. Immunosuppressive therapy (IST) is administered in the remaining cases. 105 children in 11 hematology centers of the Polish Pediatric Hematology Group (PPHG) were given IST treatment. 27 patients (12 girls and 15 boys aged 6–16 years) received antithymocyte rabbit globulin (ATG) in the first course of therapy. ATG was administered according to PPHG and Working Party SAA Group of EBMT protocol: ATG 3.75mg/kg iv for 5 days, cyclosporin A (CSA) 5mg/kg orally from day 1 to day 180 and granulocyte-stimulating factor during deep neutropenia. Remission of the disease was assessed on days 84, 112 and 180 from start of therapy on the basis of blood and bone marrow examination. On day 180 remission was obtained in 17 of 27 patients (62.9%), complete remission (CR) in 11 children (40.7%) and partial remission in 6 children (22.2%) There was no response to treatment (NR) in 10 patients (37%). In the no response group, 1 child died and 4 had bone marrow transplants from unrelated donors. The remaining 5 patients received a second course of IST therapy, and 2 of them had CR. In the group of 27 patients, 4 died (14.8%). 2 of them died early (day 52 and day 72 of therapy) due to septicemia and central nervous system (CNS) hemorrhage. The other 2 died late (day 99 and day 365of therapy) due to CNS hemorrhage. Observation time of patients ranged from 1 to 13 years. During this time we noted one relapse. MDS/AML or PNH was not observed in any of the patients. The probability of 13 year survival is 85.1% in our group. Our data suggest that initial SAA treatment with ATG is safe and effective in children. Further studies are needed, however, to assess long-term effectiveness of ATG in IST.
Results of Immunosuppressive Therapy in Children with Severe Aplastic Anaemia (SAA). Report Polish Pediatric Hematology Group (Warszawa, Lublin, Krakow, Wroclaw, Zabrze, Bydgoszcz, Poznan, Lodz, Bialystok, Szczecin, Gdansk).
Authors: Pawelec Katarzyna , Matysiak Michal , Niewiadomska Edyta , Kowalczyk Jerzy , Stefaniak Jolanta , Balwierz Walentyna , Zaleska-Czepko Ewa , Chybicka Alicja , Szmyd Krzysztof , Sonta-Jakimczyk Danuta , Bubala Halina , Wysocki Mariusz , Kurylak Andrzej , Wachowiak Jacek , Kaczmarek-Karnold Malgorzata , Stolarska Malgorzata , Krawczuk-Rybak Maryna , Leszczynska Elzbieta , Urasinski Tomasz , Peregud-Pogorzelski Jaroslaw , Balcerska Anna , Wlazlowski Marek
Published: October 2019

Introduction: Bone marrow transplantation (BMT) from HLA identical family donors is the treatment chosen, according indication of Polish Pediatric Hematology Group, for children with severe aplastic anemia (SAA). When no donor is available, combined immunosuppressive therapy (IST) is given.

Material and method: SAA was recognized in 105 children (42 girls, 73 boys) aged 2–18 years in eleven pediatric hematological center in Poland between 1993–2006 years. All patients received IST according Polish Pediatric Hematology and Working Party SAA of the EBMT protocol: antithymocyte globulin horse or rabbit, cyclosporine A (CSA), prenisolon and in 79 patients G-CSF or GM-CSF was additionally administered. Hematological response was evaluated on day 84 and 180 of therapy.

Results and conclusion: On day 180 remission was obtained in 80 from 105 patients (76.1%), complete remission (CR) in 53 patients (51.5%), partial remission (PR) in 27 (24.7%), There was no response to treatment (NR) in 25 patients (23.8%). Period of observation was from 10 months to 13 years. During this time relapse occurred in 28 patients (25.7%). Patients with NR and relapse were treated second course of IST or BMT from matched bone marrow donors. Unrelated BMT was performed in 15 patients with NR after IST. We observed 22 deaths (8 early during the first 3 months of IST) in all group. During the 13 year of observation we notice one case with late clonally complication (PNH), one case of transformation to AML and one to MDS. Probably of 13 year survival was 75.2% in our group. Our results are effective and compared to other hematological centers.
CD4+CD25+ Regulatory T Cells in Patients with Acute Lymphocytic Leukemia.
Authors: Cao Xiangshan , Xie Xiaobao , Li Aaihua , Qiu Guoqiang
Published: October 2019

Human CD4+CD25+ regulatory T (Treg) cells suppress antigen-specific T cell immune response and contribute to peripheral tolerance. However, little is known about the role of Treg cells in the pathogenesis of acute lymphocytic leukemia (ALL). In this study, the proportion of CD4+CD25+ Treg cells in the peripheral blood of three groups of people [the patients with ALL who had never received any chemotherapy before, the patients with ALL who had achieved partial remission (PR) or complete remission (CR) after chemotherapy and the healthy donors] was evaluated by flow cytometry. The expression level of Foxp3 mRNA of each group was examined by real-time RT-PCR. In vitro, the peripheral blood mononuclear cells (PBMC) from healthy donors were cultured in the serum of ALL patients without chemotherapy; each group was divided into sub-groups according to various serum doses. After co-cultured for 72 hours, the cells of all the groups were harvested separately and further tested the number of CD4+CD25+ Treg cells and the mRNA expression of Foxp3. The results showed that both the percentage of CD4+CD25+ T cells and the mRNA expression level of Foxp3 in patients with chemotherapy were significantly higher than those of the healthy donors and patients without chemotherapy (P<0.01 and P<0.01). Although the population of CD4+CD25+ T cells in ALL patients without chemotherapy was almost the same to that in the healthy donors (P>0.05), the expression of Foxp3 mRNA in former was higher (P<0.05) .The serum derived from ALL patients without chemotherapy remarkably increased the proportion of CD4+CD25+ T cells and the expression level of Foxp3 mRNA in co-cultured system, both of which were statistically higher than those in controls (P<0.05 and P<0.01) and did not vary with the serum dose. These results suggest that the chemotherapy may exert an influence on the production of CD4+CD25+ Treg cells and CD4+CD25+ Treg cells may play an important role in the pathogenesis of ALL.
Platelet Counts Remain Elevated in Two Patients with Idiopathic Thrombocytopenic Purpura after Cessation of Oral Eltrombopag.
Authors: Cheng Gregory
Published: October 2019

Idiopathic thrombocytopenic purpura (ITP) is a disease of inadequate platelet production and increased platelet destruction. Two randomized, double-blind, placebo-controlled trials (TRA100773 A/B) reported that eltrombopag, the first-in-class, oral, platelet growth factor, results in a dose-dependent increase in platelet counts in adult patients with chronic ITP. After treatment of eltrombopag was stopped, platelet counts returned to baseline levels in most patients within 2 weeks, reflecting the normal platelet lifespan (range, 10–14 days). This abstract presents data on two patients enrolled in TRA100773 A/B in whom a prolonged platelet count elevation was observed after cessation of eltrombopag treatment. A 79-year old Asian female (Patient 1) was diagnosed with ITP in September 2003. Prior ITP therapy included prednisolone and Danazol simultaneously in 1997. Prednisolone was stopped after 10 months. Danazol was continued until March 2005 (Day -50). The patient relapsed in March 2005 and received dexamethasone (5 days), IVIg (once), vincristine, and cyclosporin A without a significant response. Eltrombopag treatment was started in May 2005 leading to an increase in platelet counts that remained elevated above 100,000/uL after treatment with eltrombopag was discontinued (Table). Patient 2, a 49-year old Asian female, was diagnosed with ITP in 1997. She relapsed after two courses of dexmethasone 40 mg, QD × 4, administered in September 2003 with a 3-month response and in January 2006 with a 1-month response. Therapy with eltrombopag was initiated in March 2006. As was seen with Patient 1, platelet counts increased after administration of eltrombopag and remained elevated after cessation of eltrombopag (Table). To the best of our knowledge, this is the first report of an ongoing prolonged elevation of platelet counts after cessation of thrombopoietic medication, such as eltrombopag.

Platelet Counts (/uL) Patient 1 Patient 2 *Eltrombpag withdrawn in both patients. Screening 26,000 69,000 Day 1 26,000 26,000 Day 8 193,000 136,000 Day 15* 591,000 283,000 Day 22 664,000 --- Day 50 --- 260,000 Day 57 330,000 116,000 Day 71 112,000 100,000 Day 85 116,000 111,000
Haemostatic Actions of the Folkloric Medicinal Plant Extract, Ankaferd Blood Stopper.
Authors: Goker Hakan , Haznedaroglu Ibrahim C. , Ercetin Sevil , Kirazli Serafettin , Akman Umit , Ozturk Yusuf , Firat Cahit H.
Published: October 2019

Ankaferd Blood Stopper® (ABS), a standardized mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica has been used as a haemostatic agent. However, the essential ‘mechanism of action’ of ABS is currently unknown. The aim of this study is to search the essential mechanism underlying the haemostatic actions of ABS. In our study, ABS induced a very rapid (less than 1 second) formation of a protein network within the plasma and serum. Individual clotting factors namely factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XIII are not affected during the consecutive measurements. Plasma fibrinogen activity and antigen decreased from 302 mg/dl to 10 mg/dl, and fibrinogen antigen decreased from 299 mg/dl to 30 mg/dl, in parallel to the prolongation of thrombin time (TT). Biochemical tests also revealed that total protein, albumin, and globulin levels significantly decreased with the interactions of ABS. Red blood cells come together to form vital erythrocyte mass blocks in the presence of ABS. Vital physiological red blood cell aggregation after the exposure to Ankaferd Bloood Stopper in less than one second is depicted in Figure 1. The network of ABS could cover the entire physiological haemostatic process without unequally disturbing individual clotting factors. The basic mechanism of action for ABS appears to be the formation of an encapsulated protein network representing focal points as a niche for vital erythrocyte aggregation. ABS is a novel effective haemostatic agent that has the therapeutic potential for the management of hemorrhage in medical practice. Clinical trials with that promising medicine can provide the development of a new drug particularly active in pathological haemostasis.

Figure Figure
A Pharmacokinetic Study of the Plasma-Derived Factor IX AlphaNine® and Its Comparison with the Recombinant Factor IX BeneFIX®, in Previously Treated Patients with Severe Hereditary Hemophilia B.
Authors: Cortina Vicente R. , Lissichkov T. , Zavilska K. , Matysiak M. , Gercheva L. , Aznar José A.
Published: October 2019

Objectives The objective of the present study was two fold: first, to determine the pharmacokinetic (PK) profile of the plasma-derived FIX concentrate AlphaNine® in patients with congenital severe haemophilia B (FIX:C 2%). To do this, two PK studies were carried out one six months after the first. The second objective was a comparison of the Alphanine® PK profile with the recombinant Factor IX, BeneFIX®.

Patients and methods The first study was a prospective, five-center, open-label, comparative, PK study carried out in 25 severe hemophilia B patients who received 2 single doses of 65–75 IU/kg of AlphaNine® within 6 months (t=0 and t=6). The following parameters were assessed: in vivo recovery, half-life, AUC, mean residence time and clearance. As an extension of the study, a single dose of 65–75 IU/kg of BeneFIX® was administered in 9 out of 25 patients, after a wash-out period of 7–15 days.

Results Table 1 summarizes the results obtained when comparing AlphaNine® within a period of time of 6 months (PK1 vs PK2) in 25 patients. Table 2 shows the results obtained when comparing the in vivo recovery of AlphaNine ® vs BeneFIX ® in the 9 patients studied.

Conclusions These results confirm that AlphaNine® PK has similar profile as other plasma derived FIX products presently available to treat Hemophilia B patients. In addition, our results show that the recombinant FIX studied, BeneFIX® has a reduced in vivo recovery when is compared to AlphaNine®.

Table 1 Parameter AlphaNine® (PK1) t=0 m AlphaNine® (PK2) t=6 m Results are expressed as Mean (SD) In vivo recovery (IU/dl:IU/kg) 1.0 (0.2) 1.2 (0.4) Half-life (h) 34.5 (6.2) 33.7 (5.4) Clearance (ml/min) 0.07 (0.01) 0.07 (0.01) AUC0-inf (IUxh/dl) 1602 (312) 1644 (360) MRT0-inf (h) 35.8 (5.4) 34.6 (5.2) Table 2 Parameter AlphaNine® (PK2) BeneFIX® Results are expressed as Mean (SD); * p<0.05 for the comparison of the in vivo recovery for the BeneFIX® group with the AlphaNine® PK2 In vivo recovery (IU/dl:IU/kg) 1.3 (0.5) 0.8 (0.2)*